Compositions and methods for improving cardiac regeneration

Abstract

The compositions and methods described herein relate to the administration of small molecule inhibitors in combination with a genetic construct expressing KDM4D to promote cardiomyocyte proliferation.

Description

COMPOSITIONS AND METHODS FOR IMPROVING CARDIAC REGENERATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 533,377 filed August 18, 2023, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 1, 2024 is named 034186-000105WOPT_SL.xml and is 2,243 bytes in size.TECHNICAL FIELD

[0003] The technology described herein relates to compositions and methods for improving cardiac regeneration and / or proliferation, e.g., for treating cardiac injury.BACKGROUND

[0004] Ischemic heart disease leading to heart failure is one of the leading causes of death. Although adult human hearts are unable to replace lost cardiomyocytes (CMs) after injury, substantial cardiac regeneration is seen in lower vertebrate and mammalian models. Current therapeutic interventions in response to cardiac injury have focused on strategies that either salvage myocardium or restore the proliferative capacity of cardiomyocytes to promote cardiac regeneration.

[0005] However, adult mammalian hearts show a limited regenerative capacity. To date, a very limited number of new cardiac myocytes (CM) can be generated in adult hearts, but the amount is insufficient to restore contractile dysfunction after myocardial infarction (MI). Attempts to improve this cardiomyocyte proliferation have had limited success and do not approach the proliferative ability of fetal or newborn cardiomyocytes.SUMMARY

[0006] The inventors have found that combination of: a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D provides surprising synergy in promoting cardiomyocyte proliferation. As shown in Fig. 4D, the use of a JAK inhibitor of PI3K inhibitor alone does not increase cardiomyocyte proliferation, while a genetic construct that directs the expression of KDM4D induces a -2 fold increase in cardiomyocyte proliferation. The combination of agents provides a ~4 fold increase in cardiomyocyte proliferation, which is a synergistic increase. The alternatives, e.g., inhibitors of the Hippo pathway, provide only a ~1.5 fold increase in cardiomyocyte proliferation.

[0007] Accordingly, the technology described herein relates to the discovery of agents that canpromote the proliferation of cardiomyocytes in vivo. Such agents and compositions comprising them can be used, for example, to promote repair of cardiac tissue damaged by injury or disease.

[0008] Accordingly, in one aspect, described herein is a method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with a Janus kinase (JAK) inhibitor and a genetic construct that directs the expression of lysine-specific demethylase 4D (KDM4D). The JAK inhibitor and the genetic construct are contacted with the cardiomyocyte or administered in amounts sufficient to promote cardiomyocyte proliferation.

[0009] In one embodiment of this or other aspects described herein, the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the JAK inhibitor and the genetic construct to the subject. In another embodiment of this or other aspects described herein, the cardiomyocyte is human.

[0010] In another embodiment of this or other aspects described herein, the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945. In another embodiment of this or other aspects described herein, the JAK inhibitor is ruxolitinib.

[0011] In another embodiment of this or other aspects described herein, the genetic construct that directs the expression of KDM4D comprises an adeno-associated virus (AAV) vector. In another embodiment of this or other aspects described herein, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV 12. In another embodiment of this or other aspects described herein, the AAV vector is AAV9. In another embodiment of this or other aspects described herein, the adeno- associated virus expressing KDM4D is administered at a total dosage within the range of lxl09vg to about lxl016vg.

[0012] In another embodiment of this or other aspects described herein, the JAK inhibitor is administered simultaneously with the genetic construct that directs the expression of KDM4D. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of KDM4D. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of 10 pM.

[0013] In another embodiment of this or other aspects described herein, the method further comprises contacting the cardiomyocyte with a phosphatidylinositol-3 -kinase (PI3K) inhibitor. In another embodiment of this or other aspects described herein, the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765. In another embodiment of this or other aspects described herein, the PI3K inhibitor is PHT-427. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.In another embodiment of this or other aspects described herein, the PI3K inhibitor is at a dosage of lOpM.

[0014] In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered simultaneously with the genetic construct expressing KDM4D. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered simultaneously with the adenovirus expressing KDM4D. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at least 1 hour after the administration of the genetic construct expressing KDM4D. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at least 1 hour after the administration of the adenovirus expressing KDM4D.

[0015] In another aspect, described herein is a method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with a phosphatidylinositol-3 -kinase (PI3K) inhibitor and a genetic construct that directs the expression of KDM4D.

[0016] In one embodiment of this or other aspects described herein, the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the PI3K inhibitor and the genetic construct to the subject. In another embodiment of this or other aspects described herein, the cardiomyocyte is human.

[0017] In another embodiment of this or other aspects described herein, the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765. In another embodiment of this or other aspects described herein, the PI3K inhibitor is PHT-427. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage lOpM.

[0018] In another embodiment of this or other aspects described herein, the genetic construct that directs the expression of KDM4D comprises an adeno-associated virus (AAV) vector. In another embodiment of this or other aspects described herein, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV 12. In another embodiment of this or other aspects described herein, the AAV vector is AAV9. In another embodiment of this or other aspects described herein, the adeno- associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.

[0019] In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered simultaneously with the genetic construct that directs the expression of KDM4D. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of KDM4D.

[0020] In another embodiment of this or other aspects described herein, the subject has cardiac damage or a cardiac disease or disorder. In another embodiment of this or other aspects describedherein, the subject has suffered a myocardial infarction. In another embodiment of this or other aspects described herein, the cardiac disease or disorder is selected from atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.

[0021] In another aspect, described herein is a kit for promoting cardiomyocyte proliferation, the kit comprising an adeno-associated virus expressing KDM4D, a JAK inhibitor, and instructions for use. In another embodiment of this or other aspects described herein, the kit further comprises a PI3K inhibitor.

[0022] In another embodiment of this or other aspects described herein, the kit is for use in treating a cardiac disease or disorder. In another embodiment of this or other aspects described herein, the kit is for use in treating a cardiovascular disease or disorder.

[0023] In another embodiment of this or other aspects described herein, the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.In another embodiment of this or other aspects described herein, the JAK inhibitor is ruxolitinib.

[0024] 4 In another embodiment of this or other aspects described herein, the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765. In another embodiment of this or other aspects described herein, the PI3K inhibitor is PHT-427.

[0025] In another embodiment of this or other aspects described herein, the adeno-associated virus expressing KDM4D is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In another embodiment of this or other aspects described herein, the adeno-associated virus expressing KDM4D is AAV9.

[0026] In another aspect, described herein is method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof an amount of a JAK inhibitor and an amount of a genetic construct that directs the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.

[0027] In one embodiment of this or other aspects described herein, the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945. In another embodiment of this or other aspects described herein, the JAK inhibitor is ruxolitinib. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the JAK inhibitor is lOpM.

[0028] In another embodiment of this or other aspects described herein, the subject in need thereof is human.

[0029] In another embodiment of this or other aspects described herein, the genetic construct that directs expression of KDM4D comprises an AAV vector. In another embodiment of this or other aspects described herein, the AAV is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In another embodiment of this or other aspects described herein, the AAV vector is AAV9. In another embodiment of this or other aspects described herein, the adeno-associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.

[0030] In another embodiment of this or other aspects described herein, the method further comprises administering a PI3K inhibitor to the subject. In another embodiment of this or other aspects described herein, the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A- 769662, and XL765. In another embodiment of this or other aspects described herein, the PI3K inhibitor is PHT-427.

[0031] In another aspect, described herein is a method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof an amount of a PI3K inhibitor and an amount of a genetic construct that directs the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.

[0032] In one embodiment of this or other aspects described herein, the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765. In another embodiment of this or other aspects described herein, the PI3K inhibitor is PHT-427. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage of lOpM.

[0033] In another embodiment of this or other aspects described herein, the genetic construct that directs expression of KDM4D comprises an AAV vector. In another embodiment of this or other aspects described herein, the AAV is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In another embodiment of this or other aspects described herein, the AAV vector is AAV9. In another embodiment of this or other aspects described herein, the adeno-associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.

[0034] In another embodiment of this or other aspects described herein, the subject in need thereof is human.

[0035] In another embodiment of this or other aspects described herein, the method further comprises administering a JAK inhibitor to the subject. In another embodiment of this or other aspects described herein, the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945. In another embodiment of this or other aspects described herein, theJAK inhibitor is ruxolitinib. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of 10 pM.

[0036] In another aspect, described herein is a method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject an amount of a JAK inhibitor and an amount of a genetic construct directing the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.

[0037] In one embodiment of this or other aspects described herein, the subject has suffered ischemic injury to cardiac tissue.

[0038] In another embodiment of this or other aspects described herein, the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945. In another embodiment of this or other aspects described herein, the JAK inhibitor is ruxolitinib. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.

[0039] In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of lOpM.

[0040] In another embodiment of this or other aspects described herein, the subject in need thereof is human.

[0041] In another embodiment of this or other aspects described herein, the genetic construct that directs the expression of KDM4D comprises an adeno-associated virus (AAV) vector. In another embodiment of this or other aspects described herein, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV 12. In another embodiment of this or other aspects described herein, the AAV vector is AAV9. In another embodiment of this or other aspects described herein, the adeno- associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.

[0042] In another embodiment of this or other aspects described herein, the method further comprises administering a PI3K inhibitor to the subject. In another embodiment of this or other aspects described herein, the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A- 769662, and XL765. In another embodiment of this or other aspects described herein, the PI3K inhibitor is PHT-427. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the PI3K inhibitor is lOpM.

[0043] In another aspect, described herein is a method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject an amount of a PI3Kinhibitor and an amount of a genetic construct directing the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.

[0044] In one embodiment of this or other aspects described herein, the subject has suffered ischemic injury to cardiac tissue.

[0045] In another embodiment of this or other aspects described herein, the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765. In another embodiment of this or other aspects described herein, the PI3K inhibitor is PHT-427. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the PI3K inhibitor is administered at a dosage of lOpM.

[0046] In another embodiment of this or other aspects described herein, the subject in need thereof is human.

[0047] In another embodiment of this or other aspects described herein, the genetic construct directing the expression of KDM4D comprises an AAV vector. In another embodiment of this or other aspects described herein, the AAV is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In another embodiment of this or other aspects described herein, the AAV is AAV9. In another embodiment of this or other aspects described herein, the AAV expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.

[0048] In another embodiment of this or other aspects described herein, the method further comprises administering a JAK inhibitor. In another embodiment of this or other aspects described herein, the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG- 490, and cx-4945. In another embodiment of this or other aspects described herein, the JAK inhibitor is ruxolitinib. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive. In another embodiment of this or other aspects described herein, the JAK inhibitor is administered at a dosage of lOpM.BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIGs. 1A-1G shows KDM4D extends the neonatal regenerative window. (FIG. 1A-1B) Quantification and (FIG. 1C) representative serial section images of hearts subjected to MI at P7 were analyzed by Sirius red / fast green staining of transverse sections 21 days after surgery, and KDM4DTg / + heart. Values are the mean ± SE. Numbers in the bottom left comer of the serial sections indicate the distance in pm from the infarct site, with 0 being the site of the ligature placement. Scale bar: 2mm. (FIG. ID) High-resolution confocal microscopy images quantification demonstrating the effects of MI on BrdU staining at 21 days post-MI performed on P7 control and KDM4DTg / + hearts. Inset: higher magnification image. The quantitative analysis represents thecounting of multiple fields from control and KDM4DTg / + hearts. Scale bar: 50pm. (FIG. IE) High- resolution confocal microscopy images demonstrating the effects of MI on pH3 staining at seven days post-MI performed on P7 control (sham), control (MI), KDM4DTg / + (sham), and KDM4DTg / + (MI) hearts. Inset: higher magnification image. Quantitative analysis represents counting of multiple fields from control (sham), control (MI), KDM4DTg / + (sham), and KDM4DTg / + (MI) hearts. Scale bar: 50pm. (FIG. IF) Representative images and quantification of the effects of MI on cell size in control (sham), control (MI), KDM4DTg / + (sham) and KDM4DTg / + (MI) heart. Hearts were subjected to LAD ligation at seven days old and analyzed by Wheat germ agglutinin staining (W GA) staining of transverse sections 21 days after surgery. Values are the mean ± SE. Scale bar: 50pm. (FIG. 1G) qRTPCR analysis of cell cycle gene RNA expression in CMs 7 days post-MI or sham operation performed in control and KDM4DTg / + mice. All data were normalized to Gapdh. Sample Number: (FIG. 1A-1C) > 5n per group. (FIG. ID) >3 animals per group. (FIG. 1E-1G) >5 animals per group. Statistics: (FIG. 1A-1B) Two-tailed t-test, control vs. KDM4DTg / +, *P < 0.05. (FIG. 1D-1G) Two- way ANOVA / Tukey's test, *P < 0.05, **P < 0.01.

[0050] FIGs. 2A-2F shows KDM4D induction reduces infarct scar size post-MI. (FIG. 2A) 4x magnification image of trichrome stained hearts (left). Quantification of infarct scar size (right) at two weeks post-MI in control vs. iKDM4D mice. (FIG. 2B) Expression of CM and cell cycle genes in isolated ACMs measured by qRT-PCR, fold induction vs. control (sham), expression normalized to Gapdh. (FIG. 2C) BrdU staining quantification of cardiac sections of control (w / Dox) and iKDM4D (w / Dox) two weeks post-MI. Scale bar: 50pm. (FIG. 2D) Representative images (left) of 2,3,5- triphenyl tetrazolium chloride (TTC) staining to visualize ischemic zone showed more significant areas of ischemic damage in induced control hearts than in induced iKDM4D hearts 48 hours after MI. Quantification (right) of infarct scar size (% myocardium) at 48hrs post-MI in control vs. iKDM4D animals. (FIG. 2E) Expression of genes responsible for apoptosis and survival regulation in isolated ACMs measured by qRT-PCR (up), fold induction vs. control (sham), expression normalized to Gapdh. Immunoblotting of ACM lysates (down) showing an increase in anti-apoptotic APAK protein in iKDM4D hearts 48 hrs post-MI vs. controls. (FIG. 2F) Representative images of TUNEL staining in 48 hrs post-MI cardiac sections in control and iKDM4D animals (up). Quantification of TUNEL positive CMs per mm2 (down). Scale bar: 100pm (H) Schematic representation of the proposed model. H3K9me3 represses cellular APAK levels, which in turn leads to increases in p53- dependent apoptosis. However, a decrease in H3K9me3 levels via KDM4D induction subsequently leads to increased APAK expression resulting in a reduction in p53-dependent apoptosis. Sample Number: (FIG. 2A-2C) >5 animals per group. (FIG. 2D) Ctrl (+Dox) = 3, iKDM4D (+Dox) = 3; (FIG. 2E) Ctrl (sham) = 3, iKDM4D (sham) = 4, Ctrl (MI) = 4, iKDM4D (MI) = 5; (protein) Ctrl= 3, iKDM4D= 3. (FIG. 2F) Ctrl (+Dox) = 3, iKDM4D (+Dox) = 3. Statistics: (FIG. 2A) Two-tailed t-test, Ctrl (+Dox) vs. iKDM4D (+Dox), *P < 0.05. (FIG. 2B) Two-way ANOVA / Tukey's test, *P < 0.05 vs. Ctrl (sham), fP < 0.05 vs. iKDM4D (sham), JP < 0.05 vs. Ctrl (MI). (FIG. 2C) Two-tailed t-test,Ctrl (+Dox) vs. iKDM4D (+Dox), P > 0.05. (FIG. 2D) Two-tailed t-test, Ctrl (+Dox) vs. iKDM4D (+Dox), *P < 0.05. (FIG. 2E) Two-way ANOVA / Tukey's test, *P < 0.05 vs. Ctrl (sham), f < 0.05 vs. iKDM4D (sham), JP < 0.05 vs. Ctrl (MI). (FIG. 2F) Two-tailed t-test, Ctrl (+Dox) vs. iKDM4D (+Dox), *P < 0.05.

[0051] FIG. 3A-3I examines a screening for small molecules which promotes NRVM cycling with KDM4D. (FIG. 3A) Schematic shows the timing of viral vector and compounds treatment for small molecule screening. (FIG. 3B) Scatter plot results of small molecule screening. The x-axis shown the fold change of EDU positive NRVM number between compounds+Ad-KDM4D group and Ad-GFP control. Fold change >2 were highlighted with different colors and shapes. Top 10 candidates (fold change>3) were highlight with name. (FIG. 3C) The list of top 10 candidates. The six compounds with red color were tested for downstream experiments. (FIG. 3D) EdU+incorporation in NRVMs treated with or without compounds and Ad-KDM4D in 24-well plates. (FIG. 3E) phospho-H3+immunostaining of NRVMs treated with indicated interventions in 24-well plates. (FIG. 3F) Total cell number was measured by hemocytometer on NRVMs treated as indicated in 24-well plates. (FIG 3G, 3H, 31) RT-qPCR analysis shows the expression of cell cycle genes after 5 days of Ad-KDM4D and 3 days of compounds treatment in NRVMs. Statistics: Data are presented as the means ± SEM. In FIG. 3D, FIG. 3E, and FIG. 3F, * showed statistical significance atP<0.05 vs control (Ad- GFP+DMSO); # represented statistical significance at P<0.05 vs Ad-GFP+compounds. In FIG. 3G, FIG. 3H, and FIG. 31, * mean statistical significance atP<0.05; ** showed statistical significance at P<0.01; *** represented statistical significance at P0.001; **** mean statistical significance at P<0.0001. One-way ANOVA followed by Tukeys’ Test; n=3 for all the experiments.

[0052] FIGs. 4A-4D demonstrates improved heart function after two weeks of Ruxolitinib treatment in iKDM4D mice with ischemia reperfusion injury. (FIG. 4A) Schematic shows the timing of compounds and doxycycline treatment in iKDM4D mice with I / R. (FIG. 4B) 4x magnification image of trichrome stained hearts. Quantification of infarct scar size (right) at two weeks post-I / R in control vs. iKDM4D mice. (FIG. 4C) Echocardiography results show the ejection fraction (EF) and Fractional shortening (FS) after two weeks of I / R injury. (FIG. 4D) Representative immunofluorescence images show EdU-positive, Ki67-positive, and pH3 positive cardiomyocytes (left) bar=100pM. Quantification of EdU-positive and Ki67-positive cardiomyocytes (right) at two weeks post-I / R in mice treated as indicated. Statistics: Data are presented as the means ± SEM. * mean statistical significance atP<0.05; ** showed statistical significance at P<0.01; *** represented statistical significance at P<0.001; **** mean statistical significance at P<0.0001. Two-way ANOVA followed by Tukeys’ Test; each dot represents one animal, n>4 for each group.

[0053] FIGs. 5A-5D show together KDM4D induction and Ruxolitinib mitigate acute myocardial injury 48hrs post-I / R. (FIG. 5A) Schematic shows the timing of compounds and doxycycline treatment in iKDM4D mice at 48hrs post-I / R. (FIG. 5B) Representative images (up) of2,3,5-triphenyl tetrazolium chloride (TTC) staining to visualize ischemic zone in iKDM4D hearts with different treatment at 48 hrs post-I / R. Quantification (down) of infarct size (% myocardium) at 48hrs post-I / R in animals treated with indicated interventions. (FIG. 5C) Representative immunofluorescence images show EdU-positive, Ki67-positive, and pH3 positive cardiomyocytes (left) bar=10pM. Quantification of EdU-positive, Ki67-positive, and pH3-positive cardiomyocytes (right) at 48hrs post-I / R in mice treated as indicated. (FIG. 5D) Representative images of TUNEL staining at 48 hrs post-I / R cardiac sections in animals treated as indicated (left) bar=50pM. Quantification (right) of TUNEL positive CMs percentage. Statistics: Data are presented as the means ± SEM. * mean statistical significance at <0.05; ** showed statistical significance at <0.01; *** represented statistical significance at <0.001; **** mean statistical significance at / ).0001. Two- way ANOVA followed by Tukeys’ Test; each dot represents one animal, n>3 for each group

[0054] FIGs. 6A-6I show improved heart function after eight weeks of Ruxolitinib treatment in iKDM4D mice with ischemia reperfusion injury. (Fig. 6A) Schematic shows the timing of compounds and doxycycline treatment in iKDM4D mice at eight weeks post-I / R. (Figs. 6B to 6E) Echocardiography (Fig. 6B) results show the LVEDD (Fig. 6C), EF (Fig. 6D), and FS (Fig. 6E) after eight weeks of I / R injury. (Fig. 6F) Quantification of HW / BW in vehicle vs iKDM4D+Ruxolitinib mice. (Fig. 6G) Calculated ACM volume and (Fig. 6H) ACM number in vehicle vs iKDM4D+Ruxolitinib mice. (Fig. 61) Shown are the representative images (left) of dispersed CMs from vehicle and iKDM4D+Ruxolitinib mice heart showing the number of nuclei in cardiomyocytes. bar=I00pM. Quantification (right) of ploidy. For each mouse heart, at least 100 dispersed CMs were counted, and confocal Z-stacks were acquired using a Nikon AIR confocal microscope. ACM volume and nuclei number in CMs were measured by Imaris Cell Imaging Software (Imaris 9.2.1, Bitplane, Oxford Instruments Group). Statistics: Data are presented as the means ± SEM. n=5 for each group. * mean statistical significance at <0.05; ** showed statistical significance at <0.01; *** represented statistical significance at P<0.001; **** mean statistical significance at P<0.0001. Two-tailed t-test by Prism 9.4, vehicle vs. iKDM4D + Ruxolitinib.

[0055] FIGs. 7A-7F depicts Pl mice display reduced scar and increased CM cell cycling 21 days after MI. (FIG. 7A) Staining of hearts with 2,3,5-triphenyl tetrazolium chloride (TTC) to visualize ischemic zone showed equivalent areas of ischemic damage in Pl (n=5) and P7 (n=4) hearts 72hrs after MI. Values are the mean ± SE. NS, not significant. Scale bar: 1mm. The dashed area shows the infarct zone. (FIG. 7B) Sirius Red / Fast Green staining and quantification of infarct size at P21 (n=3 per group). Scale bar: 1mm. Values are the mean ± SE. *P < 0.05. (FIG. 7C-7E) Immunostaining (above) and quantification (below) of cell cycle markers (FIG. 7C) BrdU at P21, (FIG. 7D) pH3 7- days after MI, and (FIG. 7E) Aurora Kinase B (AurkB) 7-days after MI. Insets show cycling CMs double positive for CM and cell cycle markers. Quantitative analysis of cell cycle markers represents the counting of multiple fields from three independent samples per group. Values are the mean ± SE. *P < 0.05, **P < 0.01. Scale bars: (FIG. 7C and 7E) 50pm, (FIG. 7D) 100pm. (FIG. 7F) CM RNAexpression analysis demonstrating the effects of surgery on cell cycle genes 7 days post-MI or sham operation performed on Pl and P7 hearts. All data normalized by Gapdh. Pl (sham): n=6, Pl (MI): n=7, P7 (sham): n=7, P7 (MI): n=6. Values are the mean ± SE. *P < 0.05, **P < 0.01

[0056] FIGs. 8A-8D depicts inducible KDM4D mouse model construction, induction protocol, and cardiomyocyte phenotype. (FIG. 8A) Schematic showing breeding strategy resulting in iKDM4D mice, and KDM4D induction in ACMs. (FIG. 8B) Timeline showing protocol for ACM-specific KDM4D expression and endpoints. (FIG. 8C) HW / BW quantification in induced control and iKDM4D (sham or MI) mice. (FIG. 8D) Left, WGA staining in post-MI induced control and iKDM4D PFA-fix hearts, bar = 100pm. Right, quantification of the ACM cross-sectional area. Sample Number: (FIG. 8C, 8D) 3= 5 animals per group. Statistics: (FIG. 8C) Two-way ANOVA / Tukey's test, P > 0.05 vs. control (+Dox / sham); (FIG. 8D) Two-tailed t-test, control (post- MI) vs. iKDM4D (post-MI), P > 0.05.

[0057] FIGs. 9A-9D shows increased cardiomyocyte proliferation after two weeks of Ruxolitinib treatment in uninjured iKDM4D mice. (FIG. 9A) Schematic shows the timing of Ruxolitinib and doxycycline treatment in normal iKDM4D mice (FIG. 9B) EdU+incorporation in iKDM4D mice treated with or without Ruxolitinib and doxycycline. (FIG. 9C) Ki67 immunostaining of mouse heart treated as indicated in vivo. (FIG. 9D) phospho-H3+immunostaining of mouse heart treated with indicated interventions in vivo. Statistics: Data are presented as the means ± SEM. * mean statistical significance at <0.05; ** showed statistical significance at <0.01; *** represented statistical significance at O.OOl; **** mean statistical significance at O.OOOl. Two-way ANOVA followed by Tukeys’ Test; n=3 for each group.

[0058] FIG. 10A-10D shows Ruxolitinib regulates CM proliferation through WNT signaling pathway. (FIG. 10A) Cell signaling phospho-array assay using hESC derived CMs. Schematic shows the timing of virus vector and Ruxolitinib treatment. (FIG. 10B) The array images. (FIG. 10C) The heat map shows the protein abundance compared to the control. The fold change >1.4 from each comparison were listed. (FIG. 10D) Immunoblotting of 14-3-3 using NRVM lysates. n=3 for each group.DETAILED DESCRIPTION

[0059] The inventors have found that combination of: a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D provides surprising synergy in promoting cardiomyocyte proliferation. It is contemplated herein that the expression of KDM4D potentiates the cardiomyocytes to the activity of the JAK inhibitor and / or PI3K inhibitor, thereby providing a synergistic effect, where the individual agents do not provide improved cardiomyocyte proliferation. In some embodiments, the technology described herein relates to compositions and methods for improving cardiac regeneration by administering a JAK inhibitorand / or a PI3K inhibitor along with an adeno-associated virus expressing KDM4D to improve cardiomyocyte proliferation.Definitions

[0060] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0061] Definitions of common terms in cellular and molecular biology, and biochemistry can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0062] 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). The sequences for KDM4D are known for a number of species, e.g., human KDM4D sequences are available in the NCBI database under Gene ID: 55693, and human KDM4D polypeptide sequence is provided hereinas SEQ ID NO: 1. In one embodiment, KDM4D has the amino acid sequence shown in SEQ ID NO: 1. It is specifically contemplated that KDM4D proteins from other species, that perform the same enzymatic reaction, and sequences encoding them could be used in methods as described herein.

[0063] Without wishing to be bound by theory, it is contemplated that other KDM4’s do not promote cardiomyocyte proliferation, as other KDM4’s have different histone demethylation patterns.

[0064] A KDM4D polypeptide can be a variant (e.g, mutant), e.g., with one or more mutations that permit it to be longer acting in vivo, for example, longer acting than an unmutated or wild-type KDM4D. In some embodiments, a polypeptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example, transferrin (WO06096515A2), albumin (Yeh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0065] Mutant KDM4D polypeptides can be designed from any desired KDM4D polypeptide including, but not limited to, human alleles of KDM4D. In some embodiments, the KDM4D polypeptide to be mutated is a human KDM4D polypeptide. For example, a “KDM4D polypeptide” can include the human polypeptide (SEQ ID NO: 1) as well as homologs from other species, including but not limited to bovine, dog, cat chicken, murine, rat, porcine, ovine, turkey, horse, fish, baboon and other primates.

[0066] In order to retain the therapeutic benefit of a KDM4D polypeptide, it is specifically contemplated herein that the mutant KDM4D polypeptide retains the ability to catalyze the removal of methyl marks from histone lysine residues. In other embodiments, the mutant KDM4D polypeptide retains at least 50% of the activity of the wild-type KDM4D polypeptide as assessed by measuring the removal of methyl marks from histone lysine residues. In other embodiments, the mutant KDM4D polypeptide retains at least 50% of the activity of the wild -type KDM4D polypeptide as assessed by measuring cardiomyocyte proliferation, e.g., as measured in an appropriate animal model. In other embodiments, the mutant KDM4D polypeptide retains at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, activity or retains activity that is at least substantially similar to the wild-type KDM4D polypeptide from which it is derived. It is further contemplated herein that the mutant KDM4D can comprise increased activity over the wild-type KDM4D, for example, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more activity compared to the wild-type KDM4D polypeptide.

[0067] In one embodiment, a mutant KDM4D polypeptide as described herein catalyzes the removal of methyl marks from histone lysine residues. Amino acid insertions and deletions are specifically contemplated herein in a mutant KDM4D polypeptide provided that such insertions or deletions do not impair the ability of the mutant KDM4D polypeptide to catalyze the removal of methyl marks from histone lysine residues. In certain embodiments, an insertion comprises at least one additional residuebut does not exceed 20 additional residues, for example, 1-18 residues, 1-16 residues, 1-15 residues, 1- 14 residues, 1-12 residues, 1-10 residues, 1-9 residues, 1-8 residues, 1-7 residues, 1-6 residues, 1-5 residues, 1-4 residues, 1-3 residues or 1-2 residues are inserted. In other embodiments, a deletion comprises removal of at least one residue but does not exceed 10 residues, for example, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or 2 residues are deleted.

[0068] In one embodiment, a mutant KDM4D polypeptide described herein comprises 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer, i.e., does not comprise any mutations, including substitutions, deletions or insertions.

[0069] It is also contemplated herein mutations can be introduced to improve in vivo half-life, enhance activity, or reduce side effects in a subject. Conservative substitution variants that maintain the desired activity of a mutant KDM4D polypeptide can include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wild-type is guided by, for example, sequence alignment with KDM4D homologs or paralogs from other species. Amino acids that are identical between such homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of cardiomyocyte proliferation as described herein. Further discussion of the structure of KDM4D can be found, e.g. in Labbe et al Am J Transl Res 6: 1-15 (2014); which is incorporated by reference herein in its entirety.

[0070] In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide -encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., can promote cardiomyocyte proliferation at least 50% as well as its corresponding mutant KDM4D polypeptide or the wild-type KDM4D from which it is derived. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar aminoacid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, that has more than 100% of the activity of e.g. , the mutant KDM4D polypeptide or the wild-type KDM4D polypeptide from which it is derived, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[0071] One method of identifying amino acid residues which can be substituted is to align, for example, human KDM4D to a KDM4D homolog from one or more non-human species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, e.g., SEQ ID NO: 1 or a nucleic acid encoding one of those amino acid sequences. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set.

[0072] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure. Typically conservative substitutions for one another include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (L), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0073] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0074] Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Khudyakov et al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0075] In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 97% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 98% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises a sequence with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, a KDM4D protein comprises the sequence of SEQ ID NO: 1.

[0076] As used herein, a Janus Kinase (JAK) inhibitor is a type of immune modulating medication, which inhibits the activity of one or more of the Janus kinase family of enzymes (JAK1, JAK2, JAK3, TYK2).

[0077] As used herein, “proliferating” and “proliferation” refer to an increase in the number of cells in a population (growth) by means of cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to theenvironment, including growth factors and other mitogens. Cell proliferation can also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.

[0078] As used herein, a phosphoinositide 3-kinase (PI3K) inhibitor is a class of drug that functions by inhibiting one or more of the phosphoinositide 3-kinase enzymes, which are part of the PI3K / AKT / mT0R pathway.

[0079] “Treatment” of a cardiac disorder, a cardiac disease, event, or a cardiac injury (e.g., myocardial infarction) as referred to herein refers to therapeutic intervention that enhances cardiac function, and / or enhances cardiomyocyte proliferation in a treated area, thus improving the function of e.g., the heart. That is, cardiac “treatment” is oriented to the function of the heart (e.g., enhanced function within an infarcted area or as a whole), and / or other site treated with the agents or compositions described herein. Effective treatment need not cure or directly impact the underlying cause of the heart disease or disorder to be considered effective treatment.

[0080] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to a myocardial infarction. In some embodiments of any of the aspects, the methods described herein can be prophylactic relative to a second or further myocardial infarction. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0081] The terms “patient”, “subject” and “individual” are used interchangeably herein, and refer to an animal, particularly a human, to whom treatment, including prophylactic treatment is provided. The term “subject” as used herein refers to human and non-human animals. The term “non-human animals” and “non-human mammals” are used interchangeably herein includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. In one embodiment of any of the aspects, the subject is human. In another embodiment, of any of the aspects, the subject is an experimental animal or animal substitute as a disease model. In another embodiment, of any of the aspects, the subject is a domesticated animal including companion animals (e.g., dogs, cats, rats, pigs, guinea pigs, hamsters etc.). A subject can have previously received a treatment for a cardiovascular disease or cardiac event, or has never received treatment for a cardiovascular disease or a cardiac event. A subject can have previously been diagnosed with having a cardiovascular disease, or has never been diagnosed with a cardiovascular disease.

[0082] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0083] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0084] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0085] The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0086] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0087] The terms “increased," “increase," “increases,” or “enhance" or “activate" are all used herein to generally mean an increase of a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms “increased", “increase" or “enhance" or “activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold increase, at least about a 1000-fold increase or more as compared to a reference level.

[0088] As used herein, a “reference level” refers to the level of a marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., an untreated cell or tissue, a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., cell, tissue, or a biological sample obtained from a patient prior to being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition disclosed herein).

[0089] As used herein, an “appropriate control” refers to an untreated, otherwise identical cell, subject, organism, or population (e.g., a cell, tissue, or biological sample that was not contacted by an agent or composition described herein) relative to a cell, tissue, biological sample, or population contacted or treated with a given treatment.

[0090] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0091] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0092] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0093] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather thanlimitation.

[0094] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0095] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0096] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise . Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g. " is derived from the Latin exempli gratia, and is used herein to indicate a non limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0097] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0098] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0099] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al.(eds.), The Dictionary of Cell and Molecular Biology, 5thEdition, published by Academic Press, 2013 (ISBN 0123849314, 978-0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell and Molecular Biology, 3rdEdition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898);Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8thEdition,published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C.King et al. (eds.), A Dictionary of Genetics, 8thEdition, published by Oxford University Press, 2013 (ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2ndEdition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8thEdition, published by W.H. Freeman, 2016 (ISBN 1464183392, 978- 1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10thEdition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10thEdition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0100] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0101] Other terms are defined herein within the description of the various aspects of the invention.

[0102] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on theinformation available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0103] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0104] As used herein, the term "inhibitor" refers to any compound, natural or synthetic, which can antagonize or reduce the activity of a target protein or signaling pathway. An inhibitor can be, for example, a peptide, a protein, an antibody, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound. An inhibitor may attenuate or prevent the activity of a target protein either directly or indirectly.

[0105] Direct inhibition can be obtained, for instance, by binding to a protein and preventing the protein from interacting with an endogenous molecule (i.e. ligand), such as an enzyme, an ion, a hormone, a growth factor, a cytokine, a substrate, or other binding partner, thereby diminishing the activity of the protein. For instance, an inhibitor may bind an enzyme active site and sterically preclude binding of an endogenous substrate at this location, thus decreasing the enzymatic activity of the protein. Direct inhibitors can also act directly on the corresponding gene and / or mRNA of a named target protein. In some embodiments of any of the aspects, an inhibitor of a named target described herein is a direct inhibitor of that named target.

[0106] Alternatively, indirect inhibition can be obtained, for instance, by binding to a second protein that promotes the activity of a named target protein by inducing a conformational change or catalyzing a chemical modification of the named target protein. For instance, indirect inhibition of a named target protein may be achieved by binding and inactivating a kinase that catalyzes the phosphorylation of, and thus activates, the named target protein. An indirect inhibitor can also act on a protein which is downstream of the named target and thereby arrogate the activity of thenamed target protein. For instance, if the named target protein is a kinase, an indirect inhibitor can be a phosphatase that acts on the protein phosphorylated by the kinase, or a protein that promotes or contributes to the degradation of the protein phosphorylated by the kinase. In some embodiments of any of the aspects, an inhibitor of a named target described herein is an indirect inhibitor of that named target.JAK Inhibitors

[0107] In some embodiments, a JAK inhibitor is an indirect inhibitor. The components of the JAK signaling pathway are known in the art and an indirect inhibitor can act on one or more of these pathway components, e.g., the proteins, genes, or mRNAs of these pathway components. For example, the JAK signaling pathway is described in the art in Tanaka et al. Nature Reviews Rheumatology 18: 133-145 (2022); which is incorporated by reference herein in its entirety. Assays for measuring JAK inhibition are known in the art, for example, one or more JAKs can be incubated with an inhibitor, a substrate, and ATP. The JAK-catalyzed phosphorylation of the substrate will consume ATP. Once the reaction is performed the amount of remaining ATP can be measured, e.g., using a kinase detection reagent that detects the ATP by a luciferase / luciferin reaction. Greater JAK inhibition will result in more ATP being detected. Kits for such reactions are commercially available, e.g., JAK Assay Kits (using KINASE-GLO MAX™ detection reagent), from BPS Bioscience (San Diego, CA).

[0108] In some embodiments of any of the aspects, a JAK inhibitor is a direct JAK inhibitor, e.g., it functions by physical interaction with at least one JAK protein and thereby inhibits the activity of the at least one JAK protein.

[0109] Janus Kinase (JAK) proteins comprise four main domains. There is a FERM domain, which exists at the N-terminus and is important for JAKs binding to cytokine receptors, a Src2-homology-2 (SH2) domain, a pseudo-kinase domain, and a kinase domain at the C-terminus. This domain is responsible for the enzymatic activity of the kinase. There are four members of the JAK protein family, JAK1, JAK2, JAK3, and Tyrosine Kinase 2 (TYK2). In some embodiments of any of the aspects, a JAK inhibitor inhibits all 4 JAK proteins. In some embodiments of any of the aspects, a JAK inhibitor is specific for inhibition of one or a subset of the JAK proteins, e.g., it does not inhibit all four JAK proteins. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAKE In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK2. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK3. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits TYK2.

[0110] In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK1 and JAK2. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK1 and JAK3. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK1 andTYK2. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK2 and JAK3. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK2 and TYK2. In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits JAK3 and TYK2.

[0111] In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK1. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK2. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK3. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits TYK2.

[0112] In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK1 and JAK2. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK1 and JAK3. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK1 and TYK2. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK2 and JAK3. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK2 and TYK2. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits JAK3 and TYK2.

[0113] In some embodiments of any of the aspects, a JAK inhibitor specifically inhibits at least one of JAK1 or JAK2. In some embodiments of any of the aspects, a direct JAK inhibitor specifically inhibits at least one of JAK1 or JAK2.

[0114] JAK inhibitors can be divided into non-selective and selective inhibitors of JAKs. They can also be classified based on their binding mode and type of interactions with the amino acids in JAKs into reversible (competitive) and irreversible (covalent) inhibitors.

[0115] Competitive JAK inhibitors form reversible (non-covalent) binding interactions with the amino acids in one or more of the four JAKs. The binding interactions formed by this type of JAK inhibitor includes hydrogen bonds and hydrophobic interactions. The class of reversible JAK inhibitors can also be classified into two sub-classes: Type I JAK inhibitors and Type II JAK inhibitors.

[0116] Type I JAK inhibitors bind to the ATP -binding site of the JAKs under the active conformation of the kinase domain. This includes clinically approved drugs such as filgotinib, which acts and is classified as a selective JAK1 inhibitor, while fedratinib exhibits selective inhibition of JAK2. On the other hand, tofacitinib and peficitinib act by blocking multiple JAKs. The ability of type I JAK inhibitors to bind to multiple kinases and act as non-selective inhibitors could be due to the highly conserved structure of the ATP -binding site in the four JAKs.

[0117] Type II JAK inhibitors also bind to the ATP -binding site of the kinase domain in the inactive conformation of JAKs. NVP-BBT594 and NVP-CHZ868 are representative examples of type II inhibitor, which target JAK2.

[0118] The allosteric JAK inhibitors include small molecule inhibitors that bind to a site other thanthe ATP-binding site in JAKs. Among these inhibitors, deucravacitinib (BMS-986165) acts as a selective allosteric inhibitor of TYK2. In addition, LS104, and ON044580 are examples of JAK2 allosteric inhibitors.

[0119] Irreversible JAK3 Inhibitors that target JAK3 have also been reported. The mechanism of action of these inhibitors depends on the covalent interaction with the unique Cys909 residue in JAK3

[0043] , The chemical structure of these inhibitors has a covalent-bond forming group such as acrylamide and a-cyanoacrylamide, which can bind covalently with Cys909 residue. Additional information regarding the groups of JAK inhibitors can be found in Shawky et al.. A Comprehensive Overview of Globally Approved JAK Inhibitors. Pharmaceutics. 2022 May 6; 14(5): 1001. doi: 10.3390 / pharmaceuticsl4051001. PMID: 35631587; PMCID: PMC9146299.

[0120] JAK inhibitors that target the JAK proteins include, but are not limited to Ruxolitinib, Tofacitinib, Oclacitinib, Baricitinib, Peficitinib, Upadacitinib, Fedratinib, Delgocitinib, Filgotinib, Abrocitinib, Pacritinib, Deucravacitinib, Ritlecitinib, Cerdulatinib, Gandotinib, Lestaurtinib, Momelotinib, Pacritinib, Deucravacitinib, Cucurbitacin I, and CHZ868.

[0121] In some embodiments of any of the aspects, the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868. In some embodiments of any of the aspects, the JAK inhibitor is selected from the group consisting of: tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

[0122] In some embodiments of any of the aspects, the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945. In some embodiments of any of the aspects, the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, nilotinib, AG-490, and cx-4945. In some embodiments of any of the aspects, the JAK inhibitor is ruxolitinib.

[0123] In some embodiments of any of the aspects, the JAK inhibitor is not ruxolitinib.

[0124] Additional information on structure of JAK inhibitors and administration of JAK inhibitors can be found in U.S. Patent 7,031,023, U.S. Patent 8,808,764, U.S. Patent 10,617,690 and U.S. Patent 10,973,913, which are incorporated by reference herein in their entireties.PI3K Inhibitors

[0125] In some embodiments, a PI3K inhibitor is an indirect inhibitor. The components of the PI3K signaling pathway are known in the art and an indirect inhibitor can act on one or more of thesepathway components, e.g., the proteins, genes, or mRNAs of these pathway components. For example, the PI3K signaling pathway is described in the art in Yu et al. Archives of Toxicology 97:635-650 (2023); which is incorporated by reference herein in its entirety. Assays for measuring PI3K inhibition are known in the art, for example, one or more PI3Ks can be incubated with an inhibitor, a substrate (e.g., P PS and / or PIP2:3PS), and ATP. The PI3K-catalyzed phosphorylation of the substrate will consume ATP and generate ADP. Once the reaction is performed, any remaining ATP can be depleted, and the generated ADP measured, e.g., using a kinase detection reagent that converts the ADP back to ATP and detects the ATP by a luciferase / luciferin reaction. Greater PI3K inhibition will result in less ADP being detected. Kits for such reactions are commercially available, e.g., PI3K-GLO™ Kits from Promega (Madison, WI). PI3K inhibition can also be measured using antibodies specific for phosphorylated versions of PI3K targets. For example, antibodies specific for phosphorylated AKT. Kits for such assays are commercially available, e.g., the MUSE™ PI3K Activation Kits from Cytek (Fremont, California).

[0126] In some embodiments of any of the aspects, a PI3K inhibitor is a direct PI3K inhibitor, e.g., it functions by physical interaction with at least one PI3K protein and thereby inhibits the activity of the at least one PI3K protein. Such phosphoinositide 3-kinase (PI3K) inhibitors function by inhibiting one or more of the phosphoinositide 3-kinase (PI3K) enzymes, which are part of the PI3K / AKT / mT0R pathway. This signal pathway regulates cellular functions such as cell growth and survival.

[0127] There are different classes and isoforms of Phosphatidylinositol 3-kinases (PI3Ks). Class 1 PI3Ks have a catalytic subunit known as pl 10, with four types (isoforms) - pl 10 alpha (PIK3CA), pl 10 beta (PIK3CB), pl 10 gamma (PIK3CG) and pl 10 delta (PIK3CD). Most PI3K inhibitors that are currently approved inhibit one or more pl 10 isoforms of the class I PI3Ks. Inhibiting different pl 10 isoforms can have different effects, e.g. PTEN -negative tumors may be more sensitive to PIK3CB inhibitors. Additional information regarding the classes and isoforms of PI3Ks can be found in Ito et al.. Therapeutic potential of phosphatidylinositol 3-kinase inhibitors in inflammatory respiratory disease. J Pharmacol Exp Ther. 2007 Apr;321(l): 1-8. doi:10.1124 / jpet.106.111674. Epub 2006 Oct 4. PMID: 17021257, which is incorporated by reference herein in its entirety.

[0128] In some embodiments of any of the aspects, a PI3K inhibitor inhibits all classeses and isoforms of PI3K proteins. In some embodiments of any of the aspects, a PI3K inhibitor is specific for inhibition of one or a subset of the PI3K proteins, e.g., it does not inhibit every class and / or isoform of PI3K. In some embodiments of any of the aspects, a PI3K inhibitor specifically inhibits Class 1 PI3Ks. In some embodiments of any of the aspects, a PI3K inhibitor specifically inhibits Class 2 PI3Ks. In some embodiments of any of the aspects, a PI3K inhibitor specifically inhibits Class 3 PI3Ks.

[0129] In some embodiments of any of the aspects, a PI3K inhibitor specifically inhibits Class 1 pl 10a isoform PI3Ks. In some embodiments of any of the aspects, a PI3K inhibitor specifically inhibits Class 1 pl iop isoform PI3Ks. In some embodiments of any of the aspects, a PI3K inhibitor specifically inhibits Class 1 pl 10a isoform and Class 1 pl 10 isoform PI3Ks. In some embodiments of any of the aspects, a PI3K inhibitor specifically inhibits at least one of Class 1 pl 10a isoform PI3Ks and Class 1 pl iop isoform PI3Ks.

[0130] In some embodiments of any of the aspects, a direct PI3K inhibitor inhibits all class and isoforms of PI3K proteins. In some embodiments of any of the aspects, a direct PI3K inhibitor specifically inhibits Class 1 PI3Ks. In some embodiments of any of the aspects, a direct PI3K inhibitor specifically inhibits Class 2 PI3Ks. In some embodiments of any of the aspects, a direct PI3K inhibitor specifically inhibits Class 3 PI3Ks.

[0131] In some embodiments of any of the aspects, a direct PI3K inhibitor specifically inhibits Class 1 pl 10a isoform PI3Ks. In some embodiments of any of the aspects, a direct PI3K inhibitor specifically inhibits Class 1 pl iop isoform PI3Ks. In some embodiments of any of the aspects, a direct PI3K inhibitor specifically inhibits Class 1 pl 10a isoform and Class 1 pl iop isoform PI3Ks. In some embodiments of any of the aspects, a direct PI3K inhibitor specifically inhibits at least one of Class 1 pl 10a isoform PI3Ks and Class 1 pl iop isoform PI3Ks.

[0132] PI3K inhibitors that target PI3K include, but are not limited to idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, Umbralisib, Buparlisib, Dactolisib, Parsaclisib, Paxalisib, Taselisib, Zandelisib, Inavolisib, Apitolisib, Bimiralisib, Eganelisib, Fimepinostat, Gedatolisib, Linperlisib, Nemiralisib, Pictilisib, Pilaralisib, Samotolisib, Seletalisib, Serabelisib, Sonolisib, Voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, and ZSTK474.

[0133] In some embodiments of any of the aspects, the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TGI 00- 115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765. In some embodiments of any of the aspects, the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, duvelisib, copanlisib, umbralisib, buparlisib, IPI-549, AZD8186, and GSK2636771. In some embodiments of any of the aspects, the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765. In some embodiments of any of the aspects, the PI3K inhibitor is PHT-427

[0134] Additional information on structure of PI3K inhibitors and administration of PI3K inhibitors can be found in U.S. Patent 9,949,979, U.S. Patent 10,842,791, U.S. Patent 11,607,414, U.S. Patent10,206,924, and U.S. Patent Publication US20190022156, which are incorporated by reference herein in their entireties.Genetic Constructs

[0135] The methods, kits, and combinations described herein relate to the use of a JAK inhibitor and / or PI3K inhibitor in combination with a genetic construct encoding lysine -specific demethylase 4D (KDM4D). The methods, kits, and combinations described herein relate to the use of a JAK inhibitor and / or PI3K inhibitor in combination with a genetic construct that directs the expression of KDM4D, e.g., a KDM4D polypeptide. As used herein, “directs the expression” refers to a construct or polynucleotide that encodes at least one polypeptide and which further comprises the regulatory sequences necessary to drive expression of the encoded polypeptide, e.g., a promoter.

[0136] A genetic construct can be a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0137] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0138] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized forexpression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0139] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0140] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0141] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.Viral Vectors

[0142] In some embodiments, KDM4D activity can be increased in or delivered to cardiac tissue using a viral vector encoding the KDM4D polypeptide. In some embodiments of any of the aspects, the genetic construct that directs the expression of KDM4D comprises a viral vector. In some embodiments of any of the aspects, the genetic construct that directs the expression of KDM4D is provided in a viral vector. A number of different viral vectors are known, including viral vectors appropriate for delivery of transgenes to tissues in vivo.

[0143] “Delivery vectors” are used to deliver their nucleic acid cargo into a cell, typically to express the nucleic acid in the cell. A vector can include, but is not limited to, plasmids, mini -chromosomes, phage, naked DNA, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150;5,707,828; 5,759,828; 5,888,783, and 5,919,670, and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). In one embodiment, delivery vectors include, without limitation viral vectors. A variety of viral vectors are known in the art (e.g., those derived from herpesvirus, Epstein-Barr virus, retrovirus, baculovirus, adenovirus, or parvovirus such as adeno-associated virus). Non-viral delivery vectors are also known in the art and their use is also encompassed by the instant invention.

[0144] Viral vectors can include, for example, an adenovirus, and adeno-associated virus (AAV) vectors, such as a recombinant AAV vector (rAAV). In some embodiments of any of the aspects, a viral vector is an adenoviral vector. In some embodiments of any of the aspects, a viral vector is an AAV. In some embodiments of any of the aspects, a viral vector is an adenoviral vector or an AAV. In some embodiments of any of the aspects, a viral vector is a rAAV.

[0145] Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. Integration of virally carried genes into the host genome can also occur.

[0146] One type of delivery vector is adeno-associated virus (AAV). AAV itself is a non- pathogenic-dependent parvovirus that needs helper viruses for efficient replication. AAV is a protein shell surrounding and protecting a small, single -stranded DNA genome of approximately4.8 kilobases (kb). AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. Its singlestranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP I / VP2 / VP3). which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization (Samulski RJ, Muzyczka N. AAV -mediated gene therapy for research and therapeutic purposes. Annu Rev Virol. 2014; 1( 1):427— 451. doi: 10.1146 / annurev-virology-031413- 085355). It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1: 1: 10 (VP1:VP2:VP3). The aap gene encodes the assembly -activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly (Naumer M, et al., J Virol. 2012;86(23): 13038-13048. doi: 10. 1128 / JVI.01675-12). While AAP is essential for nucleolar localization of VP proteins and capsid assembly in AAV2, the subnuclear localization of AAP varies among 11 other serotypes and is nonessential in AAV4, AAV5, and AAV11 (Earley LF, et al. Adeno- associated Virus (AAV) assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5, and 11. J Virol. 2017;91(3): 1— 21. doi: 10. 1128 / jvi.01980-16).

[0147] AAV has been utilized as a viral vector for gene therapy because of its safety and simplicity. AAV has a broad host and cell type tropism capable of transducing both dividing and nondividing cells.

[0148] Alternatively, in some contexts, the term “vector,” “virus vector,” “delivery vector” (and similar terms) may be used to refer to the vector genome (e.g., vDNA) in the absence of the virion and / or to a viral capsid that acts as a transporter to deliver molecules tethered to the capsid or packaged within the capsid.

[0149] AAV sequences may be obtained from a variety of sources. For example, a suitable AAV sequence may be obtained as described in WO 2005 / 033321 or from known sources, e.g., the American Type Culture Collection, or a variety of academic vector core facilities. Alternatively, suitable sequences are synthetically generated using known techniques with reference to published sequences. In some embodiments, the AAV vector can be selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. In some embodiments of any of the aspects, the AAV vector is AAV9.

[0150] Components for AAV production (e.g., adenovirus Ela, Elb, E2a, and / or E4ORF6 gene products, rep or a fragment(s) thereof, cap, the expression cassette, as well as any other desired helper functions), may be delivered to the packaging host cell separately, or in combination, in the form of any genetic element which transfer the sequences carried thereon. As used herein, a genetic element (vector) includes, e.g., naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in a non-viral delivery vehicle (e.g., a lipid-based carrier), virus, etc., which transfers the sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. See, e.g., K. Fisher et al, J Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

[0151] Regulatable promoters allow control of gene expression by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Regulatable promoters and systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system [WO 98 / 10088]; the ecdysone insect promoter [No et al., Proc. Natl. Acad. Set. USA, 93:3346-3351 (1996)], the tetracycline-repressible system [Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system [Gossen et al., Science, 268: 1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)], the RU486-inducible system [Wang et al., Nat. Biotech.,15:239-243 (1997) and Wang etal., Gene Ther., 4:432-441 (1997)] and the rapamycin-inducible system [Magari et al., J Clin. Invest., 100:2865-2872 (1997)]. Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0152] In certain instances, a native promoter is used. The native promoter may be used when it is desired that expression of the gene product should mimic the native expression. The native promoter may be used when expression of a desired transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. Other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0153] In some embodiments, a cardiac-specific promoter is used. A “cardiac -specific promoter,” in the context of the present invention, refers to a wild-type or recombinant promoter that selectively drives expression of a gene under its control in cardiac cells. Examples of cardiac-specific promoters include the a-MHC5.5 promoter, the a-MHCse promoter, the human cardiac actin promoter, a cTnC promoter, a cTnT promoter, and the cTnT455 promoter described herein. Additional information on the types of and use of a cardiac-specific promoter can be found in International Application No.: PCT / US2015 / 023066, which is incorporated by reference herein in its entirety.

[0154] A recombinant AAV vector can be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The transgene can comprise one or more regions that encode one or more isolated polypeptides as described herein (e.g., KDM4D). The transgene can comprise a region encoding, for example, an isolated polypeptide as described herein (e.g., KDM4D) and / or an expression control sequence (e.g., a poly- A tail).

[0155] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. In some embodiments, where the vector is an AAV vector, the dosage of the vector may be from IxlO10gc / kg to IxlO15gc / kg or more, suitably from IxlO12gc / kg to IxlO14gc / kg, suitably from 5xl012gc / kg to 5xl013gc / kg. In some embodiments, the amount of the viral vector is between IxlO11and IxlO16plaque forming units (pfii).

[0156] In one embodiment, an rAAV is administered at a total dose within the range of IxlO9vg to about IxlO16vg, IxlO10vg to about IxlO16vg, IxlO11vg to about IxlO16vg, IxlO12vg to about IxlO16vg, lxl013vg to about lxl016vg, lxlO14vg to about lxl016vg, lxl015vg to about lxl016vg, lxl09vg to about lxl015vg, lxl09vg to about lxlO14vg, lxl09vg to about lxl013vg, lxl09vg to about IxlO12vg, lxl09vg to about IxlO11vg, lxl09vg to about lxlOlovg, IxlO11vg to about lxl013vg, lxl012vg to about IxlO14vg, or IxlO13vg to about IxlO15vg.Kits and Combinations

[0157] In some embodiments of any of the aspects, a method, composition, combination, or kit described herein relates to a JAK inhibitor and a genetic construct that directs the expression of KDM4D. In some embodiments of any of the aspects, a method, composition, combination, or kit described herein relates to a PI3K inhibitor; and a genetic construct that directs the expression of KDM4D. In some embodiments of any of the aspects, a method, composition, combination, or kit described herein relates to a JAK inhibitor; a PI3K inhibitor; and a genetic construct that directs the expression of KDM4D.

[0158] In one aspect of any of the embodiments, described herein is a combination of: a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D. In one aspect of any of the embodiments, described herein is a combination of: a) a JAK inhibitor; and b) a genetic construct that directs the expression of KDM4D. In one aspect of any of the embodiments, described herein is a combination of: a) a PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D. In one aspect of any of the embodiments, described herein is a combination of: a JAK inhibitor; a PI3K inhibitor; and a genetic construct that directs the expression of KDM4D.

[0159] As used herein “combination” refers to a group of two or more substances or elements for use together, e.g., for use in reducing cardiac damage or injury and / or promoting cardiomyocyte proliferation. The two or more substances can be present in the same formulation in any molecular or physical arrangement, e.g., in an admixture, in a solution, in a mixture, in a suspension, in a colloid, in an emulsion. The formulation can be a homogeneous or heterogenous mixture. In some embodiments of any of the aspects, the two or more substances can be comprised by the same or different superstructures, e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like, and said superstructure is in solution, mixture, admixture, suspension with a solvent, carrier, or some of the two or more substances. Alternatively, the two or more substances can be present in two or more separate formulations, e.g., in a kit or package comprising multiple formulations in separate containers, to be mixed or brought into contact with each other when cell growth and / or culture is to be performed.

[0160] In some embodiments, the elements of a combination are provided in the same formulation. In some embodiments, the elements of a combination are provided in separate formulations or containers.

[0161] In one aspect of any of the embodiments, described herein is a kit comprising: a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D. In one aspect of any of the embodiments, described herein is a kit comprising: a) a JAK inhibitor; and b) a genetic construct that directs the expression of KDM4D. In one aspect of any of the embodiments, described herein is a kit comprising: a) a PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D. In one aspect of any of the embodiments, described herein is a kit comprising: a JAK inhibitor; a PI3K inhibitor; and a genetic construct that directs the expression of KDM4D.

[0162] A kit is an assemblage of materials or components, including at least one element or reagent described herein. The exact nature of the components configured in the kit depends on its intended purpose. In some embodiments of any of the aspects, a kit includes instructions for use. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit, e.g., to administer to a patient. Still in accordance with the present invention, “instructions for use” may include a tangible expression describing the preparation of at least one element described herein, such as dilution, mixing, or dosing instructions, and the like, typically for an intended purpose. Optionally, the kit also contains other useful components, such as, measuring tools, diluents, buffers, syringes, pharmaceutically acceptable carriers, or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0163] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging may also preferably provide an environment that protects from light, humidity, and oxygen. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, polyester (such as polyethylene terephthalate, or Mylar) and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of a composition containing a volume of at least one reagent described herein. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.

[0164] In some embodiments of any of the aspects, a kit or combination described herein is for use in treating a cardiac disease or disorder. In some embodiments of any of the aspects, a kit or combination described herein is for use in treating a cardiovascular disease or disorder.Cardiovascular Diseases

[0165] The inventors have found that the combination of a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D provides a synergistic effect on cardiomyocyte proliferation. Accordingly, described herein is a method for promoting cardiomyocyte proliferation comprising contacting a cardiomyocyte with a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D. In some embodiments of any of the aspects, the method is an ex vivo or in vitro method. In some embodiments of any of the aspects, the cardiomyocyte is in a subject in vivo, and the contacting comprises administering the a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D to the subject.

[0166] In some embodiments of any of the aspects, the cardiomyocyte is a human cardiomyocyte. In some embodiments of any of the aspects, the subject or patient is a human.

[0167] The ability to induce significant levels of cardiomyocyte proliferation can provide a therapeutic effect, e.g., in cardiac diseases and disorders and / or cardiovascular diseases and disorders, e.g., by repairing or replacing damaged or diseased tissue. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating cardiac damage in a subject in need thereof, the method comprising administering a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D, to the subject. In one aspect of any of the embodiments, described herein is a method of reducing cardiac damage in a subject in need thereof, the method comprising administering a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D, to the subject. In one aspect of any of the embodiments, described herein is a method of slowing the rate of cardiac damage in a subject in need thereof, the method comprising administering a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D, to the subject.

[0168] In one aspect of any of the embodiments, described herein is a method of treating a cardiac disease or disorder in a subject in need thereof, the method comprising administering a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D, to the subject. In one aspect of any of the embodiments, described herein is a method of treating a cardiovascular disease or disorder in a subject in need thereof, the method comprising administering a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of KDM4D, to the subject.

[0169] In some embodiments of any of the aspects, the subject has cardiac damage.

[0170] In some embodiments of any of the aspects, the subject has a cardiac disease or disorder. A cardiac disease is a disease that affects the heart of a subject. Non-limiting examples of cardiac diseases include atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation,ischemia, fibrillation, and polymorphic ventricular tachycardia. Symptoms of cardiac disease can include but are not limited to syncope, fatigue, shortness of breath, chest pain, and palpitations. A cardiac disease is generally diagnosed by a physical examination, blood tests, and / or an electrocardiogram (EKG).

[0171] In some embodiments of any of the aspects, the subject has a cardiovascular disease or disorder. A cardiovascular disease is a disease that affects the heart and / or circulatory system of a subject. Non-limiting examples of cardiovascular diseases include atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia. Symptoms of cardiovascular disease can include but are not limited to syncope, fatigue, shortness of breath, chest pain, and palpitations. A cardiovascular disease is generally diagnosed by a physical examination, blood tests, and / or an electrocardiogram (EKG). An abnormal EKG is an indication that the subject has an abnormal cardiac rhythm or cardiac arrhythmia. Methods of diagnosing arrhythmias are known in the art.

[0172] In some embodiments of any of the aspects, the subject has suffered a myocardial infarction.

[0173] In some embodiments of any of the aspects, the subject has suffered ischemic injury to cardiovascular tissue. In some embodiments of any of the aspects, the subject has suffered ischemic injury to cardiac tissue.

[0174] In some embodiments of any of the aspects, the subject has or is at risk for having a cardiovascular disease or a cardiac event. The term, “cardiac event” refers to an incident of a myocardial injury, myocardial infarction, ventricular fibrillation, stenosis, arrhythmia, or the like.

[0175] Cardiac electrophysiological and contractile function is a tightly controlled process. When ion channel regulation or contractile function is disrupted in a cardiac cell or tissue, this can result in cardiac arrhythmias that can sometimes be deadly. Cardiac diseases remain a leading cause of death worldwide.Dosage and Administration

[0176] Pharmaceutical or therapeutic compositions comprising a JAK inhibitor and / or a PI3K inhibitor and / or a vector expressing KDM4D to promote cardiomyocyte proliferation can contain a physiologically tolerable carrier, wherein the therapeutic agent is dissolved or dispersed therein as an active ingredient(s). In a preferred embodiment, the pharmaceutical composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes. As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceuticallyacceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological or pharmaceutical composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition comprising a therapeutic agent for promoting cardiomyocyte proliferation can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[0177] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment or prevention of cardiac graft rejection depends on a number of factors, and can be determined by standard clinical techniques.

[0178] A pharmaceutical composition as described herein can be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0179] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients can be prepared as appropriate oily or water-based injection suspensions.

[0180] Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0181] Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

[0182] In some embodiments, a therapeutic agent can be delivered in an immediate release form. In other embodiments, the therapeutic agent can be delivered in a controlled-release system or sustained- release system. Controlled- or sustained-release pharmaceutical compositions can have a common goal of improving drug therapy over the results achieved by their non-controlled or non-sustained-release counterparts. Advantages of controlled- or sustained-release compositions include extended activity of the therapeutic agents, reduced dosage frequency, and increased compliance. In addition, controlled- or sustained-release compositions can favorably affect the time of onset of action or other characteristics, such as blood levels of the therapeutic agent, and can thus reduce the occurrence of adverse side effects. Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

[0183] In one embodiment, a pump can be used for administration (Langer, Science 249: 1527-1533 (1990); Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N. Engl. J. Med 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25:351 (1989); and Howard et al., J. Neurosurg. 71: 105 (1989)).

[0184] The appropriate dosage range for a given therapeutic agent depends upon the potency, and includes amounts large enough to produce the desired effect, e.g., reduction in at least one symptom of cardiac graft rejection. The dosage of the therapeutic agent should not be so large as to cause unacceptable or life-threatening adverse side effects or should be used under close supervision by a medical professional. Generally, the dosage will vary with the type of agent, and with the age, condition, and sex of the patient. The dosage can be determined by one of skill in the art and can also be adjusted by the individual physician in the event of any complication.

[0185] Administration of the doses recited above or as employed by a skilled clinician can be repeated for a limited and defined period of time. In some embodiments, the doses are given once a day, or multiple times a day, for example, but not limited to three times a day. Typically, the dosage regimen is informed by the half-life of the agent as well as the minimum therapeutic concentration of the agent in blood, serum or localized in a given biological tissue. In a preferred embodiment, the doses recited above are administered daily for several weeks or months. The duration of treatment depends upon the subject’s clinical progress and continued responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.

[0186] A therapeutically effective amount is an amount of an agent that is sufficient to produce a statistically significant, measurable change in cardiomyocyte proliferation or in a given symptom of cardiac injury). Such effective amounts can be gauged in clinical trials as well as animal studies for a given agent or treatment regimen.

[0187] In some embodiments of any of the aspects, the amount of a reagent administered is an amount sufficient to promote cardiomyocyte proliferation, e.g., to increase cardiomyocyte proliferation as compared to the rate of proliferation prior to administration.

[0188] Agents useful in the methods and compositions described herein can be administered by an appropriate route that delivers to cardiac tissue or to a site of cardiac injury. In various embodiments, administration can be local or systemic

[0189] Therapeutic compositions containing at least one therapeutic agent can be conventionally administered in a unit dose. The term "unit dose" when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of a therapeutic agent calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.

[0190] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system or tissue to utilize the active ingredient, and degree of therapeutic effect desired. An agent can be targeted by means of a targeting moiety, such as e.g., an antibody or targeted liposome technology.

[0191] Precise amounts of active ingredients required to be administered depend on the judgment of the practitioner and are particular to each individual. Suitable regimes for administration are also variable, but can involve an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion of agents, such as JAK inhibitors or PI3K inhibitors, sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

[0192] In some embodiments of any of the aspects, the a) at least one of: a JAK inhibitor and PI3K inhibitor is administered simultaneously with b) the genetic construct that directs the expression ofKDM4D. In some embodiments of any of the aspects, the a) at least one of: a JAK inhibitor and PI3K inhibitor is administered concurrently with b) the genetic construct that directs the expression of KDM4D. In some embodiments of any of the aspects, the a) at least one of: a JAK inhibitor and PI3K inhibitor is administered at least 1 hour after b) the genetic construct that directs the expression of KDM4D. In some embodiments of any of the aspects, the a) at least one of: a JAK inhibitor and PI3K inhibitor is administered at least 2 hours after b) the genetic construct that directs the expression of KDM4D. In some embodiments of any of the aspects, the a) at least one of: a JAK inhibitor and PI3K inhibitor is administered at least 6 hours after b) the genetic construct that directs the expression of KDM4D.

[0193] In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 1 nMto lOOpM, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 10 nM to lOpM, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 100 nM to lOOpM, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 100 nM to lOpM, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 100 nM to IpM, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 10 nM to IpM, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 10 pM.

[0194] In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 1 mg / kg - 1,000 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 10 mg / kg - 500 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 50 mg / kg -400 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 100 mg / kg - 300 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 100 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 100 mg / kg -250 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 150 mg / kg - 200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 160 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 170 mg / kg -190 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered at a dosage of 180 mg / kg daily.

[0195] In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 1 mg / kg - 1,000 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 10 mg / kg - 500 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 50 mg / kg -400 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 100 mg / kg -300 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 100 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 100 mg / kg -250 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 150 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 160 mg / kg - 200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 170 mg / kg -190 mg / kg daily, inclusive. In some embodiments of any of the aspects, the JAK inhibitor is administered orally at a dosage of 180 mg / kg daily.

[0196] In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 1 mg / kg - 1,000 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 10 mg / kg - 500 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 50 mg / kg -400 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 100 mg / kg -300 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 100 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 100 mg / kg -250 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 150 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 160 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 170 mg / kg -190 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 180 mg / kg daily.

[0197] In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 1 mg / kg - 1,000 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 10 mg / kg - 500 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 50 mg / kg - 400 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 100 mg / kg -300 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 100 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 100 mg / kg -250 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 150 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 160 mg / kg -200 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor isadministered orally at a dosage of 170 mg / kg -190 mg / kg daily, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered orally at a dosage of 180 mg / kg daily.

[0198] In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 1 nM to lOOpM, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 10 nM to lOpM, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 100 nM to lOOpM, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 100 nM to lOpM, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 100 nM to IpM, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 10 nM to IpM, inclusive. In some embodiments of any of the aspects, the PI3K inhibitor is administered at a dosage of 10 pM.Combination Therapies

[0199] Treatment of a subject with both a JAK inhibitor and a vector encoding KDM4D, a PI3K inhibitor and a vector encoding KDM4D, or a JAK Inhibitor, a PI3K inhibitor and a vector encoding KDM4D can be considered a considered a “combination therapy.” In other embodiments, the therapeutically effective agents (e.g., a JAK inhibitor or a PI3K inhibitor and a vector encoding KDM4D) described herein are administered to a subject concurrently with an additional combination therapy, such as an immunosuppressive agent. Exemplary immunosuppressive agents can include, but are not limited to, methylprednisolone, betamethasone, cortisone, dexamethasone, hydrocortisone, prednisolone, prednisone, or triamcinolone. An exemplary treatment regimen for cardiac graft rejection comprises 500mg methylprednisolone administered IV until separation from cardiopulmonary bypass, followed by 125 mg methylprednisolone administered IV every 8 hours for the first 24 hours and then the subject is switched to oral prednisone at a dose of 0. 1-10 mg / kg PO daily.

[0200] As used herein, the term “concurrently” is not limited to the administration of the two or more agents at exactly the same time, but rather, it is meant that they are administered to a subject in a sequence and within a time interval such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered alone). For example, the combination of therapeutics can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic effect, preferably in a synergistic fashion. The agents can be administered separately, in any appropriate form and by any suitable route. When each of the therapeutic agents in a combination are not administered in the same pharmaceutical composition, it is understood that they can be administered in any order to a subject in need thereof. For example, the first therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours,4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second therapeutic agent, to a subject in need thereof (or vice versa), as long as both agents are effective at the same time. In other embodiments, the delivery of either therapeutic agent ends before the delivery of the other agent / treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the therapeutic agents used in combination are more effective than would be seen with either agent alone. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with either therapeutic agent alone. The effect of such a combination can be partially additive, wholly additive, or greater than additive.

[0201] When administered in combination, one or more of the therapeutic agents can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of the given agent typically used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of a first therapeutic agent when administered in combination with a second therapeutic agent is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the first agent when used individually. In other embodiments, the amount or dosage of a first therapeutic agent, when administered in combination with a second therapeutic agent, that results in a desired effect (e.g., improved cognitive functioning) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of the first (or second) agent required to achieve the same therapeutic effect when administered alone.

[0202] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with a Janus kinase (JAK) inhibitor and a genetic construct that directs the expression of lysine-specific demethylase 4D (KDM4D).2. The method of paragraph 1, wherein the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the JAK inhibitor and the genetic construct to the subject.3. The method of paragraph 1 or paragraph 2, wherein the cardiomyocyte is human.4. The method of any one of paragraphs 1-3, wherein the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.5. The method of any one of paragraphs 1-4, wherein the JAK inhibitor is ruxolitinib.6. The method of any one of paragraphs 1-5, wherein the genetic construct that directs the expression of KDM4D comprises a viral vector.7. The method of paragraph 6, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.8. The method of paragraph 7, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.9. The method of any of paragraphs 6-8, wherein the AAV vector is AAV9.10. The method of any one of paragraphs 2-9, wherein the JAK inhibitor is administered simultaneously with the genetic construct that directs the expression of KDM4D.11. The method of any one of paragraphs 2-10, wherein the JAK inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of KDM4D.12. The method of any one of paragraphs 1-11, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.13. The method of paragraph 12, wherein the JAK inhibitor is administered at a dosage of 10 pM.14. The method of any one of paragraphs 1-13, wherein the adeno-associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.15. The method of any one of paragraphs 1-14, further comprising contacting the cardiomyocyte with a phosphatidylinositol-3-kinase (PI3K) inhibitor.16. The method of paragraph 15, wherein the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765.17. The method of paragraph 15 or 16, wherein the PI3K inhibitor is PHT-427.18. The method of any one of paragraphs 15-17, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.19. The method of any one of paragraphs 15-18, wherein the PI3K inhibitor is lOpM.20. The method of any one of paragraphs 15-19, wherein the PI3K inhibitor is administered simultaneously with the adenovirus expressing KDM4D.21. The method of any one of paragraphs 15-19, wherein the PI3K inhibitor is administered at least 1 hour after the administration of the adenovirus expressing KDM4D.22. A method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with a phosphatidylinositol-3-kinase (PI3K) inhibitor and a genetic construct that directs the expression of KDM4D.23. The method of paragraph 22, wherein the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the PI3K inhibitor and the genetic construct to the subject.24. The method of paragraph 22 or paragraph 23, wherein the cardiomyocyte is human.25. The method of any one of clams 22-24, wherein the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765.26. The method of any one of paragraphs 22-25, wherein the PI3K inhibitor is PHT-427.27. The method of any one of paragraphs 22-26, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.28. The method of any one of paragraphs 22-27, wherein the PI3K inhibitor is administered at a dosage of lOpM.29. The method of any one of paragraphs 22-28, wherein the genetic construct that directs the expression of KDM4D comprises a viral vector.30. The method of paragraph 29, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.31. The method of paragraph 30, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.32. The method of any one of paragraphs 29-31, wherein the AAV vector is AAV9.33. The method of any one of paragraphs 22-32, wherein the PI3K inhibitor is administered simultaneously with the genetic construct that directs the expression of KDM4D.34. The method of any one of paragraphs 22-32, wherein the JAK inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of KDM4D.35. The method of any one of paragraphs 22-34, wherein the adeno-associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.36. The method of any one of paragraphs 1-35, wherein the subject has cardiac damage or a cardiac disease or disorder.37. The method of paragraph 36, wherein the subject has suffered a myocardial infarction.38. The method of paragraph 36, wherein the cardiac disease or disorder is selected from atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.39. A kit for promoting cardiomyocyte proliferation, the kit comprising a viral vectorexpressing KDM4D, a JAK inhibitor, and instructions for use.40. The kit of paragraph 39, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.41. The kit of paragraph 39, further comprising a PI3K inhibitor.42. The kit of any one of paragraphs 39-41, for use in treating a cardiac disease or disorder.43. The kit of any one of paragraphs 39-41, for use in treating a cardiovascular disease or disorder.44. The kit of any one of paragraphs 39-43, wherein the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.45. The kit of any one of paragraphs 39-44, wherein the JAK inhibitor is ruxolitinib.46. The kit of any one of paragraphs 39-45, wherein the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765.47. The kit of any one of paragraphs 39-46, wherein the PI3K inhibitor is PHT-427.48. The kit of any one of paragraphs 39-47, wherein the adeno-associated virus expressing KDM4D is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11 and AAV 12.49. The kit of any one of paragraphs 39-49, wherein the adeno-associated virus expressing KDM4D is AAV9.50. A method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof an amount of a JAK inhibitor and an amount of a genetic construct that directs the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.51. The method of paragraph 50, wherein the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.52. The method of paragraph 50 or 51, wherein the JAK inhibitor is ruxolitinib.53. The method of any one of paragraphs 50-52, wherein the subject in need thereof is human.54. The method of any one of paragraphs 50-53wherein the genetic construct that directs expression of KDM4D comprises a viral vector.55. The method of paragraph 55, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.56. The method of paragraphs 54 or 55, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.57. The method of any one of paragraphs 54-56, wherein the AAV vector is AAV9.58. The method of any one of paragraphs 50-57, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.59. The method of any one of paragraphs 50-58, wherein the JAK inhibitor is lOpM.60. The method of any one of paragraphs 53-59, wherein the adeno-associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.61. The method of any one of paragraphs 50-60, further comprising administering a PI3K inhibitor to the subject.62. The method of paragraph 61, wherein the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765.63. The method of paragraph 61 or 62, wherein the PI3K inhibitor is PHT-427.64. A method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof an amount of a PI3K inhibitor and an amount of a genetic construct that directs the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.65. The method of paragraph 64, wherein the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765.66. The method of paragraph 64 or 65, wherein the PI3K inhibitor is PHT-427.67. The method of any one of paragraphs 64-66, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.68. The method of any one of paragraphs 64-67, wherein the PI3K inhibitor is administered at a dosage of lOpM.69. The method of any one of paragraphs 64-68, wherein the genetic construct that directs expression of KDM4D comprises a viral vector.70. The method of paragraph 69, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.71. The method of paragraphs 69 or 70, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.72. The method of any one of paragraphs 69-71, wherein the AAV vector is AAV9.73. The method of any one of paragraphs 69-72, wherein the adeno-associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.74. The method of any one of paragraphs 64-73, wherein the subject in need thereof is human.75. The method of any one of paragraphs 64-74, further comprising administering a JAK inhibitor to the subject.76. The method of paragraph 75, wherein the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.77. The method of paragraph 75 or 76, wherein the JAK inhibitor is ruxolitinib.78. The method of any one of paragraphs 75-77, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.79. The method of any one of paragraphs 75-78, wherein the JAK inhibitor is administered at a dosage of 10 pM.80. A method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject an amount of a JAK inhibitor and an amount of a genetic construct directing the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.81. The method of paragraph 80, wherein the subject has suffered ischemic injury to cardiac tissue.82. The method of paragraph 80 or 81, wherein the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.83. The method of any one of paragraphs 80-82, wherein the JAK inhibitor is ruxolitinib.84. The method of any one of paragraphs 80-83, wherein the subject in need thereof is human.85. The method of any one of paragraphs 80-84, wherein the genetic construct that directs the expression of KDM4D comprises a viral vector.86. The method of paragraph 85, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.87. The method of paragraphs 85 or 86, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.88. The method of any one of paragraphs 85-87, wherein the AAV vector is AAV9.89. The method of any one of paragraphs 80-88, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.90. The method of any one of paragraphs 80-89, wherein the JAK inhibitor is administered at a dosage of lOpM.91. The method of any one of paragraphs 85-90, wherein the adeno-associated virus expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.92. The method of any one of paragraphs 80-91, further comprising administering a PI3K inhibitor to the subject.93. The method of paragraph 92, wherein the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765.94. The method of paragraph 92 or 93, wherein the PI3K inhibitor is PHT-427.95. The method of any one of paragraphs 92-94, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.96. The method of any one of paragraphs 92-95, wherein the PI3K inhibitor is administered at a dosage of lOpM.97. A method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject an amount of a PI3K inhibitor and an amount of a genetic construct directing the expression of KDM4D sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.98. The method of paragraph 97, wherein the subject has suffered ischemic injury to cardiac tissue.99. The method of paragraph 97 or 98, wherein the PI3K inhibitor is selected from the group consisting of cx-4945, PHT-427, A-769662, and XL765.100. The method of any one of paragraphs 97-99, wherein the PI3K inhibitor is PHT-427.101. The method of any one of paragraphs 97-100, wherein the subject in need thereof is human.102. The method of any one of paragraphs 97-101, wherein the genetic construct directing the expression of KDM4D comprises a viral vector.103. The method of paragraph 102, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.104. The method of paragraph 103, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.105. The method of paragraphs 103 and 104, wherein the AAV is AAV9.106. The method of any one of paragraphs 97-105, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.107. The method of any one of paragraphs 97-106, wherein the PI3K inhibitor is administered at a dosage of lOpM.108. The method of any one of paragraphs 97-107, wherein the AAV expressing KDM4D is administered at a total dose within the range of lxl09vg to about lxl016vg.109. The method of any one of paragraphs 97-108, further comprising administering a JAK inhibitor.110. The method of paragraph 109, wherein the JAK inhibitor is selected from the group consisting of tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.111. The method of paragraph 109 or 110, wherein the JAK inhibitor is ruxolitinib.112. The method of any one of paragraphs 109-111, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.113. The method of any one of paragraphs 109-112, wherein the JAK inhibitor is administered at a dosage of lOpM.

[0203] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with: a Janus kinase (JAK) inhibitor; and a genetic construct that directs the expression of a lysine-specific demethylase 4D (KDM4D) polypeptide.2. The method of paragraph 1, wherein the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the JAK inhibitor and the genetic construct to the subject.3. The method of paragraph 1 or paragraph 2, wherein the cardiomyocyte is human.4. The method of any one of paragraphs 1-3, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.5. The method of any one of paragraphs 1-4, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.6. The method of any one of paragraphs 1-5, wherein the JAK inhibitor is ruxolitinib.7. The method of any one of paragraphs 1-6, wherein the genetic construct that directs the expression of KDM4D comprises a viral vector.8. The method of paragraph 7, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.9. The method of paragraph 8, wherein the AAV vector is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 andAAV12.10. The method of any of paragraphs 7-9, wherein the AAV vector is AAV9.11. The method of any one of paragraphs 2-10, wherein the JAK inhibitor is administered simultaneously with the genetic construct that directs the expression of a KDM4D polypeptide.12. The method of any one of paragraphs 2-10, wherein the JAK inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of a KDM4D polypeptide.13. The method of any one of paragraphs 1-12, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.14. The method of paragraph 13, wherein the JAK inhibitor is administered at a dosage of 10 pM.15. The method of any one of paragraphs 1-14, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.16. The method of any one of paragraphs 1-15, further comprising contacting the cardiomyocyte with a phosphatidylinositol-3-kinase (PI3K) inhibitor.17. The method of paragraph 16, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.18. The method of paragraph 16, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.19. The method of paragraph 16, wherein the PI3K inhibitor is PHT-427.20. The method of any one of paragraphs 16-19, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.21. The method of any one of paragraphs 16-20, wherein the PI3K inhibitor is administered at a dosage of lOpM.22. The method of any one of paragraphs 16-21, wherein the PI3K inhibitor is administered simultaneously with the genetic construct expressing a KDM4D polypeptide.23. The method of any one of paragraphs 16-22, wherein the PI3K inhibitor is administered at least 1 hour after the administration of the genetic construct expressing a KDM4D polypeptide.24. A method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with: a phosphatidylinositol-3 -kinase (PI3K) inhibitor; and a genetic construct that directs the expression of a KDM4D polypeptide.25. The method of paragraph 24, wherein the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the PI3K inhibitor and the genetic construct to the subject.26. The method of paragraph 24 or paragraph 25, wherein the cardiomyocyte is human.27. The method of any one of clams 24-26, wherein the PI3K inhibitor is selected from the group consisting of:idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.28. The method of any one of clams 24-26, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.29. The method of any one of paragraphs 24-26, wherein the PI3K inhibitor is PHT-427.30. The method of any one of paragraphs 24-29, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.31. The method of any one of paragraphs 24-30, wherein the PI3K inhibitor is administered at a dosage of lOpM.32. The method of any one of paragraphs 24-31, wherein the genetic construct that directs the expression of a KDM4D polypeptide comprises a viral vector.33. The method of paragraph 32, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.34. The method of paragraph 33, wherein the AAV vector is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.35. The method of any one of paragraphs 33-34, wherein the AAV vector is AAV9.36. The method of any one of paragraphs 24-35, wherein the PI3K inhibitor is administered simultaneously with the genetic construct that directs the expression of a KDM4D polypeptide.37. The method of any one of paragraphs 24-35, wherein the PI3K inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of a KDM4D polypeptide.38. The method of any one of paragraphs 24-37, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.39. The method of any one of paragraphs 1-38, wherein the subject has cardiac damage or a cardiac disease or disorder.40. The method of paragraph 39, wherein the subject has suffered a myocardial infarction.41. The method of paragraph 39, wherein the cardiac disease or disorder is selected from atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.42. A kit for promoting cardiomyocyte proliferation, the kit comprising:a viral vector expressing a KDM4D polypeptide; and a JAK inhibitor.43. The kit of paragraph 42, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.44. The kit of paragraph 42, further comprising a PI3K inhibitor.45. A kit for promoting cardiomyocyte proliferation, the kit comprising: a viral vector expressing a KDM4D polypeptide; and a PI3K inhibitor.46. The kit of paragraph 45, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.47. The kit of paragraph 45, further comprising a JAK inhibitor.48. The kit of any one of paragraphs 42-47, further comprising instructions for use.49. The kit of any one of paragraphs 42-48, for use in treating a cardiac disease or disorder.50. The kit of any one of paragraphs 42-49, for use in treating a cardiovascular disease or disorder.51. The kit of any one of paragraphs 42-50, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.52. The kit of any one of paragraphs 42-50, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.53. The kit of any one of paragraphs 42-50, wherein the JAK inhibitor is ruxolitinib.54. The kit of any one of paragraphs 42-53, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.55. The kit of any one of paragraphs 42-53, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.56. The kit of any one of paragraphs 42-53, wherein the PI3K inhibitor is PHT-427.57. The kit of any one of paragraphs 42-56, wherein the adeno-associated virus expressing a KDM4D polypeptide is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.58. The kit of any one of paragraphs 42-57, wherein the adeno-associated virus expressing a KDM4D polypeptide is AAV9.59. A method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof: an amount of a JAK inhibitor; and an amount of a genetic construct that directs the expression of a KDM4D polypeptide; sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.60. The method of paragraph 59, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.61. The method of paragraph 59, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.62. The method of paragraph 59, wherein the JAK inhibitor is ruxolitinib.63. The method of any one of paragraphs 59-62, wherein the subject in need thereof is human.64. The method of any one of paragraphs 59-63, wherein the genetic construct that directs expression of a KDM4D polypeptide comprises a viral vector.65. The method of paragraph 64, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.66. The method of paragraphs 64 or 65, wherein the AAV is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.67. The method of any one of paragraphs 59-66, wherein the AAV vector is AAV9.68. The method of any one of paragraphs 59-67, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.69. The method of any one of paragraphs 59-68, wherein the JAK inhibitor is administered at a dosage lOpM.70. The method of any one of paragraphs 59-69, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.71. The method of any one of paragraphs 59-70, further comprising administering a PI3K inhibitor to the subject.72. The method of paragraph 61, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.73. The method of paragraph 61, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.74. The method of paragraph 61, wherein the PI3K inhibitor is PHT-427.75. A method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof: an amount of a PI3K inhibitor; and an amount of a genetic construct that directs the expression of a KDM4D polypeptide; sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.76. The method of paragraph 75, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.77. The method of paragraph 75, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.78. The method of paragraph 75, wherein the PI3K inhibitor is PHT-427.79. The method of any one of paragraphs 75-78, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.80. The method of any one of paragraphs 75-79, wherein the PI3K inhibitor is administered at a dosage of lOpM.81. The method of any one of paragraphs 75-80, wherein the genetic construct that directs expression of a KDM4D polypeptide comprises a viral vector.82. The method of paragraph 81, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.83. The method of paragraph 82, wherein the AAV is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 andAAV12.84. The method of any one of paragraphs 82-83, wherein the AAV vector is AAV9.85. The method of any one of paragraphs 82-84, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.86. The method of any one of paragraphs 75-85, wherein the subject in need thereof is human.87. The method of any one of paragraphs 75-86, further comprising administering a JAK inhibitor to the subject.88. The method of paragraph 87, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.89. The method of paragraph 87, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.90. The method of paragraph 87, wherein the JAK inhibitor is ruxolitinib.91. The method of any one of paragraphs 87-90, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.92. The method of any one of paragraphs 87-90, wherein the JAK inhibitor is administered at a dosage of 10 pM.93. A method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject: an amount of a JAK inhibitor; and an amount of a genetic construct directing the expression of a KDM4D polypeptide; sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.94. The method of paragraph 93, wherein the subject has suffered ischemic injury to cardiac tissue.95. The method of paragraph 93 or 94, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.96. The method of paragraph 93 or 94, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.97. The method of any one of paragraphs 93 or 94, wherein the JAK inhibitor is ruxolitinib.98. The method of any one of paragraphs 93-97, wherein the subject in need thereof is human.99. The method of any one of paragraphs 93-98, wherein the genetic construct that directs the expression of a KDM4D polypeptide comprises a viral vector.100. The method of paragraph 99, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.101. The method of paragraph 100, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.102. The method of any one of paragraphs 100-101, wherein the AAV vector is AAV9.103. The method of any one of paragraphs 93-102, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.104. The method of any one of paragraphs 93-103, wherein the JAK inhibitor is administered at a dosage of lOpM.105. The method of any one of paragraphs 100-104, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.106. The method of any one of paragraphs 93-105, further comprising administering a PI3K inhibitor to the subject.107. The method of paragraph 106, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.108. The method of paragraph 106, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.109. The method of paragraph 106, wherein the PI3K inhibitor is PHT-427.110. The method of any one of paragraphs 106-109, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.111. The method of any one of paragraphs 106-110, wherein the PI3K inhibitor is administered at a dosage of lOpM.112. A method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject an amount of a PI3K inhibitor and an amount of a genetic construct directing the expression of a KDM4D polypeptide sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.113. The method of paragraph 112, wherein the subject has suffered ischemic injury to cardiac tissue.114. The method of paragraph 112 or 113, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.115. The method of paragraph 112 or 113, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.116. The method of paragraph 112 or 113, wherein the PI3K inhibitor is PHT-427.117. The method of any one of paragraphs 112-116, wherein the subject in need thereof is human.118. The method of any one of paragraphs 112-117, wherein the genetic construct directing the expression of a KDM4D polypeptide comprises a viral vector.119. The method of paragraph 118, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.120. The method of paragraph 119, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.121. The method of paragraph 119, wherein the AAV is AAV9.122. The method of any one of paragraphs 112-121, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.123. The method of any one of paragraphs 112-122, wherein the PI3K inhibitor is administered at a dosage of lOpM.124. The method of any one of paragraphs 119-123, wherein the AAV expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.125. The method of any one of paragraphs 112-124, further comprising administering a JAK inhibitor.126. The method of paragraph 125, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.127. The method of paragraph 125, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.128. The method of paragraph 125, wherein the JAK inhibitor is ruxolitinib.129. The method of any one of paragraphs 125-128, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.130. The method of any one of paragraphs 125-129, wherein the JAK inhibitor is administered at a dosage of lOpM.131. A combination of: a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of a KDM4D polypeptide for use in a method of reducing cardiac damage or injury in a subject in need thereof.132. The combination of paragraph 131, comprising a JAK inhibitor.133. The combination of paragraph 131, comprising a PI3K inhibitor.134. The combination of any one of paragraphs 131-133, comprising a JAK inhibitor and a PI3K inhibitor.135. The combination of paragraphs 131, 132, or 134, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.136. The combination of paragraphs 131, 132, or 134, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.137. The combination of paragraphs 131, 132, or 134, wherein the JAK inhibitor is ruxolitinib.138. The combination of any one of paragraphs 131, 132, or 134-137, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.139. The combination of any one of paragraphs 131, 132, or 134-138, wherein the JAK inhibitor is administered at a dosage lOpM.140. The combination of any one of paragraphs 131, or 133-139, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.141. The combination of any one of paragraphs 131, or 133-139, wherein the PI3K inhibitor is selected from the group consisting of:cx-4945, PHT-427, A-769662, and XL765.142. The combination of any one of paragraphs 131, or 133-139, wherein the PI3K inhibitor is PHT- 427.143. The combination of any one of paragraphs 131 or 133-142, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.144. The combination of any one of paragraphs 131 or 133-143, wherein the PI3K inhibitor is administered at a dosage lOpM.145. The combination of any one of paragraphs 131-144, wherein the genetic construct that directs expression of a KDM4D polypeptide comprises a viral vector.146. The combination of paragraph 145, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.147. The combination of paragraph 146, wherein the AAV is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 andAAV12.148. The combination of any one of paragraphs 146-147, wherein the AAV vector is AAV9.149. The combination of any one of paragraphs 146-148, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.150. The combination of any one of paragraphs 131-149, wherein the subject in need thereof is human.151. The combination of any one of paragraphs 131-150, wherein the subject has suffered ischemic injury to cardiac tissue.152. The combination of any one of paragraphs 131-151, the subject has cardiac damage or a cardiac disease or disorder.153. The combination of paragraph 152, the cardiac disease or disorder is selected from atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.154. The combination of any one of paragraphs 131-152, the subject has suffered a myocardial infarction.

[0204] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESEXAMPLE 1:

[0205] Introduction

[0206] The limited regenerative capacity of adult mammalian hearts following an injury is a major contributor to the development of heart failure. Although a very limited number of new cardiac myocytes (CM) can be generated in adult hearts, it is insufficient to restore contractile dysfunction after myocardial infarction (MI). Increasing evidence suggests that these new CMs are derived from the proliferation of pre-existing CMs. This regenerative capacity seen in newborn mice is lost by postnatal day 7 (P7). Ischemic injury in P7 mice leads to fibrotic scarring similar to that seen after injury in adult hearts. Reduced regenerative capacity in P7 and adult CMs (ACMs) correlates with onset of terminal differentiation in most CMs, which is characterized by permanent cell cycle arrest. Consequently, therapeutic interventions in response to adult hearts injury have focused on strategies that either salvage myocardium or restore the proliferative capacity of ACMs to promote adult heart regeneration.

[0207] The molecular basis of the limited proliferative capacity of ACMs is poorly understood. However, when subjected to a growth stimulus, CMs preferentially experience an increase in cell size (hypertrophy) rather than dividing. Interestingly, despite the lack of division, stimulating mitogenic pathways in ACMs can lead to the induction of Gl / S phase genes, DNA synthesis, and increases in DNA content. However, the G2 / M and cytokinesis genes remain silent. This inability to upregulate late cell cycle genes is consistent with the inability of ACMs to complete the later phases of the cell cycle. It is believed that stable silencing of gene expression is often controlled by epigenetics, which regulates gene activity through remodeling of chromatin structure. One methylation commonly associated with transcriptional silencing is the trimethylation of lysine 9 on histone H3 (H3K9me3). To explore the role of H3K9me3 in ACMs, a transgenic mouse model was developed with specific depletion of H3K9me3 in CMs using lysine demethylase 4D (KDM4D), a member of the histone demethylase family of enzymes. Constitutive, CM-specific expression delayed CM cell cycle exit and increased ACM expression of G2 / M and cytokinesis genes in vivo.

[0208] Recent studies indicate that small molecules can promote CM proliferation and protect heart from injury. Of the 18600 chemicals screened, TT-10 was identified as a YAP-TEADs activator capable of inducing quiescent cultured CMs to re-enter the cell cycle(15). Additionally, Cardiomogen, a Wnt inhibitor targeting -catenin and reducing Tcf / Lef-mediated transcription in cultured cells, was found to increase CM proliferation and expedites wound healing, thereby accelerates cardiac muscle regeneration in Zebrafish(16). Slow-release CHIR99021 and FGF1 were also identified to enhance CM cell cycling and enable cardioprotection by reducing infarct size by 20%-30% in mouse or pig models (17). Although highly upregulated compared to that in controls, the cell cycle activity in treated ACMs was still much less than in wildtype fetal CMs (14).

[0209] In this current study, the therapeutic potential of KDM4D was investigated for the treatment of MI. It was found that induction of KDM4D specifically in ACM improves cardiac function and reduces infarct scar size post MI. This is due to the KDM4D’s anti-apoptotic effect, since the less CMproliferative response of KDM4D in adult myocardium. Additionally, the effects of small molecules and epigenetic manipulations through KDM4D were evaluated, either alone or in combination, on CM proliferation and regeneration in both in vitro and in vivo models. The small molecule screening identified Ruxolitinib, a selected JAK signaling pathway inhibitor, as a potent promoter of CM proliferation when used in combination with KDM4D in vitro. Moreover, the oral administrated with Ruxolitinib in the iKDM4D mice subjected to myocardial infarction (MI) promoted CM proliferation and protection, leading to an improvement in cardiac dysfunction. The findings provide a treatment strategy that complements current therapies for patients with MI. Notably, the in vivo study is the first to propose that KDM4D regulates not only cell proliferation but also plays a role in controlling ACM’s responses to injury (18-20).

[0210] Materials and methods

[0211] Animals

[0212] All animal experiments were conducted in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocol #4290-01, the institutional guidelines at the University of Washington, and National Institute of Health Guide for the Care and Use of Uaboratory Animals. The inducible KDM4D mouse model used in this study was previously developed in the lab(14, 21). Tet- responsive-KDM4D mice were bred to the repressible aMHC-rtTA (iKDM4D; tet-on) mice(22, 23). This iKDM4D model enables the control of KDM4D expression by administrating doxycycline- containing chow (Harlan TD.00502) at the indicated times.

[0213] To create the neonatal cardiac injury model, MI was induced by occluding the left anterior descending coronary artery (UAD) in 1-day old (Pl) or P7 mice(24). Neonates were anesthetized by cooling on an ice bed and underwent lateral thoracotomy at the fourth intercostal space after skin incision. MI was induced by tying a tapered needle (BV-1) attached to a 7-0 prolene suture (Ethicon; Johnson & Johnson) through the mid-ventricle below the origin of the UAD. Following UAD ligation, thoracic wall incisions were sutured with a 7-0 nonabsorbable proline suture for Pl or 6-0 for P7, and the skin wound was closed using skin adhesive. Sham -operated mice underwent the same procedure without UAD ligation. After surgery, neonates were warmed for several minutes under a heating lamp until recovery. The entire procedure lasted approximately 10 minutes. For the adult cardiac injury model, MI was induced via UAD occlusion in adult mice (10-16 weeks), as previously described (25). Sham-operated mice underwent the same procedure but without UAD ligation. Eittermate controls were used for all experiments involving transgenic mice. DNA synthesis of CMs was quantified by 5- Bromo-2 -Deoxyuridine (BrdU) incorporation after a pulse at postoperative day 0, 7, and 14 with a 0.035 ml of a 20-mg / ml solution of BrdU (Sigma- Aldrich, MO) dissolved in sterile 0.9% saline solution (Hospira, Lake Forest, IL).

[0214] To investigate the additive effect of KDM4D and small molecules on cardioprotection after ischemic injury in vivo, myocardial ischemia-reperfusion (ER) was performed as previously described(26). Adult mice (10-16weeks) were randomly assigned in a 1: 1 ratio to receive vehicle or compounds after the I / R procedure. Briefly, myocardial ischemia was induced by temporarily exteriorizing the heart via a left thoracic incision while placing a silk suture slipknot approximately 1.5 mm below the tip of the LAD. After 45 min MI, the slipknot was released to allow for reperfusion. The occlusion was confirmed by a change in color (paler appearance) of the anterior wall of the left ventricle. Twelve to sixteen hours post I / R procedure, the mice were administrated an oral solution (0.5%- 1 % CMC-Na) with or without small molecules. DNA synthesis of CMs was quantified by a 5-Ethynyl-2'- deoxyuridine (EdU) incorporation with a 50 mg / mL dissolved in oral solution. The hearts were harvested 48 hours, 2 weeks, or 8 weeks after surgery.

[0215] For neonatal rat ventricle cardiomyocyte (NRVM) cultures, time-pregnant Sprague Dawley outbred rats (TP 19) were purchased from Harlan / ENVIGO, and CMs were isolated as previously described (27). Neonatal rats were euthanized through decapitation, while adult mice were sacrificed by isoflurane overdose.

[0216] Small molecular screening assay

[0217] NRVMs were seeded at a density of 4000 cells per well in the 384-well plates coated with fibronectin. The cells were cultured in 20 pL of M199 culture medium (Medium 199 500ml, HEPEs lOmM, MEM Non-Essential Amino Acids l x, glucose 1.75g, L-glutamine 2mM, Vitamin B12 2mg, and penicllin 50,000 units) supplemented with 10% FBS for the first 24 hours and then replaced with serum-free M199 medium. The cells were then infected with either Ad-CMV-GFP-h-KDM4D (Ad- KDM4D) or control Ad-CMV-GFP-h-control (Ad-GFP) at MOI 100. Both vectors were purchased from Vector Biolabs (Malvern, PA). Forty -eight hours after virus infection, the NRVMs were treated with a protein kinase inhibitor library (295 compounds from Selleckchem.com) using three replicates of each compound at a concentration of I OpM. with DMSO as an internal control. After another 48 hours, the cells were treated with lOnM EdU in serum -free medium. After 24 hours, the cells were fixed and assayed for EdU incorporation using the Click-iT EdU Alexa Fluor 647 HCS Assay (Thermo Scientific: C10356). Image acquisition was performed using an automated, high-content screening fluorescent microscope (InCell Analyzer 1000, GE Healthcare) at 4x magnification. One image was acquired per well and per replicates. Image analysis was performed using Image J.

[0218] The top-rank candidates were chosen for verification in a 24-well plates format with NRVMs plated at a density of 1 x 10A5 cells per well on the fibronectin-coated plates. The NRVMs were cultured and processed according to the same protocol as the primary screening. Final compound concentrations ranged from 1 nM to 100 pM. Fluorescent immunochemistry, image acquisition, and analysis were conducted in the same manner as the primary screen, using three replicates for each compound concentration.

[0219] Histology and Immunostaining

[0220] NRVMs or heart sections were fixed with 4% PFA in PBS for 10 mins, followed bypermeabilization with 0.2% Triton X-100 in PBS for 20 mins and blocking with PBS containing 5% normal donkey serum (NDS) for 1 hour at room temperature. For immunostaining, the cells were incubated overnight at 4°C with the following antibodies diluted in the blocking buffer: anti-a-actinin (Thermo Scientific: 701914) was used to identify CMs, anti-BrdU (rat monoclonal; Abeam) or Click- iT EdU Alexa Fluor 647 HCS Assay (Thermo Scientific: C10356) to identify cells in the S phase of cell cycle, anti-phospho-H3 (Thermo Scientific: PA5-17869) to identify cells in the M phase of cell cycle, anti-Ki67 antibody (BioLegend: 151206) to identify cells with active cell cycle. Subsequently, the cells were then washed three times with PBS containing 5% BSA and stained with the appropriate secondary antibodies for 45 mins at room temperature. Finally, the cells were treated with 4', 6- diamidino-2-phenylindole dihydrochloride (DAPI) and wheat germ agglutinin (WGA) for 30 minutes and observed under a high-resolution Nikon fluorescence microscope.

[0221] Quantitative real-time PCR

[0222] Total RNA was isolated using TRIzol™ Reagent (Invitrogen), followed by purification 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 carried out using the Sybrgreen PCR master mix (Applied Biosystems) according to the manufacturer’s instructions. The qPCR cycling was conducted on the AB7900H. Primer sequences for qPCR are listed in the supplemental material.

[0223] In vivo compounds administration

[0224] To investigate the combined effects of KDM4D and small molecules on CM proliferation and regeneration in vivo, adult iKDM4D mice (aged 10-16 weeks) were administrated with oral solution (0.5%-l% CMC-Na (Sodium carboxymethyl cellulose) and EdU (50mg / kg / day)) along with small molecules from Adooq BioScience by oral gavage using a 24-gauge reusable feeding needle (Fine Science Tools) for the indicated period. The vehicle mice were given the oral solution without compounds. The iKDM4D mice were also fed Doxycycline-containing chow to induce KDM4D expression in ACMs. At the endpoint, the mice were sacrificed, and their hearts were harvested for the experiments.

[0225] Assessment of ML

[0226] To evaluate the infarct size, TTC staining was performed 2 days after LAD or I / R injury. To distinguish infarct from viable myocardium, the 2mm heart slices were incubated in a 1% TTC in phosphate buffer (pH 7.4) for 10 minutes at 37 °C. The hearts were then fixed in 10% neutral buffered formaldehyde for 20 minutes at room temperature and photographed. The percentage of viable (red) and dead (white) myocardium was quantified using Image J software.

[0227] Fibrotic areas were evaluated 21 days after LAD or 14 days after I / R by analyzing paraffin serial sections cut at 50 pm intervals from the mid-papillary muscle plane of the hearts. Trichrome staining was performed, and the myocardial area and LV chamber area were manually traced inImage J and area was calculated using the “measure” tool. The scar area and the total area of myocardium were also traced manually in Image J and measured automatically by the computer. Scar size was expressed as a percentage and was calculated by dividing the sum of scar areas from all sections by the sum of entire myocardial area from all sections (including those without infarct scar) and multiplying by 100.

[0228] Apoptosis was visualized in paraffin sections by using a TUNEL staining kit (Life Technologies Cl 0618) according to the manufacturer’s guidelines. After TUNEL labeling, the tissue sections were stained for WGA, Hoechst, and phalloidin, and imaged as described in the procedures for Immunostaining.

[0229] 2-1) Echocardiography

[0230] Under 0.5% isoflurane, adult mice EKG and heart function were assessed using Visual Sonics Vevo 2100. Parasternal short axis images at the plane of the papillary muscle were collected in B- and M-Modes. Images were collected and analyzed by a single operator who was blinded to the genotypes. Quantification of images was performed using Vevo Labs 1.7.0, according to the manufacturer’s guidelines.

[0231] Isolation of adult mouse ventricular myocytes

[0232] Cardiomyocytes were isolated using Langendorff perfusion digestion as previously described (28). The mice were injected with 200 pL of heprin (100 lU / mouse) before being anesthetized with Isoflurane. The hearts were then harvested and perfused with a Ca2+- free Tyrodes buffer (126 mM NaCl, 5.4 mM KC1, 0.33 mM NaH2PC>4, 1 mM MgCE, 10 mM HEPES, 10 mM Glucose, 20 mM Taurine, pH=7.4) supplemented with 25 pM blebbistatin (Toronto Research Chemicals Inc. and BOC Sciences) for 2 minutes at 37 °C. Next, the hearts were enzymatically digested by perfusing with 0.35 U / ml Liberase TH (Roche) prepared with blebbistatin-supplemented Ca2+- free Tyrode’s buffer for 8-12 minutes at 37 °C. The resulting tissue was mechanically dissociated in ice-cold KB solution (20 mM KC1, 10 mM KH2PO4, 70 mM Potassium Glutamate, 1 mM MgCU, 25 mM Glucose, 20 mM Taurine, 0.5 mM EGTA, 10 mM HEPES, 0. 1% Albumin, pH=7.4). The cell suspensions were then passed through a 100 pm cell strainer to remove tissue debris and purified by low-speed centrifugation (50xg for 1 minute) three times, resulting in approximately 90% pure ACMs.

[0233] CM measurement method.

[0234] ACM measurements method was described before(14). Isolated ACMs were fixed in 4% PFA for 15 minutes and stained with a-actinin to identify CMs and DAPI to visualize nuclei. Z-stack images were acquired using Nikon AIR confocal microscope. Over 150 ACMs per animal were analyzed by selecting several random regions. Imaris Cell Imaging Software (Imaris 9.2.1, Bitplane) was used to quantify the ACM volume and ploidy. The ACM number was estimated using the following formula: [mean heart volume (Heart mass / 1.06, the density of muscle tissue (29)) / meanACM volume x volume fraction of ACMs (the proportion of adult heart volume occupied by CMs)](14).

[0235] Cell Signaling Phospho-Array

[0236] Human embryo stem cell (RUES2) derived CMs (hESC-CMs) were cultured in 6-well plates and divided into three groups treating with PHT-427 for 24 hours, Ruxolitinib for 24 hours, or Ad- KDM4D for 72 hours separately. After treatment, the cells were lysed using ice-cold RIPA buffer and the cell lysates were applied to a Cell Signaling Phospho-Antibody Array (PCS300, Full Moon Biosystems, USA). The Cell Signaling Phospho-Antibody Array is a high-throughput ELISA based antibody array for qualitative profiling and screening of 304 candidate biomarkers from 16 cell signaling pathways. The array was performed as per the manufacturer's protocol with image quantification and analysis performed by Full Moon Biosystems data service.

[0237] Mass spectrometry

[0238] The cytoplasmic protein and nuclei protein from the isolated ACMs were extracted separately and then subjected to Co-IP using a KDM4D antibody (Creative Biolabs: CBMAB-0355- WJ) and Pierce™ Protein A / G Magnetic Beads (Invitrogen: 88802). Four Co-IP samples were generated, including iKDM4D-Cyto, iKDM4D-nuclei, Vehicle-Cyto, and Vehicle -nuclei. These CoIP samples were run on SDS-PAGE gel and the protein samples were then digested by trypsin. The resulting peptides were identified using a nano LC-MS / MS platform which was provided by commercial service (Creative Proteomics). The raw MS files were then analyzed and searched against a mouse protein database using Maxquant (1.6.2.6), which is a software platform commonly used for proteomic data analysis. This allowed for the identification of proteins that interacted with KDM4D in both the cytoplasmic and nuclear fractions of both the iKDM4D and Vehicle groups.

[0239] Statistical analysis

[0240] All the experiments were carried out with sufficient biological replicates to ensure the validity and reproducibility of the results. Animal or cells per group are identified in the respective figure legends. In experiments with multiple groups, analysis of variance (ANOVA) followed by Tukey’s test was used to compare group means by GraphPad Prism 9.4. Two-tailed T-test were used to analyze studies comparing two groups by GraphPad Prism. P value<0.05 was considered to represent a statistically significant difference. In all panels, numerical data are presented as mean ± SEM.

[0241] Results

[0242] KDM4D extends the postnatal regenerative window

[0243] To determine whether KDM4D expression could delay CM cell cycle exit and extend the postnatal regenerative window, MI was performed at Pl and P7 in control and CM-specific KDM4D expressing mice (KDM4DTg / +)(14). The regenerative response at Pl was associated with robust induction of CM proliferation in wildtype mice resulting in minimal scarring. In contrast, theproliferative response was lost by P7 in wildtype mice leading to substantial replacement fibrosis and scarring (Figure 7A-7E). To evaluate if the lack of proliferation post-MI at P7 is secondary to an inability to induce the necessary cell cycle factors as is seen in adult hearts, cell cycle gene expression were examined in CMs seven days after MI performed at Pl or P7 (Figure 7F). All cell cycle genes showed reduced expression in the groups receiving surgeries at P7 versus Pl. In Sham mice, the expression levels of Ccnbl, Plkl, and Aurkb were 2.4 to 3.3 -fold higher in the Pl -sham group compared to the P7-sham group ( <0.05). In Pl animals receiving either sham or MI, cell cycle genes remained elevated beyond the P7 time point in CM, with a further increase in expression of Plkl in Mi-operated mice. There was no significant induction of cell cycle genes by MI at P7.

[0244] To determine if KDM4D could restore cell cycle gene expression and neonatal CM cycling after MI at P7, the KDM4DTg / +mice were utilized. At 21 days post-MI, control hearts displayed fibrotic scar (% average scar / LV area was 12.8+3.4%, and the maximum % scar / LV area was 18.8+3.6% (Figure 1A-1C). Ventricular dilation were also observed in controls, consistent with no regeneration (Figure 1C). In contrast, KDM4DTg / +hearts showed minimal scarring with a predominance of viable myocardium (% average scar / LV area was 4.2+ 1.5%, and the maximum % scar / LV area was 8.3+2.1%, <0.05 vs. control hearts; Figure 1A-1C).

[0245] To verify that the regeneration seen after P7 MI in KDM4D hearts was related to enhanced CM proliferation, CM cycling was analyzed with a panel of cell cycle markers. In control hearts, BrdU-positive cells were mainly seen in non-CMs 21 days after MI (Figure ID). In contrast, cycling CMs were detected in KDM4DTg / +hearts after MI with BrdU-positive CMs representing approximately 15% of KDM4DTg / +CMs in both border and remote zones (Figure ID). While MI at P7 did not increase phospho-H3 positive CMs in control hearts, phospho-H3 -positive CMs were upregulated over 3-fold after MI in KDM4DTg / +hearts (Figure IE). Analysis of the CM area demonstrated there was no difference in CM size between control and KDM4DTg / +after sham surgery (Figure IF). CM hypertrophy was observed in control animals post-MI; however, there was no change in CM size in KDM4DTg / +hearts (Figure IF). Late cell cycle gene expression was increased in KDM4DTg / +CMs compared to control CMs after MI (Figure 1G).

[0246] ACM-specific KDM4D induction improves cardiac function and reduces infarct scar size post-MI.

[0247] Next, the effect of KDM4D on ACM proliferation was determined in response to ischemic injury. To overcome a potential limitation of the KDM4DTg / +transgenic model where KDM4D is constitutively express throughout development, an inducible CM-specific model was generated to allow assessment of whether KDM4D could reverse ACM cell cycle exit after CMs underwent normal terminal differentiation. CM-specific reverse tetracycline transactivator (rtTA) mouse25were mated KDM4D tet-responder (tet) line. The resulting mice (iKDM4D) displayed tightly regulated KDM4D gene expression in the heart with doxycycline treatment (Figure 8A). The mice were startedon doxycycline chow to induce KDM4D expression immediately post-op (Figure 8B). After MI, analysis of cardiac function 14-days post-MI showed reduced ejection fraction (EF) and fractional shortening (FS) in control animals. In contrast, cardiac function in iKDM4D mice was significantly preserved (EF- control 30.2% vs. iKDM4D 50.8%, P<0.05; FS- control 13.9% vs. iKDM4D 26.7%, <0.05, Table 1). Scar size was reduced by nearly half in iKDM4D induced-hearts after MI compared to control (3.1% vs. 5.8% fibrosis of the myocardium, <0.05, Figure 2A).

[0248] Table 1 Cardiac function and morphology in Ml-operated mice. Echocardiography results in 12-week old mice, 14 days post-operation. HR: Heart Rate, EF: Ejection Fraction, FS: Fractional Shorting, LVESV: Left Ventricular End-Systolic Volume, LVEDD: Left Ventricular End-Diastolic Dimension, LVPW: Left Ventricular Posterior Wall Thickness. Mean, and SEM values are shown. Sample Number: Sham, Control=4, iKDM4D=4; MI, Control=8, iKDM4D=9. Statistics: Two- way ANOVA / Tukey's test, control (MI) vs. iKDM4D (MI), * <0.05.

[0249] To determine if this preservation of cardiac function and reduction in fibrotic scarring occurred primarily through ACM hyperplasia, a panel of cell cycling markers were analyzed. There was a 3.7-fold increase in Ccndl and a 1.5-fold increase in Cdkl gene expression in iKDM4D MI hearts compared to control MI hearts ( <0.05). However, no additional increases in cytokinesis gene expression were observed post-MI (Figure 2B). BrdU incorporation also shown a slight increase of DNA synthesis in activated iKDM4D (w / Dox) hearts relative to controls (w / Dox) two weeks post-MI (Figure 2C). HW / BW and morphological analysis demonstrated that there was no significance in size differences of iKDM4D hearts or ACMs compared to controls post-MI (Figure 8C-8D).

[0250] KDM4D induction mitigates acute myocardial injury post-MI.

[0251] To investigate whether KDM4D could also be preserving myocardium, triphenyl tetrazolium chloride (TTC) staining was performed on iKDM4D and control hearts at 48 hours post-MI to identify viable and infarcted myocardial tissue. Infarct size was smaller in iKDM4D animals compared to controls (6.1% vs. 10.3% infarct size, <0.05, Figure 2D). Several genes involved in cellular apoptosis and survival, including Bcl-2, Survivin, and Apak, were significantly increased in iKDM4D CMs compared to control (3-fold, 1.7-fold, and 2.3-fold increase respectively, / ><0.05 (Figure 2E). Apak protein was also increased 48 hrs post-MI compared to control (Figure 2E). To determine if the expression of these factors reduced the apoptosis typically seen post-MI, apoptosis was quantified using the TUNEL assay. iKDM4D hearts showed a 3 ,4-fold decrease in TUNEL-positive CMs compared to control hearts (iKDM4D 0.06+CM / mm2vs. control 0.2+CM / mm2<0.05, Figure 2F). Without wishing to be bound by theory, these results suggested a role for KDM4D in regulating CM death presumably through the repression of genes responsible for the regulation of anti-apoptotic pathways.

[0252] Screening for small molecules promote NRVM cycling with KDM4D

[0253] Since the less CM proliferative response of KDM4D in adult myocardium, it was determined if the interaction of different pathways and epigenetic manipulation through KDM4D could additively increase the CM proliferation and regeneration. To achieve this, a protein kinase inhibitor library was screened, comprising 295 compounds obtained from Selleckchem.com. NRVMs were treated with Ad-KDM4D or Ad-GFP for two days, followed by a three-day exposure to the compound library to induce robust cell cycle activity (Figure 3A). Ad-KDM4D treatment for five days resulted in sufficient overexpression of KDM4D and reduced levels of H3K9me3 in NRVMs(21). EdU incorporation was visualized using the EdU Click-iT assay one day after compound treatment. It was observed that compounds from four distinct signaling pathways, including PI3K, JAK, p38 MAPK, and MAPK signaling pathway, increased the number of EdU+ NRVMs by 2-4 times in Ad-KDM4D treated cells compared to the cells treated with Ad-GFP alone (Figure 3B). The top 10 small molecule candidates identified in this screen are presented in Figure 3C.

[0254] Six compounds were further evaluated, including Tofacitinib, Ruxolitinib, Nilotinib, cx- 4945, PHT-427, and A-769662, by testing them in 24-well plates with gradient concentrations ranging from InM to lOOpM in combination with Ad-KDM4D treatment. In addition to Ad-KDM4D treatment, Tofacitinib, Ruxolitinib, and Nilotinib exhibited the highest number of EdU+ cells at 10 pM, while cx-4945, PHT-427, and A-769662 displayed the highest number of EdU+ cells at 10 nM. Ruxolitinib, PHT-427, and A-769662 in combination with Ad-KDM4D treatment increased the number of EdU+ cells by -100% compared to the DMSO control (Figure 3D). Furthermore, only PHT-427 and A-769662 led to an increase in the number of pH3+ cells compared to the DMSO control (Figure 3E). All treatments resulted in an increase in the total number of cells, further supporting their pro-proliferative effects on NRVMs (Figure 3F). Combining two compounds did notresult in any additional effect on the number of EdU+ cells, pH3+ cells, or total cell number.

[0255] Gene expression analysis was also performed of cell cycle-related genes for the lead compounds, including Ruxolitinib, PHT-427, and A-769662 (Figure 3G-3I). Treatment with Ruxolitinib and Ad-KDM4D+Ruxolitinib resulted in 2-3 fold increases in the expression of CCNE1, Arukb, and survivin ( <0.05), while treatment with A-769662 and Ad-KDM4D+A-769662 led to 1.5- 2 fold increases in the expression of the same genes ( <0.05). Treatment with Ad-KDM4D+PHT-427 increased Arukb expression by 3-fold and survivin expression by 2-fold. However, PHT-427 alone did not affect the expression of the tested cell cycle-related genes. Thus, small molecules combined with KDM4D overexpression promoted cell cycle gene expression in vitro.

[0256] Together KDM4D and Ruxolitinib increases cardiomyocyte cell cycle activity and improves systolic function in myocardial infarction heart

[0257] Next, the potential of a combination of KDM4D and top-ranked candidate, Ruxolitinib was investigated in promoting the proliferation of ACMs in uninjured mouse hearts. The study was conducted in vivo using adult mice aged between 10-16 weeks (Figure 9A). Ruxolitinib (125mg / kg, q.d.) was orally administrated to vehicle and iKDM4D mice. The iKDM4D mice were fed doxycycline chow to induce KDM4D overexpression in ACMs. EdU (50mg / kg, q.d.) was also administrated to visualize CMs that had entered S phase. The results showed that the administration of small molecules alone did not significantly impact the number of EdU+, Ki67+, and phospho-H3+ CMs (Figure 9B-9D). However, the combination of iKDM4D and Ruxolitinib increased the number of EdU+ and phospho-H3+ CMs by three-fold compared to the vehicle group ( <0.05). Furthermore, iKDM4D+Ruxolitinib group had twice as many Ki67+ CMs as the vehicle group.

[0258] To evaluate the efficacy of the combination of KDM4D and small molecules in promoting cardiac regeneration after MI, 10-16 weeks old iKDM4D mice were induced MI via ischemia / reperfiision (I / R) injury by transiently occluding the left anterior descending artery for 45 min, followed by reperfusion. The infarcted animals were administrated Ruxolitinib and EdU by oral gavage for two weeks as described above. The treated I / R hearts were analyzed for EdU incorporation, Ki67 immunostaining, phospho-H3 immunostaining, Masson trichrome staining, as well as cardiac function assessment using echocardiography analysis (Figure 4A). The results showed that the combination of KDM4D and Ruxolitinib significantly reduced scar size compared to vehicle group ( <0.01), whereas the Ruxolitinib treatment alone did not affect the scar size (Figure 4B). Echocardiography revealed that left ventricular dysfunction in vehicle mouse heart, consistent with pathologic cardiac remodeling. In contrast, iKDM4D and iKDM4D+Ruxolitinib hearts demonstrated progressive improvement in left ventricular ejection fraction and fraction shorten, indicating amelioration of left ventricular systolic heart function (Figure 4C). Image analysis revealed that the number of EdU -labeled CMs was more than 2-fold in hearts treated with Ruxolitinib or doxycycline compared to vehicle and 3 -fold in hearts treated with combinations of the two (Figure 4D). Thenumber of Ki67+ CMs increased 2-fold in doxycycline treated hearts and 3-fold in iKDM4D+Ruxolitinib hearts. The results indicated that the combination of iKDM4D and Ruxolitinib presents substantial effect on increasing CM cell cycle activity. The combination of KDM4D and Ruxolitinib ameliorated left ventricular systolic heart function and reduced scar formation after MI, which is mediated, at least in part, by the enhanced proliferation of existing CMs.

[0259] Acute effect of KDM4D and Ruxolitinib in cardiomyocyte cell cycle activity and apoptosis in myocardial infarction heart in vivo

[0260] To determine whether KDM4D and Ruxolitinib combinationally promotes heart regeneration at early stage of myocardial infarction, 10-16 weeks old iKDM4D mice were induced I / R injury and administrated Ruxolitinib and EdU through oral gavage (Figure 5A). Forty-eight hours after I / R, the infarct size was significantly decreased in the heart of iKDM4D and iKDM4D+Ruxolitinib group (Figure 5B), indicating the induction of KDM4D in CMs protected heart at early stage. Consistent with 2 weeks post-I / R results, iKDM4D+Ruxolitinib increased 2-fold of EdU+, Ki67+, and phospho-H3 CM number compared to vehicle (Figure 5C). Ruxolitinib alone also significantly increased ACM cell cycle reentry 48 hours post I / R (Figure 5C).

[0261] Ruxolitinib is known for its function in inhibiting cancer cell proliferation through apoptosis pathway (30). The in vivo studies demonstrated that KDM4D protects heart from ischemic injury at early stage of MI. Therefore, cardiomyocyte apoptosis was also assessed in the periinfarct border zone. To this end, ventricular sections in this area were subjected to TUNEL assay 48 hours after infarction. TUNEL assay revealed that iKDM4D and iKDM4D+ruxolitinib reduced the ACM apoptosis 48 hours post-I / R, indicating the effect of KDM4D in cardiac cell scarring (Figure 5D).

[0262] The effect of KDM4D and Ruxolitinib in cardiomyocyte cell cycle activity and systolic function 8 weeks post myocardial infarction

[0263] The combination of KDM4D and Ruxolitinib was demonstrated to be highly effective in promoting CM proliferation and heart regeneration, as assessed two weeks post-I / R injury. To further evaluate the long-term effects of KDM4D and Ruxolitinib on the heart, an in vivo experiment was conducted on mice, 8 weeks post-I / R injury. The iKDM4D mice, aged 10-16 weeks, were subjected to I / R injury and administered Ruxolitinib and EdU orally as described before (Figure 6A). Cardiac function assessment was performed using echocardiography analysis at 4 and 8 weeks post-I / R injury (Figure 6B). The results revealed that the iKDM4D+Ruxolitinib hearts showed progressive improvement in left ventricular EF, FS, and LVEDD, compared to vehicle group (Figure 6C), which is similar to the 2-weeks post-I / R assessment. The systolic cardiac function remained unchanged in the iKDM4D+Ruxolitinib hearts at 2 weeks, 4 weeks, and 8 weeks (Figure 6D and 6E), indicating no long-term side effects observed in the iKDM4D+Rucolitinib hearts.

[0264] To provide further evidence of KDM4D and Ruxolitinib resulted in an increase in the number of CMs, the ACMs were isolated to examine the estimated ACM volume and number. Theresults showed no significant difference in HW / BW ratio or ACM volume between the two groups (Figure 6F and 6G). However, the estimated total ACM number was significantly increased in the iKDM4D+Ruxolitinib hearts compared to the vehicle group ( <0.05) (Figure 6H). Because CM division should lead to an increase in the number of mononucleated cells, CM ploidy was measured in CMs treated with iKDM4D+Ruxolitinib (Figure 61). The number of mononucleated CMs was significantly higher in iKDM4D+Ruxolitinib hearts than in vehicle hearts (p<0.0001), providing further support that CM dividing was increased in the iKDM4D+Ruxolitinib hearts. These findings indicate that the combination of KDM4D and Ruxolitnib can induce CM proliferation and ultimately improve left ventricular systolic function in mouse model of myocardial infarction.

[0265] Interacting pathways regulating ACM proliferation and regeneration

[0266] To investigate the mechanisms underlying the induction of ACM proliferation and regeneration by iKDM4D+Ruxilitinib, a Cell Signaling Phospho-Antibody Array (PCS300, Full Moon Biosystems, USA) was employed to screen 304 candidate biomarkers from 16 cell signaling pathways (Figure 10). Results showed that Ruxolitinib primarily regulated the activity of proteins in the WNT signaling pathway (Figure 10C), with 14-3-3 theta / tau (Ser-232) levels increasing by 4-fold and Catenin beta (Thr41 / Ser45) levels increasing by 2-fold compared to the control group. Conversely, KDM4D did not affect Catenin beta (Thr41 / Ser45) levels but instead increased unphosphorylated Catemin beta by 2-fold compared to the control group. Further western blot on NRVM samples revealed that Ruxolitinib increased both 14-3-3 theta / tau (Ser-232) and total 14-3-3, whereas iKDM4D+Ruxolitinib only increased the total 14-3-3 (Figure 10D). Without wishing to be bound by theory, these findings indicate that iKDM4D+Ruxolitinib regulates CM proliferation and regeneration may proceed via WNT signaling pathway by modulating 14-3-3 activity.

[0267] Furthermore, a Co-IP coupled with nanoLC-MS / MS analysis was performed on isolated ACMs from iKDM4D and WT mice hearts to identify CM proteins that interact with KDM4D. Results revealed that protein a-Enolase (ENO1) interacted with KDM4D exclusively in iKDM4D hearts, but not in vehicle hearts. Previous studies have implicated ENO1 in cell apoptosis and proliferation, suggesting that KDM4D promotion of CM proliferation and protection of CMs from apoptosis may also involve regulating ENO1 activity(31, 32).

[0268] Discussion

[0269] The basal turnover of adult mammalian cardiac myocytes is very low(3). Rather than the generation of new CMs, the majority of cell cycle reentry results in generation of polyploid nuclei or multinucleated CMs(l). The limited self-renewal potential is a significant factor in the progression to heart disease. Therefore, enhancing endogenous CM proliferation to compensate for CMs loss after MI may be a promising strategy for treating the development of heart failure. It was originally sought to better understand the molecular blockades to ACM division, which restrict CM proliferation. However, it was discovered that KDM4D also plays a pivotal role in controlling myocyte death in theadult myocardium. The study was the first to demonstrate that induction of KDM4D promote ACMs survival after ischemic injury. Earlier research in the laboratory revealed that the combination of KDM4D reexpression and inhibition of hippo signaling pathway can additively induce CM cycling in transgenic mice through distinct transcriptional pathways(21). In this report, it was demonstrated that the combination of KDM4D and Ruxolitinib, a small molecule target to JAK signaling pathway, can stimulate CM proliferation both in vitro and in vivo, and that KDM4D protects CMs from apoptosis by increasing Apak expression and regulating EN01 activity, which inhibits p53-dependent apoptosis signaling(18). The 48-hours and 8-weeks of Ruxolitinib treatment in iKDM4D mice resulted in improved cardiac remodeling following I / R injury in mice, due to CM proliferation and direct antiapoptotic effects in vivo.

[0270] A small molecule library of 295 kinase inhibitors was screened that targets various signaling pathways to identify compounds that can stimulate CMs cell cycle reentry in Ad-KDM4D treated NRVMs. After conducting a cell-based screening, 10 top-ranked candidates were identified which belongs to PI3K, JAK, and P38 MAPK signaling pathways. Previous studies have demonstrated that these pathways promote CM proliferation and regeneration after myocardial injury(33-35). Ruxolitinib proved to be the most effective out of the 10 candidates, increasing EdU+ cell number by 50% and pH3+ cell number by 20% compared to DMSO control in Ad-KDM4D treated NRVMs. The cell cycle gene expression analysis also confirmed the immunostaining results suggesting that, in combination with KDM4D, Ruxolitinib can induce CM cell cycle activity in vitro.

[0271] Ruxolitinib is the first potent and selective JAK 1 / 2 inhibitor to enter the clinic(36). As an anticancer drug, Ruxolitinib was first approved for the treatment of myelofibrosis (MF) by the United States Food and Drug Administration (FDA) in 2011 and was subsequently used to study its anticancer effects on various solid tumors(37). To date, several studies reported significant anticancer effects of Ruxolitinib in cancer cells via the inhibition of cell proliferation, increased apoptosis, cell cycle arrest in G2 / M phase, and alteration in the expression of related proteins(38-41). In the adult zebrafish heart, Ruxolitinib suppressed the Cilliary Neurotrophic Factor (CNTF) induced cardioprotection and proliferative activity(42). However, the results demonstrated that KDM4D+Ruxolitinib treatment can promote CM proliferation and protect CM from Ruxolitinib- and ischemia- induced CM apoptosis. KDM4D+Ruxolitinib also increased NRVM viability compared to the compound alone in vitro (data not shown) and reduced the infarct size 48 hours post-I / R and scar size 2 weeks post-IR in iKDM4D mice in vivo. Additionally, KDM4D+Ruxolitinib reduced the TUNEL+ cell number compared to the control and single compound treatment 48 hours post-I / R. Without wishing to be bound by theory, these findings indicate that KDM4D+Ruxolitinib has the potential to be an effective treatment for ischemic heart injury by promoting CM proliferation and protecting CM from apoptosis.

[0272] It was found that the induction of KDM4D leads to the upregulation of pro-survivalpathways, including Bcl-2, survivin, and Apak, which may be associated with reduced apoptosis. Apak is known to regulate p53-mediate apoptosis(18), and p53 is a well-established mediator of CM injury after hypoxia or ischemia(43, 44). Ablation of p53 can protect against the development of cardiac dysfunction after ischemic injury(44, 45), as p53 mediates apoptosis(44) and blocks the cell cycle in the G1 phase(46) and G2 / M checkpoint(47, 48). Apak interacts with p53 in unstressed cells, suppressing p53-mediated apoptosis by recruiting KRAB-box-associated protein and histone deacetylase 1 to reduce the acetylation of p53. In response to stress, Apak is phosphorylated by ATM and dissociates from p53, resulting in activation of p53 and induction of cell cycle arrest and apoptosis(49). Recently, H3K9me3 was shown to directly regulate Apak, repressing gene expression as H3K9me3 accumulates along the Apak loci(50). H3K9me3 also indirectly represses Apak by stimulating ATM activity(51) through the regulation of Tip60 , which acetylates ATM(52). Without wishing to be bound by theory, Apak-induction through H3K9me3 reduction may remove both an essential barrier to cell cycle progression and proliferation while simultaneously reducing a principal driver of apoptosis after injury.

[0273] ENO1 (a-enolase) is a multifunctional protein this is dependent on its localization within the ce 11(53). Its primary function is to catalyze the dehydration of 2-phosphoglycerate to phosphoenolpyruvate in the catabolic glycolytic pathway in the mitochondria(54). Beyond its catalytic activity, ENO1 also acts as a plasminogen receptor and promotes extracellular matrix (ECM) degradation on the cell surface(55), helps organize microtubules and the interphase cytoskeleton at the centrosome(56), and maintains mitochondrial membrane stability in the cytoplasm (57). Studies have shown that ENO1 is upregulated in cardiovascular diseases, including cardiac ischemia and cardiac hypertrophy(58), and it plays a role in cell proliferation and apoptosis in both cancer cell and CMs(59). In particular, silencing of ENO 1 has been shown to attenuate Doxorubicin-induced heart injury and CM apoptosis, while overexpression of ENO 1 in NRVMs increased the apoptotic cells compared to the empty vector-transfected cells control(58). Furthermore, several studies revealed that the differentially expressed genes (DEGs) related to ENO1 largely participated in cell proliferation and p53 signaling pathway in cancer cells(60, 61). And the expression of ENO 1 is negatively correlated with p53 expression in the tumor tissues, suggesting that ENO1 regulates apoptosis through p53 signaling pathway(62, 63). The activities of ENO1 were regulated by the post-translational modifications (PTMs), such as lysine residues acetylation or methylation(63). Acetylation lysine residue 335, 256, and 285 of ENO 1 could disrupt both the electrostatic binding potential and the geometry of the binding site, lead to the loss of ENO 1 activity and suppress the glycolysis in tumor cells(63, 64). The mass spectrometry results indicate that KDM4D interacts with ENO1 in CMs, suggesting that KDM4D may remove the methyl group from lysine residues and increase ENO1 acetylation, inhibiting ENO1 activities and protecting CMs from apoptosis through the p53 signaling pathway.

[0274] The applicability of certain chemotherapeutic drugs can be limited due to the risk ofcardiotoxicity, which can lead to an increased morbidity and mortality rate(65). Cardiotoxicity can occur early or late in the course of the disease and may vary from subclinical myocardial dysfunction to irreversible heart failure or even death(66). Reciprocally, myocardial infarction also accelerates tumor growth(67), indicating the need to protect the heart from injury in cancer patients. Recent preclinical and clinical results demonstrate that MI induces reprogramming alterations in myelomonocytic cells to an immunosuppressive phenotype that was maintained in circulation and recruited to the tumor, thereby accelerates breast cancer growth(68). Ruxolitinib (Jakafi) is an FDA approved drug commercially available for the treatment of Myelofibrosis (MF), polycythemia vera (PV), steroid-refractory acute graft-versus-host disease (aGVHD), and chronic graft-versus-host disease (cGVHD). Ruxolitinib is discontinued by most patients during the first 5 years of treatment because of the severe side-effect, such as thrombosis and heart failure(69). Patients with myelofibrosis-associated pulmonary hypertension treated with Ruxolitinib for five years developed severe left ventricular dysfimction(70). Consequently, it is essential to protect heart from myocardial infarction injury in cancer patients undergoing chemotherapy. The study showed that the combination of KDM4D and Ruxolitinib decreased the TUNEL+ cell number and reduced the infarct size indicating the protective role of KDM4D in heart regeneration. The heart function remained steady during 2, 4, and 8 weeks of KDM4D+ruxolitinib treatment post I / R, indicating that KDM4D can also safeguard against Ruxolitinib-induced cardiotoxicity. As a consequence of increased life expectancy, the KDM4D+Ruxolitinib results provide a safe and novel therapeutic approach to reduce the chemotherapeutic drug -induced cardiotoxicity in cancer patients.EXAMPLE 2:

[0275] A small-molecule screening strategy to promote cardiomyocyte proliferation that protects the heart from ischemic injury

[0276] Restoring the ability of adult cardiac myocytes (ACMs) to proliferate and promote heart regeneration have been proposed as a promising therapeutic strategy. We have demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases ACM cell cycle activity, but proliferation was limited. To improve this, we performed a small-molecule screening in combination with KDM4D on neonatal rat cardiomyocytes (NRVMs), using a library of kinase inhibitors targeting different signaling pathways. The study revealed that compounds targeting the PI3K and JAK signaling pathways significantly potentiated the effects of KDM4D and promote NRVM proliferation in vitro. Specifically, a JAK inhibitor (JAKi2) in combination with Ad-KDM4D treatment resulted in approximately 100%, 34%, and 62% increase in EdU+, phospho-H3+, and total cell number compared to control, respectively. To validate these findings in vivo, JAKi2 was tested in an inducible KDM4D transgenic mouse model (iKDM4D). The animals underwent ischemia / reperfiision (I / R) to induce infarction, and JAKi2 was administrated orally for up to 8 weeks. It was found both early and late effects. 48 hours after I / R, the infarct size in iKDM4D+JAKi2 heartswas approximately 4-fold smaller than that in vehicle-treated or JAKi2 alone groups, and the combination increased EdU+, Ki67+, and phospho-H3+ CM number separately by 3.2-fold, 2.9-fold, and 6.5-fold. At 2 weeks post-I / R, the scar size was smaller in iKDM4D+JAKi2 hearts compared to vehicle-treated mice (9.0% vs 1.8%, P<0.05), and the combination resulted in a 3-fold, 5.5-fold, and 8.5-fold increases in EdU+, Ki67+, and phospho-H3+ CMs, respectively. Moreover, the iKDM4D+JAKi2 hearts demonstrated progressive improvement in left ventricular ejection fraction (26.8%, 27.3%, and 25.5%) and fraction shorten (37.9%, 38.1%, and 35.0%) at 2 weeks, 4 weeks, and 8 weeks post-I / R. The increased estimated ACM number and mononucleated CMs at 8 weeks post-I / R support that CM division was increased in the iKDM4D+JAKi2 hearts. These findings indicate that a combination therapy involving JAKi2, along with KDM4D, promotes robust CM proliferation and protects the heart after ischemic injury.

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[0278] Amino Acid Sequence of KDM4D (SEQ ID NO: 1 ): metmkskancaqnpncnimifhptkeefndfdkyiaymesqgahraglakiippkewkaretydniseiliatplqqvasgragvftqyhk kkkamtvgeyrhlanskkyqtpphqnfedlerkywknriynspiygadisgslfdentkqwnlghlgtiqdllekecgvviegvntpylyfg mwkttfawhtedmdlysinylhlgepktwyvvppehgqrlerlarelfpgssrgcgaflrhkvalisptvlkengipfnritqeagefinvtfpy gyhagfhhgfhcaeainfatprwidygkmasqcscgearvtfsmdafvrilqperydlwkrgqdravvdhmeprvpasqelstqkevqlpr raalglrqlpshwