WO2023056456A1 - Vectors and methods for improving cardiac regeneration - Google Patents

Vectors and methods for improving cardiac regeneration Download PDF

Info

Publication number
WO2023056456A1
WO2023056456A1 PCT/US2022/077401 US2022077401W WO2023056456A1 WO 2023056456 A1 WO2023056456 A1 WO 2023056456A1 US 2022077401 W US2022077401 W US 2022077401W WO 2023056456 A1 WO2023056456 A1 WO 2023056456A1
Authority
WO
WIPO (PCT)
Prior art keywords
kdm4d
cell
expression vector
cardiac
cms
Prior art date
Application number
PCT/US2022/077401
Other languages
French (fr)
Inventor
William Robb MACLELLAN
Zhenhe ZHANG
Danny EL-NACHEF
Original Assignee
University Of Washington
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Washington filed Critical University Of Washington
Publication of WO2023056456A1 publication Critical patent/WO2023056456A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C12Y114/11027—[Histone H3]-lysine-36 demethylase (1.14.11.27)

Definitions

  • CM mammalian cardiac myocytes
  • the Hippo pathway is a signaling cascade that plays an essential role in organ size control from Drosophila to mammals by regulating cell proliferation, apoptosis, and stem cell/progenitor cell fate determination.
  • the core components of the Hippo pathway are highly conserved in mammals. Inhibition of the Hippo pathway results in Yap1 translocation to the nucleus, where it can stimulate cell cycle gene transcription and proliferation.
  • Activating Yap1 in CM promotes proliferation and reverses systolic heart failure after infarction.
  • Cell cycle gene transcription is also regulated via complex epigenetic signaling.
  • methylation of histone H3 can activate or repress transcription.
  • H3K4me1 and H3K4me3 mark activate chromatin
  • H3K9me3 and H3K27me3 mark silence chromatin.
  • HMTs histone methyltransferases
  • HDMs histone demethylases
  • the disclosure is based on the surprising discovery that KDM4D synergistically induces cardiac myocyte proliferation with the Hippo- Yap1 pathways. This unexpected discovery of synergistic mechanisms enables new strategies for cardiac regeneration in treating heart disease. [0009]
  • the disclosure provides an expression vector.
  • the expression vector comprises (a) a nucleic acid sequence encoding KDM4D; (b) a nucleic acid sequence encoding an activator/effector of the Hippo-Yap pathway; (c) a promoter that effects overexpression of KDM4D and the activator/effector of the Hippo-Yap pathway; and (d) a regulatory element that inducibly represses the overexpression of KDM4D and/or the activator/effector of the Hippo-Yap pathway.
  • the promoter which in some embodiments is a tissue-specific promoter, is operably linked to the nucleic acid sequence of (a) and (b).
  • the tissue-specific promoter is a cardiac-specific promoter.
  • the regulatory element is a tetracycline responsive element.
  • the expression vector is a viral vector that infects quiescent cells.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • the activator/effector of the Hippo-Yap pathway is selected from one or more of Myc, miR-199, and si-Sav1. The latter two, miR-199 and si-Sav1 are activators of the Hippo-Yap pathway. Myc is activated downstream, and is an effector of Hippo-Yap.
  • Vectors for use in the methods described herein include viral vectors, as well as non- viral vectors, virus-like particles, bacterial vectors, bacteriophage vectors, and other vectors known in the art.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated virus (AAV) vector, or other vector suited for infecting quiescent cells.
  • AAV vector include, but are not limited to, AAV6 and AAV9.
  • a cell comprising the expression vector described herein.
  • the cell is a cardiac myocyte (CM).
  • CM cardiac myocyte
  • the cell is derived from a stem cell.
  • the cell is derived from cardiac tissue.
  • a method of proliferating cardiac myocytes in a mammal comprises administering an expression vector as described herein to the mammal.
  • the disclosure additionally provides a method of promoting cardiac regeneration.
  • the method comprises administering an expression vector described herein to a subject in need of cardiac regeneration.
  • a method of regenerating an organ or tissue comprises administering an expression vector described herein to the mammal.
  • a method for inducing cardiac myocyte (CM) hyperplasia in a mammal comprises grafting CMs to the heart of the mammal, wherein the CMs contain an expression vector as described herein.
  • the expression vector is administered by administering CMs that contain the expression vector.
  • the CMs are adult CMs (ACMs).
  • ACMs adult CMs
  • the methods of the invention can involve administration to the subject by any of a variety of means understood by those skilled in the art to be suitable for particular circumstances.
  • the administration is systemic.
  • the administration is intravenous.
  • the administration is by intra- myocardial injection.
  • the subject is typically a mammal.
  • the mammal is human.
  • the mammal is a veterinary subject.
  • FIGS.1A-1C illustrate the screening of growth factors and miRNAs that promote CM proliferation.
  • FIGS.2A-2H demonstrate the effect of KDM4D and miR-199 on NRVM proliferation.
  • FIGS.4A-4E illustrate the inducible KDM4D mouse model.
  • FIGS.5A-5G demonstrate the additive effect of KDM4D overexpression and Sav1 knockdown on ACM proliferation in vivo.
  • 5A-5D Representative photomicrographs showing EdU, phospho-H3, and Aurora B immunostaing after KDM4D induction and Sav1 knockdown for 2 weeks.
  • Nuclei were stained with DAPI, cell borders with WGA, and cycling cells with EdU, white arrow points to the EdU + CMs in (A). Nuclei were stained with DAPI, CMs with cTnT, and cycling CM with phospho-H3, white arrow points to the phospho-H3 + CMs in (B).
  • A Nuclei were stained with DAPI, CMs with cTnT, and cycling CM with phospho-H3, white arrow points to the phospho-H3 + CMs in (B).
  • 5C Aurora B (arrow) in a dividing CM.
  • Aurora B (white, arrow) in the nucleus of CMs. Nuclei were stained with DAPI, CM actin with ⁇ -actinin, and cycling CM with Aurora B in (C) and (D). Bar 10 ⁇ M.
  • E Quantification of EdU + ACMs in different groups.
  • FIGS.6A-6I depicts transcriptional analysis of Sav1-sh and iKDM4D treated cardiac myocytes in vivo.
  • (6F-6I) Expression of common cell cycle transcription factors after indicated treatments. Statistics: n 3 for each group.
  • FIGS.7A-7H Additive effects of KDM4D and Myc expression on CM proliferation.
  • Ad-GFP was used as control.
  • FIG.8 Is a schematic illustration of a proposed model for KDM4D and Hippo signaling pathway in the regulation of cell cycle activities.
  • KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle by stimulating E2F1 and FoxM1 expression, while miR-199 or Hippo pathway inhibition preferentially up-regulated genes involved in G1/S phase by stimulating Myc expression.
  • FIGS.9A-9E Is a schematic illustration of a proposed model for KDM4D and Hippo signaling pathway in the regulation of cell cycle activities.
  • KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle by stimulating E2F1 and FoxM1 expression, while miR-199 or Hippo pathway inhibition preferentially up-regulated genes involved in G1/S phase by stimulating Myc expression.
  • NRVM purity, virus infection efficiency, and miRNA transfection efficiency are shown in FIG. 1 .
  • A Timeline showing protocol for NRVM in vitro study.
  • B FACS results showing the purity of NRVM after one day of culture by staining cTnT and NKX2.5 protein.
  • C Immunostaining of ⁇ -actinin showing the purity of NRVM after 5 days of culture. Different colors were used to visualize the nuclei and the ⁇ -actinin positive cells.
  • D MOI selection by detecting GFP expression after 5 days of infection.
  • E small RNA transfection efficiency was detected by miR-Dy547 after 3 days of transfection. Left panel was the representative picture showing the miR-Dy547 transfected cells.
  • FIGS.10A-10C KDM4D expression level after Ad-KDM4D infection in NRVMs.
  • B Timeline showing the protocol for KDM4D and H3K9me3 western blot analysis.
  • FIGS.11A-11C KDM4D expression level after Ad-KDM4D infection in NRVMs.
  • FIGS.12A-12D RNA-seq in iKDM4D mouse model.
  • A The pipeline of the RNA- seq.
  • FIG.13 is a set of photomicrographs showing GFP expression detected by anti-GFP antibody in a pig that had been injected with: 1) AAV6-GFP control, 2) AAV6-mir-199, 3) AAV6-KDM4D, and 4) the combination of (2) and (3). Each injection was at a different site in one MI pig heart.
  • FIG.14 is an exemplary photomicrograph and a bar graph illustrating how quantification of the Ki67 positive CMs in MI pig shows that mir-199 treatment led to a dramatic increase in proliferation, and this effect was significantly enhanced by combination treatment with both KDM4D and mir-199.
  • This unexpected discovery of synergistic mechanisms enables new strategies for cardiac regeneration in treating heart disease.
  • lysine-specific demethylase 4D or “KDM4D” means a specific member of the KDM4 family of lysine-specific demethylases that exhibits demethylase activity specific to the methylated lysine residue at position 9 (H3K9) of heterochromatin protein 1 (HP1).
  • the KDM4D has the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence optionally further includes tags, such as, for example, a MYC tag and/or a FLAG tag, as shown in SEQ ID NO: 2.
  • tags such as, for example, a MYC tag and/or a FLAG tag, as shown in SEQ ID NO: 2.
  • activator of the Hippo-Yap pathway refers to an agent that activates Yap transcriptional activity. Examples of such agents include, but are not limited to, miR-199, and si-Sav1. Myc is an effector of Yap, but can be activated by multiple signaling pathways.
  • inducibly represses or “inducible repression” refers to regulation of gene expression whereby expression of the gene can be repressed upon introduction of an inducing condition.
  • the inducing condition can be administration of or contact with an agent that effects the repression.
  • the agent can be a corepressor, such as is found in repressible gene regulation wherein expression is on except when the corepressor is present to suppress gene expression.
  • the agent can be an inducer, such as is found in inducible gene regulation wherein expression is off except when the inducer is present to allow for gene expression.
  • a “regulatory element” refers to an element that regulates gene expression.
  • the regulatory element may induce or repress gene expression in response to the presence or absence of a condition.
  • a regulatory element whose activity is dependent on the presence or absence of a condition is referred to as a “conditional” regulatory element.
  • a “tetracycline responsive element” refers to a regulatory element that reduces expression from a tet-inducible promoter in the presence of tetracycline or a derivative thereof, e.g., doxycycline.
  • tissue-specific promoter refers to a regulatory element that promotes expression of a gene in a tissue-specific manner.
  • a cardiac-tissue-specific promoter has activity in only cardiac tissue.
  • a tissue-specific promoter can be used to restrict unwanted transgene expression in other tissues based on the tissue type as well as facilitate persistent transgene expression.
  • nucleic acid or “polynucleotide” or “oligonucleotide” refers to a sequence of nucleotides, a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • active fragment refers to a substantial portion of an oligonucleotide that is capable of performing the same function of specifically hybridizing to a target polynucleotide.
  • hybridizes means that the oligonucleotide forms a noncovalent interaction with the target DNA molecule under standard conditions.
  • Standard hybridizing conditions are those conditions that allow an oligonucleotide probe or primer to hybridize to a target DNA molecule. Such conditions are readily determined for an oligonucleotide probe or primer and the target DNA molecule using techniques well known to those skilled in the art.
  • the nucleotide sequence of a target polynucleotide is generally a sequence complementary to the oligonucleotide primer or probe.
  • the hybridizing oligonucleotide may contain nonhybridizing nucleotides that do not interfere with forming the noncovalent interaction.
  • the nonhybridizing nucleotides of an oligonucleotide primer or probe may be located at an end of the hybridizing oligonucleotide or within the hybridizing oligonucleotide.
  • an oligonucleotide probe or primer does not have to be complementary to all the nucleotides of the target sequence as long as there is hybridization under standard hybridization conditions.
  • complement and “complementary” as used herein, refers to the ability of two DNA molecules to base pair with each other, where an adenine on one DNA molecule will base pair to a guanine on a second DNA molecule and a cytosine on one DNA molecule will base pair to a thymine on a second DNA molecule.
  • Two DNA molecules are complementary to each other when a nucleotide sequence in one DNA molecule can base pair with a nucleotide sequence in a second DNA molecule.
  • the two DNA molecules 5'-ATGC and 5'-GCAT are complementary, and the complement of the DNA molecule 5'-ATGC is 5'-GCAT.
  • complement and complementary also encompasses two DNA molecules where one DNA molecule contains at least one nucleotide that will not base pair to at least one nucleotide present on a second DNA molecule. For instance the third nucleotide of each of the two DNA molecules 5'-ATTGC and 5'-GCTAT will not base pair, but these two DNA molecules are complementary as defined herein.
  • two DNA molecules are complementary if they hybridize under the standard conditions referred to above.
  • two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity.
  • the term “isolated” means that a naturally occurring DNA fragment, DNA molecule, coding sequence, or oligonucleotide is removed from its natural environment, or is a synthetic molecule or cloned product.
  • the DNA fragment, DNA molecule, coding sequence, or oligonucleotide is purified, i.e., essentially free from any other DNA fragment, DNA molecule, coding sequence, or oligonucleotide and associated cellular products or other impurities.
  • AAV Adeno-associated viruses
  • Aurora B Aurora kinase B
  • Ccnd1 Cyclin D1
  • Ccne1 Cyclin E1
  • Cdk1 Cyclin Dependent Kinase 1
  • cTnT cardiac troponin T
  • E2F1 E2F transcription factor 1
  • EdU 5-ethynyl-2’-deoxyuridine
  • FoxM1 Forkhead Box M1
  • GFP green fluorescent protein
  • H3K9me3 Histone H3 lysine 9 trimethylation
  • KDM4D Lysine Demethylase 4D
  • Myc MYC Proto-Oncogene, BHLH Transcription Factor
  • Myh6 ⁇ -myosin heavy chain
  • Myh7 ⁇ - myosin heavy chain
  • NRVM neonatal rat ventricular myocyte
  • phospho- H3 Phospho-Histone H3
  • Plk1 neonatal rat ventricular myocyte
  • phospho- H3 Phos
  • the disclosure provides an expression vector.
  • the expression vector comprises (a) a nucleic acid sequence encoding KDM4D; (b) a nucleic acid sequence encoding an activator/effector of the Hippo-Yap pathway; (c) a promoter that effects overexpression of KDM4D and the activator/effector of the Hippo-Yap pathway; and (d) a regulatory element that inducibly represses the overexpression of KDM4D and/or the activator/effector of the Hippo-Yap pathway.
  • the promoter is operably linked to the nucleic acid sequence of (a) and (b). In some embodiments, the promoter is a tissue-specific promoter.
  • the activator/effector of the Hippo-Yap pathway is selected from one or more of Myc, miR-199, and si-Sav1.
  • the latter two, miR-199 and si-Sav1 are activators of the Hippo-Yap pathway.
  • Myc is activated downstream, and is an effector of Hippo-Yap.
  • the regulatory element is a tetracycline responsive element.
  • separate promoters serve the functions described in (c) and (d) above.
  • the separate promoter are tissue-specific promoters.
  • the tissue-specific promoter(s) is a cardiac-specific promoter.
  • tissue-specific promoters include, but are not limited to, promoters specific to cardiac tissue, skeletal muscle, neurons, pancreatic islet cells, or hepatocytes.
  • a promoter that is tissue-specific promotes expression of the gene encoded by the nucleic acid sequence predominantly in the particular tissue.
  • the tissue-specific promoter is specific to cardiac tissue.
  • An ⁇ -myosin heavy chain ( ⁇ MHC) promoter is one example of a cardiac-specific promoter.
  • the tissue- specific promoter is specific to liver tissue, or hepatocytes.
  • a CBA promoter is one example of a liver-specific promoter.
  • tissue-specific promoters known in the art include the neuron-specific enolase (NSE) and tubulin ⁇ 1 promoters for neurons, ⁇ 1- antitrypsin and albumin (ALB) promoters for hepatocytes, and troponin, CMV, or myosin light chain-2 (MLC2) for cardiac myocytes.
  • NSE neuron-specific enolase
  • ALB ⁇ 1- antitrypsin and albumin
  • MLC2 myosin light chain-2
  • Representative examples of a regulatory element capable of inducibly repressing expression (or overexpression) include, but are not limited to, tetracycline responsive elements.
  • tetracycline responsive elements include, but are not limited to, tetracycline responsive elements.
  • the regulatory element enables positive regulation of KDM4D expression, while in another embodiment, the regulatory element enables negative regulation of KDM4D expression. In another example, the regulatory element enables tissue-specific and/or condition-specific regulation of KDM4D expression.
  • the expression vector is a viral vector that infects quiescent cells.
  • the viral vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • Vectors for use in the methods described herein include viral vectors, as well as non- viral vectors, virus-like particles, bacterial vectors, bacteriophage vectors, and other vectors known in the art. In one embodiment, the vector is a viral vector.
  • the viral vector is an adeno-associated virus (AAV) vector, or other vector suited for infecting quiescent cells.
  • AAV vector include, but are not limited to, AAV6 and AAV9.
  • Sequences [0056] KDM4D amino acid sequence (SEQ ID NO: 1): Cells [0060]
  • a cell comprising the expression vector described herein is useful for carrying out the methods of regenerating an organ or tissue.
  • the cells can be used for cardiac regeneration and proliferation.
  • the cell is a cardiac myocyte (CM).
  • the cell is derived from a stem cell.
  • the cell is derived from cardiac tissue.
  • the CMs are adult CMs (ACMs).
  • Methods [0061] Provided is a method of proliferating cardiac myocytes in a mammal. In some embodiments, the method comprises administering an expression vector as described herein to the mammal. The disclosure additionally provides a method of promoting cardiac regeneration. In some embodiments, the method comprises administering an expression vector described herein to a subject in need of cardiac regeneration. Further described is a method of regenerating an organ or tissue. In some embodiments, the method comprises administering an expression vector described herein to the mammal. Also described is a method for inducing cardiac myocyte (CM) hyperplasia in a mammal.
  • CM cardiac myocyte
  • the method comprises grafting CMs to the heart of the mammal, wherein the CMs contain an expression vector as described herein.
  • the expression vector is administered by administering CMs that contain the expression vector.
  • the CMs that contain the expression vector are adult CMs (ACMs).
  • ACMs adult CMs
  • the methods of the invention can involve administration to the subject by any of a variety of means understood by those skilled in the art to be suitable for particular circumstances.
  • the administration is systemic.
  • the administration is intravenous.
  • the administration is by intra- myocardial injection.
  • the subject is typically a mammal. In one embodiment, the mammal is human.
  • the mammal is a veterinary subject.
  • veterinary subjects include, but are not limited to, equine, canine, bovine, porcine, ovine, and feline subjects.
  • EXAMPLES [0064] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.
  • Example 1 Synergistic Interaction of Pathways Regulating Cardiomyocyte Proliferation [0065] Accumulating data demonstrates that new adult cardiomyocytes (CMs) are generated throughout life from pre-existing CMs, although the absolute magnitude of CM self-renewal is very low.
  • CMs new adult cardiomyocytes
  • KDM4D and miR-199 further increased total cell number of NRVMs in culture.
  • Inhibition of Hippo signaling via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1) also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression.
  • Inducibly activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone.
  • KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while miR-199 and si-Sav1 preferentially up-regulated genes involved in G1/S phase.
  • KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc.
  • KDM4D/Myc significantly increased CM proliferation but did not affect cardiac function.
  • KDM4D effects on CM proliferation are additive with the Hippo-Yap1 pathway and appear to preferentially regulate different cell cycle regulators. This has important implications for strategies that target cardiac regeneration in treating heart disease.
  • CMs cardiomyocytes
  • ACM adult CM
  • Oncostatin M (OSM) and IL-13 stimulate ACM cell cycle re-entry and improve cardiac function after myocardial infarction (MI) through Raf/MEK/Erk and STAT3/6 signaling pathway.
  • 7 Extracellular matrix (ECM) components such as periostin and agrin have also been reported to regulate CM proliferation and may provide a therapeutic target for advanced heart failure.
  • 9 10
  • the utility of these approaches remain uncertain but the concept of promoting endogenous myocardial proliferation and regeneration is a promising strategy to prevent the development of heart failure after myocardial injury. [0070]
  • the mechanisms through which these pro-growth factors exert their effects on CMs is poorly understood but several miRNAs have also been reported to promote CMs proliferation.
  • 11-14 Inhibition of miRNAs increased CM cycling and enhanced expression of Cyclin A2 (Ccna2).
  • 11 Deletion of miR-128 promotes CM proliferation by increasing expression of the chromatin modifier SUZ12, which suppresses p27, a cyclin-dependent kinase inhibitor, hence activating positive cell cycle regulators Cyclin E (Ccne1) and Cdk2.
  • SUZ12 chromatin modifier
  • Cyclin E Cyclin E
  • Cdk2 Cyclin E
  • a high-throughput screen of 875 miRNAs found at least 40 miRNAs that increased both DNA synthesis and cytokinesis in cultured neonatal mouse and rat CMs.
  • miR-590 and miR-199a could promote cell-cycle re-entry and stimulate CM proliferation in both neonatal and adult rat CMs.
  • Loss of miR-302-367 led to decreased CM proliferation during development while miR-302-367 overexpression resulted in a marked increase in CM cycling, in part through the repression of the Hippo signaling pathway.
  • 17 The majority of miRNAs that impact CM cycling appear to exert their effect through the Hippo signaling pathway. 17-19
  • the Hippo pathway signaling cascade plays an essential role in organ size control from Drosophila to mammals by regulating cell proliferation, apoptosis, and stem cell/progenitor cell fate determination. 20-22 And the core components of the Hippo pathway are highly conserved in mammals.
  • H3K4me1 and H3K4me3 mark activate chromatin
  • H3K9me3 and H3K27me3 mark silence chromatin.
  • H3K27me3 mark silence chromatin.
  • HMTs histone methyltransferases
  • HDMs histone demethylases
  • H3K9me3 has been demonstrated in numerous studies. Diverse roles for H3K9me3 have been identified in regulating apoptosis 36 , autophagy 37 , development 38 , DNA repair 39, 40 , self-renewal 41 , and aging 42 , among others.
  • H3K9me3 itself is not specific to cell cycle genes, in CM H3K9me3 depletion could preferntially increase cell cycle gene expression through the disruption of specific inhibitory complexes that bind H3K9me3 or H3K9me3-adapter proteins.
  • Hippo pathway and epigenetic manipulations individually or in combination, on CM cycling and proliferation in both in vitro and in vivo settings.
  • Our data demonstrate that combining KDM4D overexpression with inhibition of the Hippo pathway significantly increases CM proliferation compared to either single treatment alone.
  • RNA-seq Isolation of adult mouse ventricular myocytes
  • cardiomyocytes were isolated using Langendorff perfusion digestion as previously described.
  • 43 RNA-seq
  • Two independent samples from each group were used for RNA-seq.
  • Library preparation and sequencing was performed by commercial service (GENEWIZ) as described in Example 2.
  • Quantitative real-time PCR [0086] RNA extraction, reverse transcription, and real-time quantitative polymerase chain reaction (qRT-PCR) were performed as described in the Example 2. Primer sequences for qPCR are detailed in Table 2.
  • miR-199 decreased the proportion of pYap1/Yap1 in NRVMs as reported 26 (Fig.2A- B).
  • Ad-KDM4D did not change Yap1 phosphyorlation (Fig.2B), suggesting different mechanisms of action (Fig.2A).
  • Ad-KDM4D preferentially increased the expression of G2/M genes including Cyclin-dependent kinase 1 (Cdk1), polo-like kinase 1 (Plk1), Aurora Kinase B (Aurkb), and Survivin (SurV) expression, but did not significantly increase G1/S phase genes (Fig.2C).
  • miR-199 increased Cyclin D1 (Ccnd1) and Cyclin E1 (Ccne1) expression compared to control, but not Cdk1, Plk1, Aurkb, or SurV expression (Fig. 2C).
  • Ccnd1 Cyclin D1
  • Cyclin E1 Cyclin E1
  • Fig. 2C There was a additive increase in expression of Plk1 and Aurkb when KDM4D and miR-199 were combined (Fig.2C).
  • FGF had no additive effect on Ad-KDM4D- or miR-199-induced cell cycle gene expression (Fig.2D).
  • si-Sav1 increased Ccnd1 and Ccne1 expression compared to control, but not Cdk1, Plk1, Aurkb, or SurV expression. FGF had no additive effect on the expression of these genes (Fig.3B).
  • si-Sav1 treatment increased EdU+ CMs, but not phospho-H3+ CMs nor total cell number (Fig.3D, 3E, &3F).
  • Table 3 28 cell cycle DEGs in iKDM4D group compared to Sav1-sh [0105] Control-vs-iKDM4D (cont’d) [0107]
  • CM-specific reverse tetracycline transactivator (rtTA) mice 44 were mated to a KDM4D tet-responder (tet) line 4 .
  • the resulting mice (iKDM4D) displayed tightly regulated KDM4D gene expression in the heart with doxycycline treatment (Fig.11A).
  • KDM4D depleted H3K9me3 and up regulated cell cycle genes (Fig.4A-4C). Late cell cycle genes Cdk1 and AurkB were up-regulated 4-fold and 6-fold, respectively (p ⁇ 0.05).
  • KDM4D induction did not result in a significant difference in HW/BW ratio in iKDM4D mice compared to control mice at two weeks (Fig.4D).
  • CMs isolated from iKDM4D hearts had an average area 30% smaller than control CMs, (3437 ⁇ m 2 ⁇ 55 ⁇ m 2 for iKDM4D and 4993 ⁇ m 2 ⁇ 350 ⁇ m 2 for ctrl; p ⁇ 0.01; Fig.4D), suggesting a total increase in the number of CMs.
  • iKDM4D and AAV9-Sav1-sh led to further increases in cell cycle activity including a 3-fold increase in DNA synthesis activity (Fig.5E), 7.8-fold increase in mitosis (Fig.5F), and ⁇ 3-fold increase in cytokinesis compared to wild- type control (Fig.5G).
  • KDM4D and Hippo induce distinct transcriptional reprogramming in ACMs
  • RNA-seq was performed on control (wildtype mice injected with AAV9-GFP), Sav1-sh (wildtype mice injected with AAV9-U6- Sav1-sh-GFP), and iKDM4D (iKDM4D mice injected with AAV9-GFP) (Fig.12A).
  • Fig.6A 295 upreguatled genes and 362 downregulated genes in iKDM4D group compared to Sav1-sh group
  • Fig.6B Among those differentially expressed genes, GO analysis identified 28 cell cycle genes (Fig.6B). There were 13 genes upregulated in the iKDM4D group of which 11 are involved in cell division, including Trnp 45 , Anln 46 , Lrrcc1 47 , Map9 48 , 6- Sep 49 , Eid1 50 , Prkcd 51 , Dab2ip 52 , Mapk12 53 , Haus8 54 , and Tacc1 55 .
  • FGF had no additive effect over KDM4D, miR-199, or si-Sav1 on the expression of these TFs (Fig. 6G&6I). Consistent with these findings, the E2F1 promoter does not have any TEAD binding sites, but the Myc promoter contains four (Fig.12C). 7 (43%) of the promoters of the 16 Sav1-sh upregulated genes contained at least one Myc binding site (Fig.6D).
  • KDM4D and Myc additively induce cardiomyocyte proliferation but does not impact cardiac function
  • NRVMs Ad-Myc and Ad- KDM4D+Ad-Myc treatments increased Myc expression leading to a 3-fold enhancement of EdU + CMs (Fig.7A-7B).
  • Ad-KDM4D treated NRVMs exhibited a ⁇ 2-fold increase in pH3 + CMs, (Fig.7C).
  • Ad-KDM4D, Ad-Myc, and Ad-KDM4D+Ad-Myc all increased total cell number after 6 days of culture compared to the control (Fig.7D).
  • HW/BW ratio was increased 17% and 25% respectively compared to wild-type littermates (p ⁇ 0.05), but dual expression of MycER and KDM4D had a ⁇ 1.6-fold increase in heart mass compared to control (p ⁇ 0.05; Fig.7E).
  • CM size was similar in these mice regardless of MycER or KDM4D expression (Fig.7F) suggesting that the increase in heart mass was due to increased CM number. This CM proliferation was supported by the finding of increased phospho-H3 + CMs in MycER+KDM4D Tg/+ hearts (Fig.7G-7H).
  • RNA-seq was performed on CM samples from iKDM4D and Sav1-sh treated mice.
  • Our RNA-Seq data indicated different transcriptional profiles between iKDM4D and Sav1-sh group.
  • KDM4D overexpression preferentially upregulated the G2/M phase genes, such as, Lrrcc1 47 , Map9 48 , and Dab2ip 52 , which are involved in mitotic spindle formation.
  • Mapk12 53 and Haus8 54 regulate the activity of Plk1 which plays an essential role in mitosis.
  • Trnp 45 , Anln 46 , 6-Sep 49 , Prkcd 51 , and Tacc1 55 also participate in the cell division.10 of the 15 upregulated cell cycle genes in the Sav1-sh group are involved in the G1/S phase. Gadd45a 58 and Crocc 64 expression are higher in the G1 phase. Rgs2 59 , Ddit3 60 , Tspyl2 61 , and Txnip 63 are involved in the G0-G1 switch or G1 checkpoint. Tfdp2 56 , Usp2 57 , Pim3 62 , and Mybl2 65 are also engaged in the G1/S phase of cell cycle.
  • KDM4D overexpression downregulated TEAD3 expression which is a transcriptional enhancer factor that plays a key role in the Hippo signaling pathway.
  • 74 KDM4D overexpression upregulated Patj and Mob1b expression which play an important role in LATS1/2 and MST1/2 phosphorylation.
  • 75, 76, 77 14 upregulated genes in the Sav1-sh group contained TEAD binding sites, and 13 of them have been linked to cell proliferation. 7 of the 13 proliferation-related genes also contained canonical Myc binding sites. Among them, Mid1ip1, Usp2, Art3, and Nr4a1 impact the G1/S phase of the cell cycle in different cell types.
  • KDM4D regulated a number of genes involved in proliferation-related pathways, including the ErbB signaling pathway, Wnt signaling pathway, and p53 signaling pathway (Fig.12D).
  • ErbB signaling activation is required for G2 checkpoint activation in human breast cancer cells.
  • 89 Inhibition of the ErbB signaling pathway induces G2/M arrest in gastric cancer cells.
  • 90 Wnt/ ⁇ -catenin signaling activity peaks during the G2/M phase, 91 and inhibition of Wnt/ ⁇ -catenin signaling leads to G2/M phase arrest.
  • Our data demonstrated that 9 downregulated genes in the iKDM4D group were enriched in p53 signaling pathways.
  • KDM4D may also regulate E2F1 activity since protein lysine methyltransferases and demethylases can modify a specific lysine residue on non-histone substrates with one or more methyl moieties, such as E2F1 101 , which impacts the activity or subcellular localization of the substrate protein.
  • 102-105 [0123]
  • Si-Sav1 or miR-199 preferentially induce G1/S phase cell cycle genes at least in part through activating Myc signaling pathway, while KDM4D promotes G2/M phase by regulating E2F1 and FoxM1 expression.
  • Yamaguchi F et al. International journal of oncology 2008;32:377-385.
  • Example 2 Supplemental Methods re Interaction of Pathways Regulating Cardiomyocyte Proliferation
  • Animals [0231] All animals were maintained and experiments performed in accordance with institutional guidelines at the University of Washington, and National Institute of Health Guide for the Care and Use of Laboratory Animals. The inducible KDM4D mouse model used in this study was generated previously in our lab. 1 Tet-responsive-KDM4D mice were bred to the inducible ⁇ MHC-tTA (KDM4D Tg/+ ; tet-off) or repressible ⁇ MHC-rTA (iKDM4D; tet- on) mice.
  • RNA vectors Ad-CMV-GFP-h-KDM4D (Ad-KDM4D), Ad-CMV-GFP-h-c-Myc (Ad- Myc), Ad-CMV-GFP-h-control (Ad-GFP), AAV9-U6-sh-Sav1-eGFP (AAV9-Sav1-sh), and its control AAV9-U6-scramble-eGFP (AAV9-C-sh) were purchased from Vector Biolabs (Malvern, PA).
  • miRNA and siRNA used in this study including hsa-miR-199a-3p mimic (miR-199), hsa-miR-590-3p mimic (miR-590), hsa-miR-302b-5p mimic/hsa-miR-302c-5p mimic (miR-302), miRIDIAN microRNA Mimic Negative Control #1 (miR-C), ON- TARGETplus SMARTpool Sav1 siRNA (si-Sav1), and ON-TARGETplus control siRNA (si- C), were purchased from Dharmacon Inc.
  • NRVMs were isolated as described before. FACS and immunostaining of ⁇ -actinin results demonstrate that NRVM purity was more than 99% (Fig.9B-9C). NRVMs were cultured and treated as shown in Figure 9A. In brief, NRVMs were plated on fibronectin- coated 24-well plates at a density of 1 ⁇ 10 ⁇ 5 per well.
  • NRVMs were seeded and cultured with 500 ⁇ L M199 culture medium (Medium 199500ml, HEPEs 10mM, MEM Non-Essential Amino Acids 1 ⁇ , glucose 1.75g, L-glutamine 2mM, Vitamin B122mg, and penicllin 50,000 units) with 10% FBS.
  • M199 culture medium Medium 199500ml, HEPEs 10mM, MEM Non-Essential Amino Acids 1 ⁇ , glucose 1.75g, L-glutamine 2mM, Vitamin B122mg, and penicllin 50,000 units
  • Ad-KDM4D Ad-KDM4D
  • Ad-Myc Ad-Myc
  • control Ad-GFP Ad-GFP
  • the NRVMs were transfected with either miRNA (25nM) or siRNA (25nM) using LipofectamineTM 3000 accordinging to the manufacturer’s protocol (Life Technologies) and/or treated with FGF (100ng/ml), EGF (100ng/ml), and OSM (50ng/ml).
  • FGF 100ng/ml
  • EGF 100ng/ml
  • OSM 50ng/ml
  • the transfection efficiency was assayed using miR-Dy547 control demonstrating 90% cells were transfected (Fig.9E).
  • the NRVMs were maintained for another 48 hours, and then switched to the 500 ⁇ L fresh serum-free medium with 5nM 5-Ethynyl-2'-deoxyuridine (EdU).
  • mice were given three intramyocardial injections using Hamilton syringe (50 ⁇ l capacity) with a 33-gauge needle to deliver a total of 2 ⁇ 10 11 viral genomes (30 ⁇ l total volume delivered) into the apex of the left ventricle. 6 24 hours after injection, the mice were treated with Doxycycline in rodent chow and EdU in drinking water (ad.lib) until the study endpoint. Two weeks after injections, the mice were sacrificed, and the hearts harvested for immunostaining and RNA extraction. [0238] Isolation of adult mouse ventricular myocytes [0239] For RNA-seq, cardiomyocytes were isolated using Langendorff perfusion digestion as previously described.
  • mice 7 8 to 10 week old iKDM4D mice were injected with AAV9-Sav1-sh or control virus (AAV9-GFP). After two weeks of injection, the mice were intraperitoneally injected with 200 ⁇ l of heprin (100 IU/mouse) before being anesthetize with Isoflurane.
  • AAV9-GFP control virus
  • a 37°C Ca 2+ - free Tyrodes buffer 126 mM NaCl, 5.4 mM KCl, 0.33 mM NaH 2 PO 4 , 1 mM MgCl 2 , 10 mM H
  • the cell suspensions were passed through a 100 ⁇ m cell strainer to remove tissue debris and then purified by low- speed centrifugation (50 ⁇ g for 1min) 3 times, resulting in ⁇ 90% pure ACMs.
  • NRVMs or heart tissue sections were fixed with 4% PFA in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 20min, and blocked with PBS containing 5% NDS (Normal Donkey Serum) for 1h at room temperature.
  • the cells were incubated overnight at 4qC with the following antibodies diluted in the blocking buffer: anti-cTnT (Thermo Scientific: MS-295-P) and Phalloidin (Thermo Scientific: R415) was used to identify CMs, Click-iT EdU Alexa Fluor 647 HCS Assay (Thermo Scientific: C10356) to identify the S phase of the cell cycle, anti-phospho-H3 (Thermo Scientific: PA5-17869) to identify the M phase of the cell cycle, anti-Aurora B antibody (Abcam: ab2254) to identify the cytokinesis phase of the cell cycle . The cells were then washed three times with PBS+5%FBS and stained for 45min at room temperature with secondary antibodies.
  • DAPI 6-diamidino-2-phenylindole dihydrochloride
  • the cells were viewed under Nikon fluorescence microscope.
  • To determine cross-sectional area (CSA) of cells 10 ⁇ m thick heart slides were stained by Anti-wheat germ agglutinin (WGA). The CSA was determined by the region within the WGA staining boundary.
  • Quantitative real-time PCR [0243] Total RNA was isolated using TRIzolTM Reagent (Invitrogen) and then purified using RNeasy Mini Kit (Qiagen). One microgram RNA was reverse transcribed using the High Capacity Reverse Transcription cDNA Synthesis Kit (Applied Biosystems).
  • RNA-seq Two independent samples from each group were used for RNA-seq. Library preparation and sequencing was performed by commercial service (GENEWIZ). Sequence reads were trimmed to remove adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. 8 The trimmed reads were mapped to the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.
  • CiiiDER was used to scan transcription factors binding site on the promoter of DEGs.
  • Protein Analysis [0247] Western blots were performed by the SDS-PAGE electrophoresis. Total cell extracts were prepared and fractionated by gel electrophoresis and transferred to nitrocellulose membranes.
  • Anti-KDM4D (ab93694; Abcam), anti-H3K9me3 (ab8898; Abcam), anti-Sav1 (105105; Abcam), Yap1 (ab81183; Abcam), pYap1 (ab76252; Abcam), Myc (ab32072; Abcam) , Anti-Histone H3 Antibody (07-690 from Sigma-Aldrich), GAPDH (ab181603; Abcam). Horseradish peroxidase anti-rabbit (ab205718; Abcam) was used as the secondary antibody. The signal was detected using the super- signal-enhanced chemiluminescence system (Pierce).
  • Example 3 Gene Therapy for Cardiac Regeneration Using an Ischemia Reperfusion Model for CM Repair
  • This Example can be used to confirm the feasibility of cardiac regeneration in large animals through stimulation of the cardiac cell proliferation via gene therapy.
  • Adeno-associated viral vector (AAV) mediated induction of cardiac proliferation This approach can be employed to test the ability of two putative inducers of the cardiac cell cycle, KDM4D and miR-199, individually and in combination to stimulate CM proliferation in swine.
  • AAV Adeno-associated viral vector
  • two na ⁇ ve farm pigs are each transfected with 1) 2x10 13 AAV6- KDM4D, 2) AAV6-miR-199, 3) combined AAV6-KDM4D and 2x10 13 AAV6-miR-199 or 4) vehicle control.
  • Direct delivery of 2x10 13 AAV is achieved via partial sternotomy with indwelling catheter placement. Delivery is by 100 ⁇ L dose (1x10 13 cells/mL) x 20 injections. This is followed by injection of BrdU at day 2 to 12 post-transfection. The heart is harvested at day 12 for histology.
  • endpoints will include cardiac proliferation (BrdU/Ki67 as marker of proliferation) and cardiac function (MRI) following MI.
  • cardiac proliferation BrdU/Ki67 as marker of proliferation
  • MRI cardiac function
  • the efficacy of cardiac proliferation following AAV gene therapy can be assessed in swine (12 subjects total, 30-day endpoint).
  • the protocol involves transfection of 4 post-MI farm pigs with 1) 2x10 13 AAV6-KDM4D, 2) AAV6-miR-199, 3) combined AAV6-KDM4D and 2x10 13 AAV6-miR-199 or 4) AAV6-GFP. Baseline MRI is obtained.
  • a 90-min percutaneous ischemia-reperfusion injury of mid-LAD and indwelling catheter and telemetry placement is performed on day -14.
  • Post-MI MRI is obtained on day -2.
  • Direct delivery of 2x10 13 AAV is achieved via partial sternotomy. This is followed by injection of BrdU day 2 to 30 post-transfection. MRI is obtained, and the heart is harvested on day 30 for histology.
  • KDM4D and mir-199 will stimulate adult CM proliferation in vivo in this large animal model. Likewise, we expect this proliferation to improve cardiac function following MI.
  • Example 4 Synergistic Effect of Combination KDM4D/mir-199 Gene Therapy for CM Repair
  • This Example demonstrates the synergistic stimulation of cardiac regeneration in a large animal via gene therapy.
  • the protocol described in Example 3 was followed in a study in which one pig was injected with: 1) AAV6-GFP control, 2) AAV6-mir-199, 3) AAV6- KDM4D, and 4) the combination of (2) and (3).
  • Each injection was at a different site in one MI pig heart. The heart was harvested at day 12 for histology.
  • GFP expression detected by anti-GFP antibody was used to confirm successful transgene delivery.
  • Fig.14 shows how quantification of Ki67 positive CMs was performed in the MI pig to measure proliferation of CMs.
  • KDM4D treatment alone did not result in more detectable proliferation than control or uninjected areas
  • mir-199 treatment led to a dramatic increase in proliferation, and this effect was significantly enhanced by combination treatment with both KDM4D and mir-199.
  • Example 5 Increasing Cardiomyocyte Proliferation via Transduction of Stem Cell-Derived CMs to Enhance Cell Engraftment
  • This Example describes how to transduce SC-derived CMs with vectors of the invention to increase the size of subsequent grafts.
  • hPSC-CMs pluripotent stem cells
  • CM proliferation increases cardiomyocyte (CM) proliferation.
  • KDM4D lysine demethylase 4D
  • Hippo-YAP pathway we found significantly increased CM proliferation compared to either treatment alone.
  • the optimal MOI can be determined by tracking GFP+ve hiPSC-CM. One can then confirm H3K9 demethylation and/or activation Hippo-YAP pathway in infected hiPSC-CM. One can then measure S-phase (EDU incorporation), M-phase (phosho-H3) and proliferation (cell number). [0278] To determine if KDM4D and/or mir199 transfected hiPSC-CMs improve cell engraftment, we will use Nod-Scid IL2R ⁇ deficient (NSG) mice. The mice will be transplanted with KDM4D and/or mir199 transfected hiPSC-CMs.
  • NSG Nod-Scid IL2R ⁇ deficient mice
  • the study will be comprised of up to 4 different treatment groups (Control, mir199, KDM4D and mir199+KDM4D). Each group will be comprised of 12 NSG mice. Engraftment will be measured using the Alu-PCR assay. [0279] Next we will generate transfected hiPSC-CMs for in vivo experiments, namely transplantation of transfected hiPSC-CMs into mouse hearts. Hearts will be harvested 14 days post-injection and Alu PCR quantified to determine engrafted human cells. [0280] We expect that KDM4D and/or mir199 transfected hiPSC-CMs will improve cell engraftment. [0281] Throughout this application various publications are referenced.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Cardiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Rheumatology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Vascular Medicine (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Materials and methods for improving cardiac regeneration are provided, which are based on the surprising discovery that KDM4D synergistically induces cardiac myocyte proliferation with the Hippo-Yap1 pathways. Described is an expression vector that comprises (a) a nucleic acid sequence encoding KDM4D; (b) a nucleic acid sequence encoding an activator/effector of the Hippo-Yap pathway; (c) a promoter that effects overexpression of KDM4D and the activator/effector of the Hippo-Yap pathway; and (d) a regulatory element that inducibly represses the overexpression of KDM4D and/or the activator/effector of the Hippo-Yap pathway. The promoter, which in some embodiments is a tissue-specific promoter, is operably linked to the nucleic acid sequence of (a) and (b). This unexpected discovery of synergistic mechanisms enables new strategies for cardiac regeneration in treating heart disease.

Description

VECTORS AND METHODS FOR IMPROVING CARDIAC REGENERATION [0001] This application claims benefit of United States provisional patent application number 63/261,978, filed October 1, 2021, the entire contents of which are incorporated by reference into this application. REFERENCE TO A SEQUENCE LISTING [0002] The content of the XML file of the sequence listing named “UW76_seq”, which is 36 kb in size, created on September 30, 2022, and electronically submitted herewith the application, is incorporated herein by reference in its entirety. BACKGROUND [0003] The vast majority of mammalian cardiac myocytes (CM) stop proliferating soon after birth and subsequent heart growth predominately comes from hypertrophy, an increase in cell size, instead of hyperplasia, an increase in cell number. Because CM proliferation is required for the heart regeneration seen in lower vertebrates and neonatal mammalian injury models, there is great interest in understanding the mechanisms regulating CM cell cycle exit and whether this cell cycle withdrawal can be reversed. [0004] Ischemic heart disease leading to heart failure is the leading cause of death in the world. Although adult human hearts are unable to replace lost CMs after injury, substantial cardiac regeneration is seen in lower vertebrate and mammalian models. Adult zebrafish and neonatal mice are able to regenerate their hearts after ≥15% has been amputated. Models of myocardial infarction (MI) in newborn mice offer a more clinically relevant injury model to demonstrate heart regeneration capacity in mammals. A common finding in these studies was the mechanism by which cardiac regeneration occurred. Blood clot formation, inflammation, and collagen deposition were seen in response to the injuries, but ultimately new CMs repopulated the lost tissue. Fate mapping studies revealed that the new cardiac myocytes came from dedifferentiation and proliferation of pre-existing cardiac myocytes, in contrast to cardiac progenitor or stem cells. However, when cardiac injury was induced in mice at a later time-point, postnatal day 7 (P7), the regenerative response was lost leading to fibrotic scarring similar to what is seen with human MIs. Thus, mammalian hearts lose their regenerative capacity early in life, a process that requires CM proliferation. [0005] The Hippo pathway is a signaling cascade that plays an essential role in organ size control from Drosophila to mammals by regulating cell proliferation, apoptosis, and stem cell/progenitor cell fate determination. The core components of the Hippo pathway are highly conserved in mammals. Inhibition of the Hippo pathway results in Yap1 translocation to the nucleus, where it can stimulate cell cycle gene transcription and proliferation. Activating Yap1 in CM promotes proliferation and reverses systolic heart failure after infarction. [0006] Cell cycle gene transcription is also regulated via complex epigenetic signaling. In particular, methylation of histone H3 can activate or repress transcription. For example, H3K4me1 and H3K4me3 mark activate chromatin, whereas H3K9me3 and H3K27me3 mark silence chromatin. These histone modifications are tightly controlled by histone methyltransferases (HMTs), and histone demethylases (HDMs) enzymes. United States patent publication 20180117125 demonstrated that depletion of H3K9me3 by KDM4D in ACMs results in remarkable transcriptomic reprogramming and preferentially leads to increased cell cycle gene expression and enhanced CM cycling. [0007] There remains a need to determine whether lysine demethylase 4D (KDM4D)- mediated CM-specific H3K9 demethylation and Hippo pathways inhibition have additive or redundant roles in promoting CM proliferation, and to provide improved means by which this cell cycle withdrawal can be reversed. There further remains a need for improved methods of treating ischemic heart disease to reduce the incidence of heart failure and related deaths. SUMMARY [0008] The present disclosure meets these needs and others by providing materials and methods for improving cardiac regeneration. The disclosure is based on the surprising discovery that KDM4D synergistically induces cardiac myocyte proliferation with the Hippo- Yap1 pathways. This unexpected discovery of synergistic mechanisms enables new strategies for cardiac regeneration in treating heart disease. [0009] In one embodiment, the disclosure provides an expression vector. In one embodiment, the expression vector comprises (a) a nucleic acid sequence encoding KDM4D; (b) a nucleic acid sequence encoding an activator/effector of the Hippo-Yap pathway; (c) a promoter that effects overexpression of KDM4D and the activator/effector of the Hippo-Yap pathway; and (d) a regulatory element that inducibly represses the overexpression of KDM4D and/or the activator/effector of the Hippo-Yap pathway. The promoter, which in some embodiments is a tissue-specific promoter, is operably linked to the nucleic acid sequence of (a) and (b). [0010] In some embodiments, the tissue-specific promoter is a cardiac-specific promoter. In some embodiments, the regulatory element is a tetracycline responsive element. In some embodiments, the expression vector is a viral vector that infects quiescent cells. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. [0011] In some embodiments, the activator/effector of the Hippo-Yap pathway is selected from one or more of Myc, miR-199, and si-Sav1. The latter two, miR-199 and si-Sav1 are activators of the Hippo-Yap pathway. Myc is activated downstream, and is an effector of Hippo-Yap. [0012] Vectors for use in the methods described herein include viral vectors, as well as non- viral vectors, virus-like particles, bacterial vectors, bacteriophage vectors, and other vectors known in the art. In one embodiment, the vector is a viral vector. In a particular embodiment, the viral vector is an adeno-associated virus (AAV) vector, or other vector suited for infecting quiescent cells. Representative examples of an AAV vector include, but are not limited to, AAV6 and AAV9. [0013] Further provided is a cell comprising the expression vector described herein. In some embodiments, the cell is a cardiac myocyte (CM). In some embodiments, the cell is derived from a stem cell. In some embodiments, the cell is derived from cardiac tissue. [0014] Also described is a method of proliferating cardiac myocytes in a mammal. In some embodiments, the method comprises administering an expression vector as described herein to the mammal. The disclosure additionally provides a method of promoting cardiac regeneration. In some embodiments, the method comprises administering an expression vector described herein to a subject in need of cardiac regeneration. Further described is a method of regenerating an organ or tissue. In some embodiments, the method comprises administering an expression vector described herein to the mammal. Also described is a method for inducing cardiac myocyte (CM) hyperplasia in a mammal. In some embodiments, the method comprises grafting CMs to the heart of the mammal, wherein the CMs contain an expression vector as described herein. [0015] In some embodiments, the expression vector is administered by administering CMs that contain the expression vector. In some embodiments, the CMs are adult CMs (ACMs). [0016] The methods of the invention can involve administration to the subject by any of a variety of means understood by those skilled in the art to be suitable for particular circumstances. In some embodiments, the administration is systemic. In other embodiments, the administration is intravenous. In some embodiments, the administration is by intra- myocardial injection. The subject is typically a mammal. In one embodiment, the mammal is human. In other embodiments, the mammal is a veterinary subject. Examples of veterinary subjects include, but are not limited to, equine, canine, bovine, porcine, ovine, and feline subjects. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIGS.1A-1C illustrate the screening of growth factors and miRNAs that promote CM proliferation. (1A) Representative photomicrographs showing EdU immunostaing after treating growth factors and/or miRNAs for 72 hours. Nuclei were stained by Dapi, CMs by a- actinin and cycling cells by EdU. Differential coloring was used to distinguish the stains. Bar=100μM. (1B) Effects of indicated growth factors on CM EdU incorporation after 72 hours treatment in NRVMs. (1C) Impact of overexpression of indicated miRNAs on CM EdU incorporation after 72 hours transfection in NRVMs. NRVMs were exposed to EdU for 24 hours. Statistics: Fold change of EdU+ NRVMs was normalized to control group; *p<0.05 in One-way ANOVA analysis followed by Tukeys’ Test; n=3 for each group. [0018] FIGS.2A-2H demonstrate the effect of KDM4D and miR-199 on NRVM proliferation. (2A) Representative western blot of cell lysates from NRVMs treated with 5 days of Ad- KDM4D and 3 days of miR-199 (representative data from one of the three replicates). (2B) Quantification of pYap1/Yap1 ratio from western blot. (2C) RT-qPCR analysis shows the expression of cell cycle genes after 5 days of Ad-KDM4D and 3 days of miR-199 treatment. (2D) The effect of FGF on KDM4D- and miR-199- stimulated cell cycle gene expression after 3 days (RT-qPCR assay). (2E) EdU+ incorporation in NRVMs treated with or without Ad- KDM4D, miR-199, and FGF. (2F) pH3+ immunostaining of NRVMs treated with indicated interventions. (2G) Total cell number was measured by hemocytometer on NRVMs treated as indicated. (2H) Representative photomicrographs showing EdU/phospho-H3 immunostaing after different treatments. EdU, bar=100μM; phospho-H3, bar=200μM. Statistics: * showed statistical significance at p<0.05 vs control (without miR-199, Ad- KDM4D, and FGF); # represented statistical significance at p<0.05 vs miR-199; $ mean statistical significance at p<0.05 vs Ad-KDM4D; & showed statistical significance at p<0.05 vs miR-199+Ad-KDM4D. One-way ANOVA followed by Tukeys’ Test; n=3 for all the experiments. [0019] FIGS.3A-3G demonstrate the effect of KDM4D and si-Sav1 on NRVM proliferation. (3A) Representative western blot of cell lysates from NRVMs after 3 days of Sav1 knockdown 199 (representative data from one of the three replicates (3B) Expression of cell cycle genes in Ad-KDM4D and si-Sav1 treated NRVMs. (3C) Effects of adding FGF to Ad- KDM4D and si-Sav1 on cell cycle gene expression. (3D) EdU+ incorporation in NRVMs treated with Ad-KDM4D and/or si-Sav1, and FGF. (3E) phospho-H3+ immunostaining in NRVMs treated with or without Ad-KDM4D, si-Sav1, and FGF. (3F) Total NRVM number after indicated treatments. (3G) Representative photomicrographs showing EdU/phospho-H3 immunostaing after different treatments. EdU, bar=100μM; phospho-H3, bar=200μM. Statistics: * showed statistical significance at p<0.05 vs control (without si-Sav1, Ad-KDM4D, and FGF); # represented statistical significance at p<0.05 vs si-Sav1; $ mean statistical significance at p<0.05 vs Ad-KDM4D; & showed statistical significance at p<0.05 vs si- Sav1+Ad-KDM4D. One-way ANOVA followed by Tukeys’ Test; n=3 for all the experiments. [0020] FIGS.4A-4E illustrate the inducible KDM4D mouse model. (4A) Immunoblotting of ACM lysates (left) showing CM-specific KDM4D protein induction in iKDM4D hearts and depletion of H3K9me3 with two weeks of induction in ACMs. Densitometry analysis (right) shows KDM4D, and H3K9me3 levels relative to PanH3 control in iKDM4D CMs with (n=3) and iKDM4D CMs without (n=3) doxycycline induction. * p<0.05. (4B) KDM4D transgene expression is robustly induced in iKDM4D ACMs fold induction vs. control (-Dox), expression normalized to Gapdh. (4C) Expression of CM and cell cycle genes in isolated ACMs measured by qRT-PCR, fold induction vs. control (-Dox), expression normalized to Gapdh. (4D) HW/BW quantification in uninduced control and iKDM4D mice (-Dox) and induced in control and iKDM4D (+Dox). (4E) Quantification of ACM area (μm2), isolated 12-wk CMs control, and iKDM4D (+Dox). Sample Number: (4A) iKDM4D (-Dox) = 3, iKDM4D (+Dox) = 3. (4B) All groups = 8 (4C) Control (+Dox) = 3, iKDM4D (+Dox) = 3 (4D-4E) Ӌ3 animals per group. Statistics: (4A) Two-tailed t-test, iKDM4D (-Dox) vs iKDM4D (+Dox), *P < 0.05. (4B)
Figure imgf000007_0001
[0021] FIGS.5A-5G demonstrate the additive effect of KDM4D overexpression and Sav1 knockdown on ACM proliferation in vivo. (5A-5D) Representative photomicrographs showing EdU, phospho-H3, and Aurora B immunostaing after KDM4D induction and Sav1 knockdown for 2 weeks. Nuclei were stained with DAPI, cell borders with WGA, and cycling cells with EdU, white arrow points to the EdU+ CMs in (A). Nuclei were stained with DAPI, CMs with cTnT, and cycling CM with phospho-H3, white arrow points to the phospho-H3+ CMs in (B). (5C) Aurora B (arrow) in a dividing CM. (5D) Aurora B (white, arrow) in the nucleus of CMs. Nuclei were stained with DAPI, CM actin with α-actinin, and cycling CM with Aurora B in (C) and (D). Bar=10μM. (5E) Quantification of EdU+ ACMs in different groups. EdU+ CM number per mm2 is shown. (5F) Quantification of phospho-H3+ ACMs in different group. Phospho-H3+ CM number per mm2 is shown. (5G) Quantification of Aurora B+ ACMs in different group. Aurora B+ CM number per mm2 is shown. Statistics: n=3 for each group. * showed statistical significance at p<0.05 vs control in WT group; # represented statistical significance at p<0.05 vs control in iKDM4D group. One-way ANOVA followed by Tukeys’ Test. [0022] FIGS.6A-6I depicts transcriptional analysis of Sav1-sh and iKDM4D treated cardiac myocytes in vivo. (6A) The global transcriptional change in the iKDM4D groups compared with Sav1-sh was visualized by a volcano plot. Each data point in the scatter plot represents a gene. The log2 fold change of each gene is represented on the x-axis and the log10 of its adjusted p-value is on the y-axis. Genes with an adjusted p-value less than 0.05 and a log2 fold change greater than 1 represent upregulated genes (dots at right). Genes with an adjusted p-value less than 0.05 and a log2 fold change less than -1 represent downregulated genes (dots at left). (6B) Fold change of 28 cell cycle genes in iKDM4D group compared to Sav1-sh. (6C) Summary of upregulated genes in Sav1-sh and iKDM4D group compared to control group. (6D) Summary of promoter binding site analysis on upregulated genes in Sav1-sh and iKDM4D groups. Percentage of genes for each binding site are shown. The number in parenthesis represents the total number of genes. (6E) Representative promoter binding site analysis. List promoters (-1500bp-500bp) of 16 upregulated genes in Sav1-sh group compared to control group. E2F1 binding site (rightward-hashed boxes); Myc binding site (leftward-hashed boxes); TEAD binding site (unhashed boxes). (6F-6I) Expression of common cell cycle transcription factors after indicated treatments. Statistics: n=3 for each group. * showed statistical significance at p<0.05 vs control (without miR-199, Ad-KDM4D, and FGF); # represented statistical significance at p<0.05 vs miR-199; $ mean statistical significance at p<0.05 vs Ad-KDM4D. One-way ANOVA followed by Tukeys’ Test. [0023] FIGS.7A-7H. Additive effects of KDM4D and Myc expression on CM proliferation. (7A) Representative western blot after 5 days of Ad-Myc infection (MOI 50, 100, and 200) in NRVMs (representative data from one of the three replicates ). (7B-7D) EdU+ incorporation, phospho-H3+ immunostaining, and total cell number in NRVMs treated with Ad-KDM4D or Ad-Myc for 5 days. Ad-GFP was used as control. (7E-7G) Quantification of HW/BW, ACM cross sectional area, and phospho-H3+ ACMs number at 10 weeks of MycER/KDM4DTg/+ mice after one week of tamoxifen treatment. (7H) Representative pictures showing phospho- H3 positive ACMs in transgenic MycER/KDM4DTg/+ mouse model. Thick section imaging of adult BiTg heart showing XY plane and reconstructed 75 μm depth of XZ and YZ orthogonal planes. White crosshairs indicate position within all 3 planes, arrows point to the phospho- H3+ ACMs, bar = 20 μm (n=3 for each genotype). Statistics: *p< 0.05 vs Control, #p<0.05 vs
Figure imgf000008_0001
One-way ANOVA followed by Tukeys’ Test. [0024] FIG.8. Is a schematic illustration of a proposed model for KDM4D and Hippo signaling pathway in the regulation of cell cycle activities. KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle by stimulating E2F1 and FoxM1 expression, while miR-199 or Hippo pathway inhibition preferentially up-regulated genes involved in G1/S phase by stimulating Myc expression. [0025] FIGS.9A-9E. NRVM purity, virus infection efficiency, and miRNA transfection efficiency. (A) Timeline showing protocol for NRVM in vitro study. (B) FACS results showing the purity of NRVM after one day of culture by staining cTnT and NKX2.5 protein. (C) Immunostaining of α-actinin showing the purity of NRVM after 5 days of culture. Different colors were used to visualize the nuclei and the α-actinin positive cells. (D) MOI selection by detecting GFP expression after 5 days of infection. (E) small RNA transfection efficiency was detected by miR-Dy547 after 3 days of transfection. Left panel was the representative picture showing the miR-Dy547 transfected cells. Right panel was the quantification of miR- Dy547 transfected cells. Data are shown as mean ± SEM (n=3 independent experiments). [0026] FIGS.10A-10C. KDM4D expression level after Ad-KDM4D infection in NRVMs. (A) KDM4D expression level increased after 3 days of Ad-KDM4D infection detected by qPCR. Sample number=1 for each treatment. (B) Timeline showing the protocol for KDM4D and H3K9me3 western blot analysis. (C) KDM4D and H3K9me3 protein expression level at different time point after Ad-KDM4D infection detected by western blot. Sample number=1 for each treatment. [0027] FIGS.11A-11C. Inducible KDM4D mouse model construction, induction protocol, and model testing after myocardial injection of AAV9-Sav1-sh and AAV9-control. (A) Schematic showing breeding strategy resulting in iKDM4D mice, and KDM4D induction in BiTg CMs. (B) Timeline showing protocol for ACM-specific KDM4D expression and endpoints. (C) The whole heart scanning showing the myocardial injection efficiency after 2 weeks (representative image from one of the three animals in each group). [0028] FIGS.12A-12D. RNA-seq in iKDM4D mouse model. (A) The pipeline of the RNA- seq. (B) The global transcriptional change in the Sav1-sh and iKDM4D groups compared with control was visualized by a volcano plot. Each data point in the scatter plot represents a gene. The log2 fold change of each gene is represented on the x-axis and the log10 of its adjusted p-value is on the y-axis. Genes with an adjusted p-value less than 0.05 and a log2 fold change greater than 1 represent upregulated genes (dots at right). Genes with an adjusted p-value less than 0.05 and a log2 fold change less than -1 represent downregulated genes (dots at left). (C) Transcription factors binding site analysis on the common cell cycle transcription factors promoter. The promoter sequence was analyzed from -1500 to +500. Rightward-hashed bars represent E2F1 binding site, leftward-hashed bars represent Myc binding site, and unhashed bars represent TEAD binding site. (D) KEGG pathway analysis between control and iKDM4D by DAVID Bioinformatics Resources. The number of genes is represented on the x-axis, and the KEGG pathways are listed on the y- axis. [0029] FIG.13 is a set of photomicrographs showing GFP expression detected by anti-GFP antibody in a pig that had been injected with: 1) AAV6-GFP control, 2) AAV6-mir-199, 3) AAV6-KDM4D, and 4) the combination of (2) and (3). Each injection was at a different site in one MI pig heart. The heart was harvested at day 12 for histology. Sections show DAPI- stained nuclei, WGA-stained cell membranes, and anti-GFP to confirm transgene delivery. While no GFP was detected in the “no injection” region, GFP was detected in all other regions (see boxed areas). Whole sections were scanned by Nikon A1 confocal at same settings; images were gained at same LUTs settings. [0030] FIG.14 is an exemplary photomicrograph and a bar graph illustrating how quantification of the Ki67 positive CMs in MI pig shows that mir-199 treatment led to a dramatic increase in proliferation, and this effect was significantly enhanced by combination treatment with both KDM4D and mir-199. DETAILED DESCRIPTION [0031] The disclosure is based on the surprising discovery that KDM4D synergistically induces cardiac myocyte proliferation with the Hippo-Yap1 pathways. This unexpected discovery of synergistic mechanisms enables new strategies for cardiac regeneration in treating heart disease. KDM4D and miR-199/si-Sav1 combinatorially promote CM cycling through distinct pathways. Si-Sav1 or miR-199 preferentially induce G1/S phase cell cycle genes at least in part through activating Myc signaling pathway, while KDM4D promotes G2/M phase by regulating E2F1 and FoxM1 expression. These additive in vitro results were replicated in vivo, demonstrating these interventions also work in adult CMs. Enhancing endogenous CM proliferation to compensate for the lost CMs after injury offers a promising strategy to prevent the development of heart failure. Definitions [0032] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified. [0033] As used herein, “lysine-specific demethylase 4D” or “KDM4D” means a specific member of the KDM4 family of lysine-specific demethylases that exhibits demethylase activity specific to the methylated lysine residue at position 9 (H3K9) of heterochromatin protein 1 (HP1). In one embodiment, the KDM4D has the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence optionally further includes tags, such as, for example, a MYC tag and/or a FLAG tag, as shown in SEQ ID NO: 2. [0034] As used herein, “activator of the Hippo-Yap pathway” refers to an agent that activates Yap transcriptional activity. Examples of such agents include, but are not limited to, miR-199, and si-Sav1. Myc is an effector of Yap, but can be activated by multiple signaling pathways. Collectively, these activators and effectors are referred to as “activators/effectors of the Hippo-Yap pathway.” [0035] As used herein, “inducibly represses” or “inducible repression” refers to regulation of gene expression whereby expression of the gene can be repressed upon introduction of an inducing condition. The inducing condition can be administration of or contact with an agent that effects the repression. The agent can be a corepressor, such as is found in repressible gene regulation wherein expression is on except when the corepressor is present to suppress gene expression. Alternatively, the agent can be an inducer, such as is found in inducible gene regulation wherein expression is off except when the inducer is present to allow for gene expression. [0036] As used herein, a “regulatory element” refers to an element that regulates gene expression. The regulatory element may induce or repress gene expression in response to the presence or absence of a condition. A regulatory element whose activity is dependent on the presence or absence of a condition is referred to as a “conditional” regulatory element. [0037] As used herein, a “tetracycline responsive element” refers to a regulatory element that reduces expression from a tet-inducible promoter in the presence of tetracycline or a derivative thereof, e.g., doxycycline. One example of a tetracycline responsive element is a tetracycline-controlled transactivator (tTA), created by fusion of the tetracycline repressor (tetR) with a transcriptional activation domain, such as the C-terminal domain of VP16 of herpes simplex virus (HSV). This is one example of conditional regulation of gene expression. [0038] As used herein, tissue-specific promoter refers to a regulatory element that promotes expression of a gene in a tissue-specific manner. For example, a cardiac-tissue-specific promoter has activity in only cardiac tissue. A tissue-specific promoter can be used to restrict unwanted transgene expression in other tissues based on the tissue type as well as facilitate persistent transgene expression. [0039] The term “nucleic acid” or “polynucleotide” or “oligonucleotide” refers to a sequence of nucleotides, a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. [0040] As used herein, the term “active fragment” refers to a substantial portion of an oligonucleotide that is capable of performing the same function of specifically hybridizing to a target polynucleotide. [0041] As used herein, "hybridizes," "hybridizing," and "hybridization" means that the oligonucleotide forms a noncovalent interaction with the target DNA molecule under standard conditions. Standard hybridizing conditions are those conditions that allow an oligonucleotide probe or primer to hybridize to a target DNA molecule. Such conditions are readily determined for an oligonucleotide probe or primer and the target DNA molecule using techniques well known to those skilled in the art. The nucleotide sequence of a target polynucleotide is generally a sequence complementary to the oligonucleotide primer or probe. The hybridizing oligonucleotide may contain nonhybridizing nucleotides that do not interfere with forming the noncovalent interaction. The nonhybridizing nucleotides of an oligonucleotide primer or probe may be located at an end of the hybridizing oligonucleotide or within the hybridizing oligonucleotide. Thus, an oligonucleotide probe or primer does not have to be complementary to all the nucleotides of the target sequence as long as there is hybridization under standard hybridization conditions. [0042] The term "complement" and "complementary" as used herein, refers to the ability of two DNA molecules to base pair with each other, where an adenine on one DNA molecule will base pair to a guanine on a second DNA molecule and a cytosine on one DNA molecule will base pair to a thymine on a second DNA molecule. Two DNA molecules are complementary to each other when a nucleotide sequence in one DNA molecule can base pair with a nucleotide sequence in a second DNA molecule. For instance, the two DNA molecules 5'-ATGC and 5'-GCAT are complementary, and the complement of the DNA molecule 5'-ATGC is 5'-GCAT. The term complement and complementary also encompasses two DNA molecules where one DNA molecule contains at least one nucleotide that will not base pair to at least one nucleotide present on a second DNA molecule. For instance the third nucleotide of each of the two DNA molecules 5'-ATTGC and 5'-GCTAT will not base pair, but these two DNA molecules are complementary as defined herein. Typically two DNA molecules are complementary if they hybridize under the standard conditions referred to above. Typically, two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity. [0043] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise. [0044] As used herein, to “prevent” or “protect against” a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease. [0045] As used herein, the term "isolated" means that a naturally occurring DNA fragment, DNA molecule, coding sequence, or oligonucleotide is removed from its natural environment, or is a synthetic molecule or cloned product. Preferably, the DNA fragment, DNA molecule, coding sequence, or oligonucleotide is purified, i.e., essentially free from any other DNA fragment, DNA molecule, coding sequence, or oligonucleotide and associated cellular products or other impurities. [0046] Abbreviations and Acronyms: [0047] AAV: Adeno-associated viruses; Aurora B: Aurora kinase B; Ccnd1: Cyclin D1; Ccne1: Cyclin E1; Cdk1: Cyclin Dependent Kinase 1; cTnT: cardiac troponin T; E2F1: E2F transcription factor 1; EdU: 5-ethynyl-2’-deoxyuridine; FoxM1: Forkhead Box M1; GFP: green fluorescent protein; H3K9me3: Histone H3 lysine 9 trimethylation; KDM4D: Lysine Demethylase 4D; Myc: MYC Proto-Oncogene, BHLH Transcription Factor; Myh6: α-myosin heavy chain; Myh7: β- myosin heavy chain; NRVM: neonatal rat ventricular myocyte; phospho- H3: Phospho-Histone H3; Plk1: Polo Like Kinase 1; Sav1: Salvador Family WW Domain Containing Protein 1; SurV: survivin; Yap1: Yes-associated protein1; α-SA: α-sarcomeric actinin. Vectors [0048] In one embodiment, the disclosure provides an expression vector. In one embodiment, the expression vector comprises (a) a nucleic acid sequence encoding KDM4D; (b) a nucleic acid sequence encoding an activator/effector of the Hippo-Yap pathway; (c) a promoter that effects overexpression of KDM4D and the activator/effector of the Hippo-Yap pathway; and (d) a regulatory element that inducibly represses the overexpression of KDM4D and/or the activator/effector of the Hippo-Yap pathway. The promoter is operably linked to the nucleic acid sequence of (a) and (b). In some embodiments, the promoter is a tissue-specific promoter. [0049] In some embodiments, the activator/effector of the Hippo-Yap pathway is selected from one or more of Myc, miR-199, and si-Sav1. The latter two, miR-199 and si-Sav1, are activators of the Hippo-Yap pathway. Myc is activated downstream, and is an effector of Hippo-Yap. [0050] In some embodiments, the regulatory element is a tetracycline responsive element. In another embodiment, separate promoters serve the functions described in (c) and (d) above. In some embodiments, the separate promoter are tissue-specific promoters. [0051] In some embodiments, the tissue-specific promoter(s) is a cardiac-specific promoter. Representative examples of tissue-specific promoters include, but are not limited to, promoters specific to cardiac tissue, skeletal muscle, neurons, pancreatic islet cells, or hepatocytes. A promoter that is tissue-specific promotes expression of the gene encoded by the nucleic acid sequence predominantly in the particular tissue. In one embodiment, the tissue-specific promoter is specific to cardiac tissue. An α-myosin heavy chain (αMHC) promoter is one example of a cardiac-specific promoter. In another embodiment, the tissue- specific promoter is specific to liver tissue, or hepatocytes. A CBA promoter is one example of a liver-specific promoter. Other examples of tissue-specific promoters known in the art include the neuron-specific enolase (NSE) and tubulin α1 promoters for neurons, α1- antitrypsin and albumin (ALB) promoters for hepatocytes, and troponin, CMV, or myosin light chain-2 (MLC2) for cardiac myocytes. [0052] Representative examples of a regulatory element capable of inducibly repressing expression (or overexpression) include, but are not limited to, tetracycline responsive elements. Those skilled in the art will appreciate alternative methods of controlled gene expression that can be adapted for use in a similar manner to regulate the expression of KDM4D, both temporally and histologically. For example, in one embodiment, the regulatory element enables positive regulation of KDM4D expression, while in another embodiment, the regulatory element enables negative regulation of KDM4D expression. In another example, the regulatory element enables tissue-specific and/or condition-specific regulation of KDM4D expression. [0053] In some embodiments, the expression vector is a viral vector that infects quiescent cells. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. [0054] Vectors for use in the methods described herein include viral vectors, as well as non- viral vectors, virus-like particles, bacterial vectors, bacteriophage vectors, and other vectors known in the art. In one embodiment, the vector is a viral vector. In a particular embodiment, the viral vector is an adeno-associated virus (AAV) vector, or other vector suited for infecting quiescent cells. Representative examples of an AAV vector include, but are not limited to, AAV6 and AAV9. [0055] Sequences [0056] KDM4D amino acid sequence (SEQ ID NO: 1):
Figure imgf000014_0001
Figure imgf000015_0001
Cells [0060] A cell comprising the expression vector described herein is useful for carrying out the methods of regenerating an organ or tissue. For example, the cells can be used for cardiac regeneration and proliferation. In some embodiments, the cell is a cardiac myocyte (CM). In some embodiments, the cell is derived from a stem cell. In some embodiments, the cell is derived from cardiac tissue. In some embodiments, the CMs are adult CMs (ACMs). Methods [0061] Provided is a method of proliferating cardiac myocytes in a mammal. In some embodiments, the method comprises administering an expression vector as described herein to the mammal. The disclosure additionally provides a method of promoting cardiac regeneration. In some embodiments, the method comprises administering an expression vector described herein to a subject in need of cardiac regeneration. Further described is a method of regenerating an organ or tissue. In some embodiments, the method comprises administering an expression vector described herein to the mammal. Also described is a method for inducing cardiac myocyte (CM) hyperplasia in a mammal. [0062] In some embodiments, the method comprises grafting CMs to the heart of the mammal, wherein the CMs contain an expression vector as described herein. In some embodiments, the expression vector is administered by administering CMs that contain the expression vector. In some embodiments, the CMs that contain the expression vector are adult CMs (ACMs). [0063] The methods of the invention can involve administration to the subject by any of a variety of means understood by those skilled in the art to be suitable for particular circumstances. In some embodiments, the administration is systemic. In other embodiments, the administration is intravenous. In some embodiments, the administration is by intra- myocardial injection. The subject is typically a mammal. In one embodiment, the mammal is human. In other embodiments, the mammal is a veterinary subject. Examples of veterinary subjects include, but are not limited to, equine, canine, bovine, porcine, ovine, and feline subjects. EXAMPLES [0064] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention. Example 1: Synergistic Interaction of Pathways Regulating Cardiomyocyte Proliferation [0065] Accumulating data demonstrates that new adult cardiomyocytes (CMs) are generated throughout life from pre-existing CMs, although the absolute magnitude of CM self-renewal is very low. Modifying epigenetic histone modifications or activating the Hippo- Yap pathway have been shown to promote adult CM cycling and proliferation. Whether these interventions work through common pathways or act independently is unknown. This Example demonstrates, for the first time, that lysine demethylase 4D (KDM4D)-mediated CM-specific H3K9 demethylation and Hippo pathways inhibition have additive or redundant roles in promoting CM proliferation. [0066] This Example shows that activating Yap1 in cultured neonatal rat ventricular myocytes (NRVM) through overexpressing Hippo pathway inhibitor, miR-199, preferentially increased S-phase CMs, while H3K9me3 demethylase KDM4D preferentially increased G2/M markers in CMs. Together KDM4D and miR-199 further increased total cell number of NRVMs in culture. Inhibition of Hippo signaling via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1) also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducibly activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while miR-199 and si-Sav1 preferentially up-regulated genes involved in G1/S phase. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Using transgenic mice over-expressing KDM4D together with Myc, we demonstrated that KDM4D/Myc significantly increased CM proliferation but did not affect cardiac function. [0067] KDM4D effects on CM proliferation are additive with the Hippo-Yap1 pathway and appear to preferentially regulate different cell cycle regulators. This has important implications for strategies that target cardiac regeneration in treating heart disease. [0068] Introduction [0069] In mammals, cardiomyocytes (CMs) proliferate rapidly during embryonic development then exit the cell cycle shortly after birth.1 However, increasing evidence demonstrates that pre-existing CMs are the source of CM renewal through proliferation in adult hearts but only do so at a very low rate throughout life.1-3 Therefore, enhancing adult CM (ACM) proliferation is an attractive strategy to compensate for the lost CMs in injured myocardium.4, 5 Numerous factors have been analyzed for their effect on CM proliferation. Fibroblast growth factor 1 (FGF1)/p38 MAP kinase inhibitor in post-infarct rats increased CM proliferation, reduced scarring, and markedly improved cardiac function.6 Oncostatin M (OSM) and IL-13 stimulate ACM cell cycle re-entry and improve cardiac function after myocardial infarction (MI) through Raf/MEK/Erk and STAT3/6 signaling pathway.7, 8 Extracellular matrix (ECM) components such as periostin and agrin have also been reported to regulate CM proliferation and may provide a therapeutic target for advanced heart failure.9, 10 The utility of these approaches remain uncertain but the concept of promoting endogenous myocardial proliferation and regeneration is a promising strategy to prevent the development of heart failure after myocardial injury. [0070] The mechanisms through which these pro-growth factors exert their effects on CMs is poorly understood but several miRNAs have also been reported to promote CMs proliferation.11-14 Inhibition of miRNAs, (miR-29a, miR-30a, and miR-141) increased CM cycling and enhanced expression of Cyclin A2 (Ccna2).11 Deletion of miR-128 promotes CM proliferation by increasing expression of the chromatin modifier SUZ12, which suppresses p27, a cyclin-dependent kinase inhibitor, hence activating positive cell cycle regulators Cyclin E (Ccne1) and Cdk2.15 A high-throughput screen of 875 miRNAs found at least 40 miRNAs that increased both DNA synthesis and cytokinesis in cultured neonatal mouse and rat CMs.16 In particular, miR-590 and miR-199a could promote cell-cycle re-entry and stimulate CM proliferation in both neonatal and adult rat CMs. Loss of miR-302-367 led to decreased CM proliferation during development while miR-302-367 overexpression resulted in a marked increase in CM cycling, in part through the repression of the Hippo signaling pathway.17 The majority of miRNAs that impact CM cycling appear to exert their effect through the Hippo signaling pathway.17-19 The Hippo pathway signaling cascade plays an essential role in organ size control from Drosophila to mammals by regulating cell proliferation, apoptosis, and stem cell/progenitor cell fate determination.20-22 And the core components of the Hippo pathway are highly conserved in mammals.23-25 The target of the Hippo pathway in mammals, the transcription factor Yes-associated protein1 (Yap1) can itself induce CM proliferation.26, 27 28 [0071] Cell cycle gene transcription is also regulated via complex epigenetic signaling. In particular, methylation of histone H3 can activate or repress transcription.29, 30 For example, H3K4me1 and H3K4me3 mark activate chromatin, whereas H3K9me3 and H3K27me3 mark silence chromatin.31, 32 These histone modifications are tightly controlled by histone methyltransferases (HMTs), and histone demethylases (HDMs) enzymes.33, 34 35 Recently, our lab demonstrated that depletion of H3K9me3 by KDM4D in ACMs results in remarkable transcriptomic reprogramming and preferentially leads to increased cell cycle gene expression and enhanced CM cycling.4 H3K9me3's function in cellular development, acting as a repressor of inappropriate lineage genes, and preserving cell integrity has been demonstrated in numerous studies. Diverse roles for H3K9me3 have been identified in regulating apoptosis36, autophagy37, development38, DNA repair39, 40, self-renewal41, and aging42, among others. Although H3K9me3 itself is not specific to cell cycle genes, in CM H3K9me3 depletion could preferntially increase cell cycle gene expression through the disruption of specific inhibitory complexes that bind H3K9me3 or H3K9me3-adapter proteins. [0072] We sought to test the combined impact of Hippo pathway and epigenetic manipulations, individually or in combination, on CM cycling and proliferation in both in vitro and in vivo settings. Our data demonstrate that combining KDM4D overexpression with inhibition of the Hippo pathway significantly increases CM proliferation compared to either single treatment alone. We found that the Hippo pathway and KDM4D activate distinct transcriptional pathways, which likely accounts for their different effects. Combinatorial targeting of epigenetic and transcriptional pathways is a potential novel strategy for myocardial regeneration. [0073] Animals [0074] For details of mouse lines used see Supplementary materials in Example 2. All animals were maintained and experiments performed in accordance with an approved Institutional Animal Care and Use Committee (IACUC protocol #4290-01), institutional guidelines at the University of Washington, and National Institute of Health Guide for the Care and Use of Laboratory Animals. Neonatal rats were sacrificed by decapitation, and adult mice were sacrificed by isoflurane overdose. [0075] Virus, miRNA, and siRNA [0076] The virus, miRNA, and siRNA used in this study are listed in Supplementary materials of Example 2. [0077] Cell culture and Transfections [0078] NRVMs were isolated as described before. FACS and immunostaining of α-actinin results demonstrate that NRVM purity was more than 99% (Fig.9B-9C). NRVMs were cultured and treated as shown in Figure 9A. [0079] In vivo gene transfer [0080] To examine the effect of KDM4D and Sav1 on CM cell cycle and proliferation in vivo, adult (8-12 weeks) iKDM4D mice were injected with AAV9-Sav1-sh or control virus (AAV9- GFP) as described in Example 2. Anesthesia was induced with 5% isoflurane and maintained at 1–3% during surgery. Mice were sacrificed by isoflurane overdose two weeks after injection and heart tissue was harvested immediately. [0081] Isolation of adult mouse ventricular myocytes [0082] For RNA-seq, cardiomyocytes were isolated using Langendorff perfusion digestion as previously described.43 [0083] RNA-seq [0084] Two independent samples from each group were used for RNA-seq. Library preparation and sequencing was performed by commercial service (GENEWIZ) as described in Example 2. [0085] Quantitative real-time PCR [0086] RNA extraction, reverse transcription, and real-time quantitative polymerase chain reaction (qRT-PCR) were performed as described in the Example 2. Primer sequences for qPCR are detailed in Table 2. [0087] Table 2: Primer Sequences Use for qPCR
Figure imgf000020_0001
[0088] Protein Analysis [0089] Western blots were performed by the SDS-PAGE electrophoresis. [0090] Statistical analysis [0091] All in vitro experiments were carried out with at least n ≥3 biological replicates, unless otherwise specified. Animal or cells per group are identified in related figure legends. In experiments with multiple groups, analysis of variance (ANOVA) followed by Tukey’s test was used to compare group means. P value <0.05 was considered to represent a statistically significant difference. In all panels, numerical data are presented as mean ± SEM. [0092] Results [0093] Screening for growth factors and miRNAs that promote CM cycling [0094] To identify factors that influence CM proliferation, we screened growth factors and miRNAs that stimulate CM cycling using NRVMs.6, 8, 16, 17 We found that all the growth factors tested increased EdU+ CM significantly except for OSM (~3-fold increases compared to control, p<0.05), but there were no differences in potency between factors (Fig.1B). Compared to the control group, both miR-199 and miR-302 treatment increased EdU+ CMs (~2.5-fold, p<0.05), while miR-590 had no significant effect. The effects of growth factors and miRs were not additive as stimulating NRVMs with FGF in addition to miRNAs did not further increase EdU+ cell number when compared to FGF or miRNA alone (Fig.1C). [0095] KDM4D and miR-199 preferentially impact different phases of cell cycle in vitro [0096] To investigate the impact of KDM4D in addition to miRs on CM proliferation, we treated NRVMs with Ad-KDM4D, miR-199, and miR-199+Ad-KDM4D. As expected Ad- KDM4D reduced the levels of H3K9me3 (Fig.2A). [0097] miR-199 decreased the proportion of pYap1/Yap1 in NRVMs as reported26 (Fig.2A- B). Ad-KDM4D did not change Yap1 phosphyorlation (Fig.2B), suggesting different mechanisms of action (Fig.2A). Ad-KDM4D preferentially increased the expression of G2/M genes including Cyclin-dependent kinase 1 (Cdk1), polo-like kinase 1 (Plk1), Aurora Kinase B (Aurkb), and Survivin (SurV) expression, but did not significantly increase G1/S phase genes (Fig.2C). In contrast, miR-199 increased Cyclin D1 (Ccnd1) and Cyclin E1 (Ccne1) expression compared to control, but not Cdk1, Plk1, Aurkb, or SurV expression (Fig. 2C). There was a additive increase in expression of Plk1 and Aurkb when KDM4D and miR- 199 were combined (Fig.2C). We also tested for additive effects of FGF stimulation in combination with Ad-KDM4D or miR-199. FGF had no additive effect on Ad-KDM4D- or miR-199-induced cell cycle gene expression (Fig.2D). We next investigated the effect of Ad-KDM4D and miR-199 on CM cycling directly by examining EdU+ incorporation, phospho- H3+ (phospho histone H3) nuclei, and total cell number. FGF and miR-199 but not Ad- KDM4D increased EdU+ CMs compared to controls (Fig.2E). FGF+miR-199 or FGF+Ad- KDM4D treatment both increased EdU+ number, but not significantly more than FGF treatment alone (Fig.2E). In contrast, Ad-KDM4D increased pH3+ CMs as well as total cell number (Fig.2F&2G). [0098] Additive effects of KDM4D overexpression and Hippo signaling inhibition on ACM proliferation in vivo [0099] This differential impact of Ad-KDM4D and miR-199 on cell cycle was unexpected. To confirm these results, we chose to test the effects of a direct regulator of the Hippo signaling pathway using siRNA to Sav1 (si-Sav1). As shown in Fig.3A, si-Sav1 reduced the ratio of pYap1 to Yap1, consistent with Yap1 activation. si-Sav1 and miR-199 had similar effects on cell cycle gene expression in NRVMs (Fig.3B). si-Sav1 increased Ccnd1 and Ccne1 expression compared to control, but not Cdk1, Plk1, Aurkb, or SurV expression. FGF had no additive effect on the expression of these genes (Fig.3B). si-Sav1 treatment increased EdU+ CMs, but not phospho-H3+ CMs nor total cell number (Fig.3D, 3E, &3F). [0100] Table 3: 28 cell cycle DEGs in iKDM4D group compared to Sav1-sh
Figure imgf000022_0001
Figure imgf000023_0001
[0105] Control-vs-iKDM4D (cont’d)
Figure imgf000024_0001
[0107] To test if KDM4D overexpression and Sav1 knockdown also combinatorially promote ACM proliferation in vivo, we firstly generated an inducible CM-specific KDM4D mouse model. CM-specific reverse tetracycline transactivator (rtTA) mice44 were mated to a KDM4D tet-responder (tet) line4. The resulting mice (iKDM4D) displayed tightly regulated KDM4D gene expression in the heart with doxycycline treatment (Fig.11A). Induction of KDM4D depleted H3K9me3 and up regulated cell cycle genes (Fig.4A-4C). Late cell cycle genes Cdk1 and AurkB were up-regulated 4-fold and 6-fold, respectively (p<0.05). KDM4D induction did not result in a significant difference in HW/BW ratio in iKDM4D mice compared to control mice at two weeks (Fig.4D). However, CMs isolated from iKDM4D hearts had an average area 30% smaller than control CMs, (3437μm2 ± 55μm2 for iKDM4D and 4993μm2 ± 350μm2 for ctrl; p<0.01; Fig.4D), suggesting a total increase in the number of CMs. [0108] Next, we performed intramyocardial injections of AAV9-Sav1-sh to inhibit the Hippo signaling pathway in our iKDM4D mice. Whole heart sections displaying GFP+ CMs were chosen for imaging and cell counting (Fig.11C). In contrast to in vitro results, both iKDM4D and AAV9-Sav1-sh increased the number of EdU+, phospho-H3+, and Aurora B+ CMs at 14 days post-injection (Fig.5). When combined, iKDM4D and AAV9-Sav1-sh led to further increases in cell cycle activity including a 3-fold increase in DNA synthesis activity (Fig.5E), 7.8-fold increase in mitosis (Fig.5F), and ~3-fold increase in cytokinesis compared to wild- type control (Fig.5G). [0109] KDM4D and Hippo induce distinct transcriptional reprogramming in ACMs [0110] To begin to understand the potential mechanisms underlying the differential effects we saw between KDM4D and Hippo pathway inhibition, RNA-seq was performed on control (wildtype mice injected with AAV9-GFP), Sav1-sh (wildtype mice injected with AAV9-U6- Sav1-sh-GFP), and iKDM4D (iKDM4D mice injected with AAV9-GFP) (Fig.12A). We identified 295 upreguatled genes and 362 downregulated genes in iKDM4D group compared to Sav1-sh group (Fig.6A). Among those differentially expressed genes, GO analysis identified 28 cell cycle genes (Fig.6B). There were 13 genes upregulated in the iKDM4D group of which 11 are involved in cell division, including Trnp45, Anln46, Lrrcc147, Map948, 6- Sep49, Eid150, Prkcd51, Dab2ip52, Mapk1253, Haus854, and Tacc155. 15 genes were upregulated in the Sav1-sh group compared to iKDM4D group of which 10 genes are involved in G1/S phase, including Tfdp256, Usp257, Gadd45a58, Rgs259, Ddit360, Tspyl261, Pim362, Txnip63, Crocc64 and Mybl265. These data confirm that KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while Sav1-sh preferentially up-regulated genes involved in G1/S phase. [0111] We also identified 16 upregulated genes and 48 downregulated genes in the Sav1-sh group compared to the control, and 492 upregulated genes and 882 downregulated genes in iKDM4D group compared to the control (Fig.6C&Fig.12B). Only 5 genes, including Dbp, BC002163, Casq1, Cacna1h, and Hlf, were upregulated in both the iKDM4D and Sav1-sh groups. We analyzed the promoters of the 16 upregulated genes in the Sav1-sh group and found that 14 (88%) of the upregulated genes contained at least one TEAD binding site, the target of Yap1/TAZ, but only 5 of them (31%) contained E2F1 binding sites, the proposed effector of KDM4Ds effects4 (Fig.6D-6E). We explored the promoters of 492 upregulated genes in the iKDM4D group and found 240 (50%) of genes contained at least one E2F1 binding site, but only 40 (8%) genes contained a TEAD binding site (Fig.6D). [0112] To further explore the expression of transcription factors (TFs) involved in cell cycle we examined the levels of Yap1 and E2F1 along with their proposed targets Myc66 and FoxM167 in NRVMs in response to si-Sav1, miR-199, and KDM4D treatment. Both si-Sav1 and miR-199 expression resulted in no significant change in E2F1, Foxm1, and Yap1 expression but a significant upregulation of Myc (p<0.05) (Fig.6F&6H). In contrast, KDM4D overexpression increased E2F1 and FoxM1 expression (p<0.05) (Fig.6F&6H). FGF had no additive effect over KDM4D, miR-199, or si-Sav1 on the expression of these TFs (Fig. 6G&6I). Consistent with these findings, the E2F1 promoter does not have any TEAD binding sites, but the Myc promoter contains four (Fig.12C). 7 (43%) of the promoters of the 16 Sav1-sh upregulated genes contained at least one Myc binding site (Fig.6D). [0113] KDM4D and Myc additively induce cardiomyocyte proliferation but does not impact cardiac function [0114] Since both miR-199 and si-Sav1 upregulated Myc expression in addition to decreasing pYap1 in NRVMs, we tested whether the combination of KDM4D and Myc also have a additive effect on cardiomyocyte proliferation. In NRVMs, Ad-Myc and Ad- KDM4D+Ad-Myc treatments increased Myc expression leading to a 3-fold enhancement of EdU+ CMs (Fig.7A-7B). Ad-KDM4D treated NRVMs exhibited a ~2-fold increase in pH3+ CMs, (Fig.7C). Ad-KDM4D, Ad-Myc, and Ad-KDM4D+Ad-Myc all increased total cell number after 6 days of culture compared to the control (Fig.7D). [0115] To test the effects of KDM4D and Myc in vivo, we generated a triple-transgenic mouse model (MycER+KDM4DTg/+). In this model, KDM4D is constitutively expressed in CM while Myc is inducibly activated in CM after tamoxifen treatment. In MycER or KDM4DTg/+ transgenic mice (10-week old mice that received tamoxifen starting at 9 weeks), HW/BW ratio was increased 17% and 25% respectively compared to wild-type littermates (p<0.05), but dual expression of MycER and KDM4D had a ~1.6-fold increase in heart mass compared to control (p<0.05; Fig.7E). CM size was similar in these mice regardless of MycER or KDM4D expression (Fig.7F) suggesting that the increase in heart mass was due to increased CM number. This CM proliferation was supported by the finding of increased phospho-H3+ CMs in MycER+KDM4DTg/+ hearts (Fig.7G-7H). Functional analysis of MycER, KDM4DTg/+, and MycER+KDM4DTg/+ hearts by echocardiography confirmed a significant increase in LV mass compared to wild-type littermates; but no difference in HR (Heart Rate), EF (Ejection Fraction), FS (Fractional shortening), CO (Cardiac Output), and LVEDD (Left Ventricular End-Diastolic Dimension) among the different mouse models and control mice (Table 1). [0116] Table 1. Cardiac function in MycER+KDM4DTg/+ triple-transgenic mice. [0117] Echocardiography results in 10-week old mice after 1 week of tamoxifen treatment. HR: Heart Rate, EF: Ejection Fraction, CO: Cardiac Output, LVEDD: Left Ventricular End- Diastolic Dimension, LV Mass: Left Ventricular Mass. Mean and SEM values are shown. Sample Number: Contol=6, Myc=8, KDM4DTg/+=6, MycER+KDM4DTg/+ =7. Statistics: One- way ANOVA/Tukey’s test, * P<0.05 vs Control, † P<0.05 vs Myc P<0.05 vs KDM4DTg/+.
Figure imgf000026_0001
[0118] Discussion [0119] Previous studies in our lab revealed that although KDM4D reexpression induced cycling in transgenic hearts, the overall the magnitude was still relatively low and cell cycle gene expression was less than that seen in fetal CMs where proliferation normally occurs.4 Thus, we hypothesized that in addition to depletion of the negative regulator H3K9me3, a stimulatory signal may also be required for more robust ACM proliferation. Enhanced Yap1 activation through inhibition of Hippo signaling similarly induces ACM cell cycle reentry, but the level was also low.28, 68 Given that KDM4D had no effect on Yap1 levels or phosphorylation status in our previous studies, we hypothesized that the Hippo signalling and epigenetic manipulation through KDM4D could have distinct mechanisms of action and therefore might have additive effects on promoting ACM proliferation. To test this hypothesis, combinatorially overexpressed KDM4D and miR-199 or si-Sav1 and found that the combination potentiates CM proliferation both in vitro and in vivo. Our data suggested that the Yap1 preferentially upregulates Myc expression while KDM4D increased E2F1 and FoxM1 expression, leading to CM proliferation. Our ability to see these selective effects was replicated in vivo. The differential effects of these factors using this in vitro model versus the in vivo effect may relate to the different developmental stage of NRVMs versus ACMs but the in vivo results in this study with KDM4D and Myc are similar to our previous studies.4, 69 This difference may be also related to the shorter temporal exposure of NRVMs to these factors resulting in less secondary effects and the impact of the culture conditions we used.70 We used serum free conditions to promote maturation of NRVMs. [0120] miR-199 overexpression in CMs increases cell cycle gene expression16 and decreases Hippo pathway genes, such as coffin 2 and TAOK1.19, 27 The ability of Hippo signaling and Yap/TAZ to preferentially promote G1/S transition has at least been implied by studies in nonmyocytes where it led to increased expression of Ccne1 and Cdc6.71 In endothelial cells, Yap1 is required for S-phase entry and knockdown of Yap1 led to accumulation of G1 phase cells and decreased the expression of G1/S phase genes.72 Yap1 is also involved in Adriamycin-induced podocyte cell cycle re-entry by regulating Ccnd1 and Cdk4 expression thereby increasing entry into S phase.73 In contrast, cardiac overexpression of KDM4D exhibits 5.8- to 21.4- fold increases of G2/M and cytokinesis genes, such as Ccnb1, Cdk1, Plk1, and Aurk B.4 These results suggested that Hippo pathway inhibition preferentially activates the expression of genes that involved in G1/S phase, but KDM4D overexpression preferentially induces the expression of genes that involved in the late phase of cell cycle. Consistent with this, both the in vitro and in vivo studies demonstrated an additive effect of KDM4D overexpression and Hippo pathway inhibition on CM proliferation. [0121] To confirm this concept; namely, that KDM4D- and Hippo pathway- regulated CM proliferation is through distinct mechanisms, RNA-seq was performed on CM samples from iKDM4D and Sav1-sh treated mice. Our RNA-Seq data indicated different transcriptional profiles between iKDM4D and Sav1-sh group. Consistent with in vitro qPCR data, KDM4D overexpression preferentially upregulated the G2/M phase genes, such as, Lrrcc147, Map948, and Dab2ip52, which are involved in mitotic spindle formation. Mapk1253 and Haus854 regulate the activity of Plk1 which plays an essential role in mitosis. Trnp45, Anln46, 6-Sep49, Prkcd51, and Tacc155 also participate in the cell division.10 of the 15 upregulated cell cycle genes in the Sav1-sh group are involved in the G1/S phase. Gadd45a58 and Crocc64 expression are higher in the G1 phase. Rgs259, Ddit360, Tspyl261, and Txnip63 are involved in the G0-G1 switch or G1 checkpoint. Tfdp256, Usp257, Pim362, and Mybl265 are also engaged in the G1/S phase of cell cycle. Regardless, our data suggest that the Hippo pathway and KDM4D regulate CM proliferation through different mechanisms. In fact, KDM4D overexpression downregulated TEAD3 expression, which is a transcriptional enhancer factor that plays a key role in the Hippo signaling pathway.74 KDM4D overexpression upregulated Patj and Mob1b expression which play an important role in LATS1/2 and MST1/2 phosphorylation.75, 76, 77 14 upregulated genes in the Sav1-sh group contained TEAD binding sites, and 13 of them have been linked to cell proliferation. 7 of the 13 proliferation-related genes also contained canonical Myc binding sites. Among them, Mid1ip1, Usp2, Art3, and Nr4a1 impact the G1/S phase of the cell cycle in different cell types.78-81 Myc can directly bind to the promoter regions of Nr4a1 and regulate its expression in hematopoietic stem cells.82 Although studies have shown that Myc is a key molecular target of Yap1 in human cancer, this is the first report in CMs.66, 83 Consistent with Myc as an effector of Yap1 in CM, overexpression of KDM4D and Myc in transgenic mice demonstrated marked cell cycle activity. Cardiac function was preserved, which has potential therapeutic relevance as persistent expression of miR-199 results in dysfunctiona and sudden arrhythmic death.27 It is possible that miR-199 may alter the expression of additional genes or factors that alter electrophysiologic properties of CM in contrast to the KDM4D/ Myc combination. [0122] In contrast to the Sav1-sh group, 51% of 492 promoters in KDM4D upregulated genes contained at least one E2F1 binding site, and only 9 genes are listed in the Yap1/TAZ/TEAD1-4 upregulated gene database.84, 85 FoxM1, a G2/M-specific transcription factor that is a known target of E2F1 was also upregulated in iKDM4D hearts.86, 87 Both E2Fs and FoxM1 play an important and well-established role in controlling the expression of genes important for the cell cycle, particularly in the control of gene expression at G2 phase of the cell cycle, encoding proteins known to function in mitosis.87, 88 KEGG analysis of the 492 upregulated genes indicated that KDM4D regulated a number of genes involved in proliferation-related pathways, including the ErbB signaling pathway, Wnt signaling pathway, and p53 signaling pathway (Fig.12D). ErbB signaling activation is required for G2 checkpoint activation in human breast cancer cells.89 Inhibition of the ErbB signaling pathway induces G2/M arrest in gastric cancer cells.90 Wnt/β-catenin signaling activity peaks during the G2/M phase,91 and inhibition of Wnt/β-catenin signaling leads to G2/M phase arrest.92 Our data demonstrated that 9 downregulated genes in the iKDM4D group were enriched in p53 signaling pathways. Among them, Sfn (stratifin) and Gadd45g (growth arrest and DNA-damage-inducible 45 gamma) play a role in blocking G2/M transition through p53-dependent arrest.93, 94 Other genes, such as Casp8, Serpine1, Shisa5, Thbs1, Mdm4, Igfbp3, and Cdkn1a, are all involved in p53-regulated apoptotic progression.95-100 Thus, our data imply that KDM4D overexpression preferentially stimulates gene expression controlling the G2/M phase of the cell cycle through E2F1 activity. In addition to regulating E2F1 expression, KDM4D may also regulate E2F1 activity since protein lysine methyltransferases and demethylases can modify a specific lysine residue on non-histone substrates with one or more methyl moieties, such as E2F1101, which impacts the activity or subcellular localization of the substrate protein.102-105 [0123] Thus KDM4D and miR-199/si-Sav1 combinatorially promote CM cycling through distinct pathways (Fig.8). Si-Sav1 or miR-199 preferentially induce G1/S phase cell cycle genes at least in part through activating Myc signaling pathway, while KDM4D promotes G2/M phase by regulating E2F1 and FoxM1 expression. Importantly, we confirmed our additive in vitro results in vivo, demonstrating these interventions also work in ACMs. Enhancing endogenous CM proliferation to compensate for the lost CMs after injury may be a promising strategy to prevent the development of heart failure. [0124] REFERENCES [0125] 1. Ali SR, et al. Proc. Nat’l Acad. Sci.2014;111:8850-8855. [0126] 2. Senyo SE, et al. Nature 2013;493:433-436. [0127] 3. Zhang Y, et al. Cell discovery 2019;5:1-15. [0128] 4. El-Nachef D, et al. Journal of molecular and cellular cardiology 2018;121:1-12. [0129] 5. Oyama K, et al. Epigenetics & chromatin 2018;11:18. [0130] 6. Engel FB, et al. Proc. Nat’l Acad. Sci.2006;103:15546-15551. [0131] 7. O’Meara CC, et al. Circulation research 2015;116:804-815. [0132] 8. Kubin T, et al. Cell stem cell 2011;9:420-432. [0133] 9. Kühn B, et al. Nature medicine 2007;13:962. [0134] 10. Bassat E, et al. Nature 2017;547:179-184. [0135] 11. Zhang Y, et al. Journal of regenerative medicine 2013;2:2. [0136] 12. Liu N, et al. Genes & development 2008;22:3242-3254. [0137] 13. Zhao Y, et al. Nature 2005;436:214-220. [0138] 14. Huang W, et al. Nature communications 2018;9:1-16. [0139] 15. Huang W, et al. Nature communications 2018;9:700. [0140] 16. Eulalio A, et al. Nature 2012;492:376-381. [0141] 17. Tian Y, et al. Science translational medicine 2015;7:279ra238-279ra238. [0142] 18. Diez-Cuñado M, et al. Cell reports 2018;23:2168-2174. [0143] 19. Torrini C, et al. Cell reports 2019;27:2759-2771. e2755. [0144] 20. Zhou Q, et al. Circulation research 2015;116:1431-1447. [0145] 21. Yu F-X, et al. Cell 2015;163:811-828. [0146] 22. Johnson R, et al. Nature reviews Drug discovery 2014;13:63-79. [0147] 23. Dong J, et al. Cell 2007;130:1120-1133. [0148] 24. Wu S, et al. Cell 2003;114:445-456. [0149] 25. Udan RS, et al. Nature cell biology 2003;5:914. [0150] 26. Leach JP, et al. Nature 2017;550:260-264. [0151] 27. Gabisonia K, et al. Nature 2019;569:418-422. [0152] 28. Liu S, et al. Science Translational Medicine 2021;13. [0153] 29. Byrd KN, et al. Proc. Nat’l Acad. Sci.2003;100:11535-11540. [0154] 30. Pesavento JJ, et al. Molecular and cellular biology 2008;28:468-486. [0155] 31. Cui K, et al. Cell stem cell 2009;4:80-93. [0156] 32. Hawkins RD, et al. Cell stem cell 2010;6:479-491. [0157] 33. Reddy MA, et al. Seminars in nephrology: Elsevier, 2013:341-353. [0158] 34. Rice JC, et al. Current opinion in cell biology 2001;13:263-273. [0159] 35. Hublitz P, et al. International Journal of Developmental Biology 2009;53:335- 354. [0160] 36. Olcina M, et al. Oncogene 2016;35:793-799. [0161] 37. Biga PR, et al. Biology open 2017;6:1720-1725. [0162] 38. Magaraki A, et al. Epigenetics & chromatin 2017;10:1-20. [0163] 39. Khoury-Haddad H, et al. Proc. Nat’l Acad. Sci.2014;111:E728-E737. [0164] 40. Ayrapetov MK, et al. Proc. Nat’l Acad. Sci.2014;111:9169-9174. [0165] 41. Pedersen MT, et al. The EMBO journal 2016;35:1550-1564. [0166] 42. Mendelsohn AR, et al. Rejuvenation Research 2015;18:371-375. [0167] 43. Oyama K, et al. Epigenetics & chromatin 2018;11:1-15. [0168] 44. Valencik ML, et al. Transgenic research 2001;10:269-275. [0169] 45. Esgleas M, et al. The EMBO journal 2020;39:e103373. [0170] 46. Magnusson K, et al. Bmc Cancer 2016;16:1-13. [0171] 47. Prosser SL, et al. Nature Reviews Molecular Cell Biology 2017;18:187-201. [0172] 48. Fontenille L, et al. Cell Cycle 2014;13:1101-1114. [0173] 49. Traikov S, et al. PloS one 2014;9:e109372. [0174] 50. Miyake S, et al. Molecular and Cellular Biology 2000;20:8889-8902. [0175] 51. Speidel JT, et al. Advances in Biological Regulation 2020:100757. [0176] 52. Yu L, et al. Oncogene 2021:1-13. [0177] 53. Soták M, et al. Annals of medicine 2014;46:221-232. [0178] 54. Goshima G, et al. The Journal of cell biology 2008;181:421-429. [0179] 55. Thakur HC, et al. Biological chemistry 2013;394:1411-1423. [0180] 56. Chen C, et al. Experimental hematology 2014;42:464-476. e465. [0181] 57. Shan J, et al. Molecular cell 2009;36:469-476. [0182] 58. Kearsey JM, et al. Oncogene 1995;11:1675-1683. [0183] 59. Linder A, et al. Scientific reports 2018;8:1-14. [0184] 60. Jauhiainen A, et al. PloS one 2012;7:e33208. [0185] 61. Tao KP, et al. PLoS One 2011;6:e21602. [0186] 62. Quan J, et al. Cellular and molecular biology 2015;61:42-50. [0187] 63. Yamaguchi F, et al. International journal of oncology 2008;32:377-385. [0188] 64. He R, et al. Cell reports 2013;4:1100-1107. [0189] 65. Musa J, et al. Cell death & disease 2017;8:e2895-e2895. [0190] 66. Choi W, et al. Cancer research 2018;78:3306-3320. [0191] 67. Millour J, et al. Molecular cancer therapeutics 2011;10:1046-1058. [0192] 68. Lin Z, et al. Circulation research 2014;115:354-363. [0193] 69. Xiao G, et al. Circulation research 2001;89:1122-1129. [0194] 70. Dambrot C et al. Journal of cellular and molecular medicine 2014;18:1509-1518. [0195] 71. Kim W, et al. Proc. Nat’l Acad. Sci.2019;116:9423-9432. [0196] 72. Shen Z, et al. PloS one 2015;10:e0117522. [0197] 73. Xie K, et al. Cell Death & Disease 2019;10:1-15. [0198] 74. Lin KC, et al. Trends in biochemical sciences 2017;42:862-872. [0199] 75. Meng Z, et al. Genes & development 2016;30:1-17. [0200] 76. Yi C, et al. Cancer cell 2011;19:527-540. [0201] 77. Gundogdu R, et al. Cells 2019;8:569. [0202] 78. Jung JH, et al. Cells 2020;9:985. [0203] 79. Davis MI, et al. Journal of Biological Chemistry 2016;291:24628-24640. [0204] 80. Shi W, et al. Journal of molecular histology 2012;43:37-47. [0205] 81. Tessem JS, et al. Proc. Nat’l Acad. Sci.2014;111:5242-5247. [0206] 82. Sheng Y, et al. Blood 2020. [0207] 83. Xiao W, et al. Biochemical and biophysical research communications 2013;439:167-172. [0208] 84. Subramanian A, et al. Proc. Nat’l Acad. Sci.2005;102:15545-15550. [0209] 85. Liberzon A, et al. Cell systems 2015;1:417-425. [0210] 86. Halasi M, et al. Biochemical pharmacology 2013;85:644-652. [0211] 87. Liao G-B, et al. Cell Communication and Signaling 2018;16:1-15. [0212] 88. Ren B, et al. Genes & development 2002;16:245-256. [0213] 89. Yan Y, et al. Oncogene 2015;34:2215-2226. [0214] 90. Mokhtari E, et al. Avicenna journal of medical biotechnology 2018;10:227. [0215] 91. Rasmussen ML, et al. Genes 2018;9:109. [0216] 92. Zhao L, et al. Molecular carcinogenesis 2016;55:778-792. [0217] 93. Hermeking H, et al. Molecular cell 1997;1:3-11. [0218] 94. Jin S, et al. Oncogene 2002;21:8696-8704. [0219] 95. Su F, et al. Oncogene 2010;29:421-431. [0220] 96. Ding H-F, et al. Journal of Biological Chemistry 2000;275:38905-38911. [0221] 97. Szołtysek K, et al. BMC genomics 2018;19:813. [0222] 98. Bourdon J-C, et al. The Journal of cell biology 2002;158:235-246. [0223] 99. Gembarska A, et al. Nature medicine 2012;18:1239-1247. [0224] 100. Grimberg A. Molecular genetics and metabolism 2000;70:85-98. [0225] 101. Hamamoto R, et al. Nature Reviews Cancer 2015;15:110-124. [0226] 102. Xie Q, et al. Journal of Receptors and Signal Transduction 2011;31:139-146. [0227] 103. Kontaki H, et al. Molecular cell 2010;39:152-160. [0228] 104. Wang G, et al. Nature cell biology 2019;21:214-225. [0229] 105. Biggar KK, et al. Nature reviews Molecular cell biology 2015;16:5-17. Example 2: Supplemental Methods re Interaction of Pathways Regulating Cardiomyocyte Proliferation [0230] Animals [0231] All animals were maintained and experiments performed in accordance with institutional guidelines at the University of Washington, and National Institute of Health Guide for the Care and Use of Laboratory Animals. The inducible KDM4D mouse model used in this study was generated previously in our lab.1 Tet-responsive-KDM4D mice were bred to the inducible αMHC-tTA (KDM4DTg/+; tet-off) or repressible αMHC-rTA (iKDM4D; tet- on) mice.2, 3 The tet-off KDM4D mice were crossed with our αMHC-MycER model4 to generate a triple-transgenic mouse model (MycER+KDM4DTg/+) that constitutively expresses KDM4D in the absence of doxycycline, with Myc induced by tamoxifen specifically in CMs. To assess the potential effects on cardiac function as a results of genetic manupliation, transthoracic echocardiography was performed prior to the endpoint as described previously.1 Both gender of the animals were used, as we found no significant difference in our previous study.1 For NRVM cultures, time-pregnant Sprague Dawley outbred rats (TP19) were purchased from Harlan/ENVIGO and CMs isolated as described.5 [0232] Virus, miRNA, and siRNA [0233] The virus vectors Ad-CMV-GFP-h-KDM4D (Ad-KDM4D), Ad-CMV-GFP-h-c-Myc (Ad- Myc), Ad-CMV-GFP-h-control (Ad-GFP), AAV9-U6-sh-Sav1-eGFP (AAV9-Sav1-sh), and its control AAV9-U6-scramble-eGFP (AAV9-C-sh) were purchased from Vector Biolabs (Malvern, PA). The miRNA and siRNA used in this study, including hsa-miR-199a-3p mimic (miR-199), hsa-miR-590-3p mimic (miR-590), hsa-miR-302b-5p mimic/hsa-miR-302c-5p mimic (miR-302), miRIDIAN microRNA Mimic Negative Control #1 (miR-C), ON- TARGETplus SMARTpool Sav1 siRNA (si-Sav1), and ON-TARGETplus control siRNA (si- C), were purchased from Dharmacon Inc. FGF Basic Recombinant Human Protein (PHG6015), EGF Recombinant Human Protein (PHG0313), and Recombinant Human Oncostatin M (HEK-293-expressed) Protein (PHC5015) were purchased from Thermo Fisher (USA). [0234] Cell culture and Transfections [0235] NRVMs were isolated as described before. FACS and immunostaining of α-actinin results demonstrate that NRVM purity was more than 99% (Fig.9B-9C). NRVMs were cultured and treated as shown in Figure 9A. In brief, NRVMs were plated on fibronectin- coated 24-well plates at a density of 1×10^5 per well. NRVMs were seeded and cultured with 500μL M199 culture medium (Medium 199500ml, HEPEs 10mM, MEM Non-Essential Amino Acids 1×, glucose 1.75g, L-glutamine 2mM, Vitamin B122mg, and penicllin 50,000 units) with 10% FBS. After 24 hours of serum medium culture, the NRVMs were infected with Ad-KDM4D, Ad-Myc, or control Ad-GFP in MOI 100 and maintained in the serum-free M199 culture medium (Fig.9D). The medium was changed to 500 μL fresh serum-free M199 culture medium at 24 hours after virus infection. At 48 hours after virus infection, the NRVMs were transfected with either miRNA (25nM) or siRNA (25nM) using Lipofectamine™ 3000 acording to the manufacturer’s protocol (Life Technologies) and/or treated with FGF (100ng/ml), EGF (100ng/ml), and OSM (50ng/ml). The transfection efficiency was assayed using miR-Dy547 control demonstrating 90% cells were transfected (Fig.9E). The NRVMs were maintained for another 48 hours, and then switched to the 500 μL fresh serum-free medium with 5nM 5-Ethynyl-2'-deoxyuridine (EdU). After 24 hours of EdU incubation, the cells were fixed for immunofluorescent staining or trypsinized with 0.05% Trypsin-EDTA for counting total cell numbers. The NRVMs were cultured 6 days in total since H3K9me3 levels were lowest after 5 days of infection (Fig.11). [0236] In vivo gene transfer [0237] To exam the effect of KDM4D and Sav1 on CM cell cycle and proliferation in vivo, adult (8-12weeks) iKDM4D mice were injected with AAV9-Sav1-sh or control virus (AAV9- GFP). A thoracotomy was performed and mice were given three intramyocardial injections using Hamilton syringe (50μl capacity) with a 33-gauge needle to deliver a total of 2×1011 viral genomes (30μl total volume delivered) into the apex of the left ventricle.6 24 hours after injection, the mice were treated with Doxycycline in rodent chow and EdU in drinking water (ad.lib) until the study endpoint. Two weeks after injections, the mice were sacrificed, and the hearts harvested for immunostaining and RNA extraction. [0238] Isolation of adult mouse ventricular myocytes [0239] For RNA-seq, cardiomyocytes were isolated using Langendorff perfusion digestion as previously described.7 8 to 10 week old iKDM4D mice were injected with AAV9-Sav1-sh or control virus (AAV9-GFP). After two weeks of injection, the mice were intraperitoneally injected with 200μl of heprin (100 IU/mouse) before being anesthetize with Isoflurane. The hearts were harvested and perfused with a 37℃ Ca2+- free Tyrodes buffer (126 mM NaCl, 5.4 mM KCl, 0.33 mM NaH2PO4, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose, 20 mM Taurine, pH=7.4) supplemented with 25 μM blebbistatin (Toronto Research Chemicals Inc. and BOC Sciences) for 1–2min, and then enzymatically digested at 37℃ with 0.35 U/ml Liberase TH (Roche) prepared with blebbistatin supplemented-Ca2+- free Tyrode’s buffer for 8–12min. Heart tissues were mechanically dissociated in ice-cold KB solution (20 mM KCl, 10 mM KH2PO4, 70 mM Potassium Glutamate, 1 mM MgCl2, 25 mM Glucose, 20 mM Taurine, 0.5 mM EGTA, 10 mM HEPES, 0.1% Albumin, pH=7.4). The cell suspensions were passed through a 100 μm cell strainer to remove tissue debris and then purified by low- speed centrifugation (50×g for 1min) 3 times, resulting in ~ 90% pure ACMs. [0240] Histology and Immunostaining [0241] NRVMs or heart tissue sections were fixed with 4% PFA in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 20min, and blocked with PBS containing 5% NDS (Normal Donkey Serum) for 1h at room temperature. For immunostaining, the cells were incubated overnight at 4qC with the following antibodies diluted in the blocking buffer: anti-cTnT (Thermo Scientific: MS-295-P) and Phalloidin (Thermo Scientific: R415) was used to identify CMs, Click-iT EdU Alexa Fluor 647 HCS Assay (Thermo Scientific: C10356) to identify the S phase of the cell cycle, anti-phospho-H3 (Thermo Scientific: PA5-17869) to identify the M phase of the cell cycle, anti-Aurora B antibody (Abcam: ab2254) to identify the cytokinesis phase of the cell cycle . The cells were then washed three times with PBS+5%FBS and stained for 45min at room temperature with secondary antibodies. This was followed by 5 min of DAPI (4′, 6-diamidino-2-phenylindole dihydrochloride). The cells were viewed under Nikon fluorescence microscope. To determine cross-sectional area (CSA) of cells, 10 μm thick heart slides were stained by Anti-wheat germ agglutinin (WGA). The CSA was determined by the region within the WGA staining boundary. [0242] Quantitative real-time PCR [0243] Total RNA was isolated using TRIzol™ Reagent (Invitrogen) and then purified using RNeasy Mini Kit (Qiagen). One microgram RNA was reverse transcribed using the High Capacity Reverse Transcription cDNA Synthesis Kit (Applied Biosystems). Quantitative PCR (qPCR) was performed using the Sybrgreen PCR master mix (Applied Biosystems) according to the manufacturer’s instructions and qPCR cycling was carried out on the AB7900. Primer sequences used for qPCR are listed in Table 2. [0244] RNA-seq [0245] Two independent samples from each group were used for RNA-seq. Library preparation and sequencing was performed by commercial service (GENEWIZ). Sequence reads were trimmed to remove adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36.8 The trimmed reads were mapped to the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.9 Unique gene hit counts were calculated by using FeatureCounts from the Subread package v.1.5.2.10 The original read counts were normalized to adjust for various factors such as variations of sequencing yield between samples. These normalized read counts were used to accurately determine differentially expressed genes (DEGs). Using DESeq2, a comparison of gene expression between the groups of samples was performed.11 The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 were called as DEGs for each comparison. A gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF v.1.1-p2.12 KEGG pathway analysis was performed by DAVID Bioinformatics Resources.13 CiiiDER was used to scan transcription factors binding site on the promoter of DEGs.14 Yap1/TEAD1-4 upregulated genes set was generated from Molecular Signatures Database v7.2.15, 16 [0246] Protein Analysis [0247] Western blots were performed by the SDS-PAGE electrophoresis. Total cell extracts were prepared and fractionated by gel electrophoresis and transferred to nitrocellulose membranes. The following primary antibodies were used: Anti-KDM4D (ab93694; Abcam), anti-H3K9me3 (ab8898; Abcam), anti-Sav1 (105105; Abcam), Yap1 (ab81183; Abcam), pYap1 (ab76252; Abcam), Myc (ab32072; Abcam) , Anti-Histone H3 Antibody (07-690 from Sigma-Aldrich), GAPDH (ab181603; Abcam). Horseradish peroxidase anti-rabbit (ab205718; Abcam) was used as the secondary antibody. The signal was detected using the super- signal-enhanced chemiluminescence system (Pierce). [0248] Statistical analysis [0249] All in vitro experiments were carried out with at least n ≥3 biological replicates, unless otherwise specified. Animal or cells per group are identified in related figure legends. In experiments with multiple groups, analysis of variance (ANOVA) followed by Tukey’s test was used to compare group means. P value <0.05 was considered to represent a statistically significant difference. In all panels, numerical data are presented as mean ± SEM. [0250] REFERENCES [0251] 1. El-Nachef D, et al. Journal of molecular and cellular cardiology 2018;121:1-12. [0252] 2. Doetschman T, et al. Circulation research 2012;110:1498-1512. [0253] 3. Sanbe A, et al. Circulation research 2003;92:609-616. [0254] 4. Xiao G, et al. Circulation research 2001;89:1122-1129. [0255] 5. Zhang Y, et al. PloS one 2010;5. [0256] 6. Leach JP, et al. Nature 2017;550:260-264. [0257] 7. Oyama K, et al. Epigenetics & chromatin 2018;11:1-15. [0258] 8. Bolger AM, et al. Bioinformatics 2014;30:2114-2120. [0259] 9. Dobin A, et al. Bioinformatics 2013;29:15-21. [0260] 10. Liao Y, et al. Bioinformatics 2014;30:923-930. [0261] 11. Love MI, et al. Genome biology 2014;15:550. [0262] 12. Subhash S, et al. BMC bioinformatics 2016;17:1-10. [0263] 13. Sherman BT, et al. Nature protocols 2009;4:44. [0264] 14. Gearing LJ, et al. PloS one 2019;14:e0215495. [0265] 15. Subramanian A, et al. Proc. Nat'l Acad. Sci.2005;102:15545-15550. [0266] 16. Liberzon A, et al. Cell systems 2015;1:417-425. Example 3: Gene Therapy for Cardiac Regeneration Using an Ischemia Reperfusion Model for CM Repair [0267] This Example can be used to confirm the feasibility of cardiac regeneration in large animals through stimulation of the cardiac cell proliferation via gene therapy. [0268] Adeno-associated viral vector (AAV) mediated induction of cardiac proliferation. This approach can be employed to test the ability of two putative inducers of the cardiac cell cycle, KDM4D and miR-199, individually and in combination to stimulate CM proliferation in swine. [0269] To quantify cardiac proliferation following AAV gene therapy in swine (8 subjects total, 12-day endpoint), two naïve farm pigs are each transfected with 1) 2x1013 AAV6- KDM4D, 2) AAV6-miR-199, 3) combined AAV6-KDM4D and 2x1013 AAV6-miR-199 or 4) vehicle control. Direct delivery of 2x1013 AAV is achieved via partial sternotomy with indwelling catheter placement. Delivery is by 100 μL dose (1x1013 cells/mL) x 20 injections. This is followed by injection of BrdU at day 2 to 12 post-transfection. The heart is harvested at day 12 for histology. [0270] To confirm the therapeutic potential for AAV mediated induction of cardiac proliferation, endpoints will include cardiac proliferation (BrdU/Ki67 as marker of proliferation) and cardiac function (MRI) following MI. [0271] The efficacy of cardiac proliferation following AAV gene therapy can be assessed in swine (12 subjects total, 30-day endpoint). The protocol involves transfection of 4 post-MI farm pigs with 1) 2x1013 AAV6-KDM4D, 2) AAV6-miR-199, 3) combined AAV6-KDM4D and 2x1013 AAV6-miR-199 or 4) AAV6-GFP. Baseline MRI is obtained. A 90-min percutaneous ischemia-reperfusion injury of mid-LAD and indwelling catheter and telemetry placement is performed on day -14. Post-MI MRI is obtained on day -2. [0272] Direct delivery of 2x1013 AAV is achieved via partial sternotomy. This is followed by injection of BrdU day 2 to 30 post-transfection. MRI is obtained, and the heart is harvested on day 30 for histology. [0273] We expect that the combination of KDM4D and mir-199 will stimulate adult CM proliferation in vivo in this large animal model. Likewise, we expect this proliferation to improve cardiac function following MI. Example 4: Synergistic Effect of Combination KDM4D/mir-199 Gene Therapy for CM Repair [0274] This Example demonstrates the synergistic stimulation of cardiac regeneration in a large animal via gene therapy. The protocol described in Example 3 was followed in a study in which one pig was injected with: 1) AAV6-GFP control, 2) AAV6-mir-199, 3) AAV6- KDM4D, and 4) the combination of (2) and (3). Each injection was at a different site in one MI pig heart. The heart was harvested at day 12 for histology. As shown in Fig.13, GFP expression detected by anti-GFP antibody was used to confirm successful transgene delivery. The whole sections were scanned by Nikon A1 confocal at same settings and images were gained at same LUTs settings. Fig.14 shows how quantification of Ki67 positive CMs was performed in the MI pig to measure proliferation of CMs. As shown in the bar graph of Fig.14, even though KDM4D treatment alone did not result in more detectable proliferation than control or uninjected areas, mir-199 treatment led to a dramatic increase in proliferation, and this effect was significantly enhanced by combination treatment with both KDM4D and mir-199. Example 5: Increasing Cardiomyocyte Proliferation via Transduction of Stem Cell-Derived CMs to Enhance Cell Engraftment [0275] This Example describes how to transduce SC-derived CMs with vectors of the invention to increase the size of subsequent grafts. [0276] There remain several challenges with the development of human cardiomyocytes from pluripotent stem cells (hPSC-CMs) into an effective therapeutic post myocardial injury. One such challenge is the low survival and engraftment rates of injected cells, which limits long term graft size. To overcome this, we describe a method of enhancing the proliferative potential of transplanted hiPSC-CM to increase graft size. As described herinabove, H3K9 demethylation using lysine demethylase 4D (KDM4D) and/or Hippo pathway inhibition, with mir199, increases cardiomyocyte (CM) proliferation. Moreover, when KDM4D overexpression is combined with inhibition of the Hippo-YAP pathway, we found significantly increased CM proliferation compared to either treatment alone. These two approaches activate different transcriptional pathways, which likely accounts for their synergistic effects. Here we describe how to confirm that increasing hiPSC-CM proliferation with KDM4 and/or mir199, at the time of cell transplantation, will improve the graft size in mice. [0277] AAV - KDM4D and mir199 infection of hiPSC-CMs. First, one can determine optimized protocols for hiPSC-CM AAV transfection. The optimal MOI can be determined by tracking GFP+ve hiPSC-CM. One can then confirm H3K9 demethylation and/or activation Hippo-YAP pathway in infected hiPSC-CM. One can then measure S-phase (EDU incorporation), M-phase (phosho-H3) and proliferation (cell number). [0278] To determine if KDM4D and/or mir199 transfected hiPSC-CMs improve cell engraftment, we will use Nod-Scid IL2Rϒ deficient (NSG) mice. The mice will be transplanted with KDM4D and/or mir199 transfected hiPSC-CMs. The study will be comprised of up to 4 different treatment groups (Control, mir199, KDM4D and mir199+KDM4D). Each group will be comprised of 12 NSG mice. Engraftment will be measured using the Alu-PCR assay. [0279] Next we will generate transfected hiPSC-CMs for in vivo experiments, namely transplantation of transfected hiPSC-CMs into mouse hearts. Hearts will be harvested 14 days post-injection and Alu PCR quantified to determine engrafted human cells. [0280] We expect that KDM4D and/or mir199 transfected hiPSC-CMs will improve cell engraftment. [0281] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains. [0282] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is: 1. An expression vector comprising: (a) a nucleic acid sequence encoding KDM4D; (b) a nucleic acid sequence encoding an activator/effector of the Hippo-Yap pathway; (c) a tissue-specific promoter that effects overexpression of KDM4D and the activator/effector of the Hippo-Yap pathway, wherein the promoter is operably linked to the nucleic acid sequence of (a) and (b); and (d) a regulatory element that inducibly represses the overexpression of KDM4D and/or the activator/effector of the Hippo-Yap pathway.
2. The expression vector of claim 1, wherein the activator/effector of the Hippo- Yap pathway is selected from one or more of Myc, miR-199, and si-Sav1.
3. The expression vector of claim 1 or claim 2, wherein the tissue-specific promoter is a cardiac-specific promoter.
4. The expression vector of any of claims 1, 2, or 3, wherein the regulatory element is a tetracycline responsive element.
5. The expression vector of any one of claims 1 to 4, which is a viral vector that infects quiescent cells.
6. The expression vector of claim 4, wherein the viral vector is an adeno- associated virus (AAV) vector.
7. A cell comprising the expression vector of claim 1.
8. The cell of claim 7, which is a cardiac myocyte (CM).
9. The cell of claim 8, wherein the cardiac myocyte (CM) is derived from a stem cell.
10. The cell of claim 8, wherein the cardiac myocyte (CM) is derived from cardiac tissue.
11. A method of proliferating cardiac myocytes in a mammal, the method comprising administering the expression vector of any one of claims 1 to 6 to the mammal.
12. A method of promoting cardiac regeneration comprising administering the expression vector of any one of claims 1 to 6 to a subject in need of cardiac regeneration.
13. A method of regenerating an organ or tissue, the method comprising administering the expression vector of any one of claims 1 to 6 to the mammal.
14. A method for inducing cardiac myocyte (CM) hyperplasia in a mammal comprising grafting CMs to the heart of the mammal, wherein the CMs contain the expression vector of any one of claims 1 to 6.
15. The method of claim 13, wherein the expression vector is administered by administering CMs that contain the expression vector.
16. The method of claim 14 or 15, wherein the CMs are adult CMs (ACMs).
17. The method of any of claims 11 to 16, wherein the administration is systemic.
18. The method of any of claims 11 to 16, wherein administration is by intra- myocardial injection.
PCT/US2022/077401 2021-10-01 2022-09-30 Vectors and methods for improving cardiac regeneration WO2023056456A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163261978P 2021-10-01 2021-10-01
US63/261,978 2021-10-01

Publications (1)

Publication Number Publication Date
WO2023056456A1 true WO2023056456A1 (en) 2023-04-06

Family

ID=85783690

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/077401 WO2023056456A1 (en) 2021-10-01 2022-09-30 Vectors and methods for improving cardiac regeneration

Country Status (1)

Country Link
WO (1) WO2023056456A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180117125A1 (en) * 2015-04-20 2018-05-03 University Of Washington Vectors and methods for regenerative therapy

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180117125A1 (en) * 2015-04-20 2018-05-03 University Of Washington Vectors and methods for regenerative therapy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FERNANDES SARAH, CHONG JAMES J.H., PAIGE SHARON L., IWATA MINEO, TOROK-STORB BEVERLY, KELLER GORDON, REINECKE HANS, MURRY CHARLES : "Comparison of Human Embryonic Stem Cell -Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair", STEM CELL REPORTS, CELL PRESS, UNITED STATES, vol. 5, no. 5, 10 November 2015 (2015-11-10), United States , pages 753 - 762, XP009545675, ISSN: 2213-6711, DOI: 10.1016/j.stemcr.2015.09.011 *
TORRINI CONSUELO, CUBERO RYAN JOHN, DIRKX ELLEN, BRAGA LUCA, ALI HASHIM, PROSDOCIMO GIULIA, GUTIERREZ MARIA INES, COLLESI CHIARA, : "Common Regulatory Pathways Mediate Activity of MicroRNAs Inducing Cardiomyocyte Proliferation", CELL REPORTS, ELSEVIER INC, US, vol. 27, no. 9, 1 May 2019 (2019-05-01), US , pages 2759 - 2771.e5, XP093060579, ISSN: 2211-1247, DOI: 10.1016/j.celrep.2019.05.005 *

Similar Documents

Publication Publication Date Title
Tao et al. LncRNA GAS5 controls cardiac fibroblast activation and fibrosis by targeting miR-21 via PTEN/MMP-2 signaling pathway
Goehe et al. hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing
Han et al. A WNT/β-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis
Lv et al. lncMGPF is a novel positive regulator of muscle growth and regeneration
Chen et al. Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy
Valkov et al. MicroRNA-1-mediated inhibition of cardiac fibroblast proliferation through targeting cyclin D2 and CDK6
Zhu et al. Adult cardiomyocyte proliferation: a new insight for myocardial infarction therapy
El-Nachef et al. Repressive histone methylation regulates cardiac myocyte cell cycle exit
US20220249623A1 (en) Vectors and methods for regenerative therapy
Al-Maqtari et al. Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells
Fisher et al. Stress-induced cell-cycle activation in Tip60 haploinsufficient adult cardiomyocytes
Judd et al. Defined factors to reactivate cell cycle activity in adult mouse cardiomyocytes
Salisch et al. Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes
Yamak et al. Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts
Oh et al. Role of the PRC2-Six1-miR-25 signaling axis in heart failure
He et al. Protective role of downregulated MLK3 in myocardial adaptation to chronic hypoxia
Zhang et al. LncRNA AZIN1-AS1 ameliorates myocardial ischemia–reperfusion injury by targeting miR-6838-5p/WNT3A axis to activate Wnt-β/catenin signaling pathway
Zhang et al. Hippo signaling and histone methylation control cardiomyocyte cell cycle re-entry through distinct transcriptional pathways
Locatelli et al. Targeting the cardiomyocyte cell cycle for heart regeneration
Hao et al. Loss of CRY2 promotes regenerative myogenesis by enhancing PAX7 expression and satellite cell proliferation
WO2023056456A1 (en) Vectors and methods for improving cardiac regeneration
JP2022543589A (en) KLF-induced myocardial regeneration
Robinson et al. MSK phosphorylation of H3S28 is required for immediate early gene induction and cardiac hypertrophy
US20220364090A1 (en) Promotion of Cardiomyocyte Proliferation and Regenerative Treatment of the Heart by Inhibition of microRNA-128
Zhang Functional Characterization of Atf3 in Skeletal Muscle Development and Regeneration

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22877634

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE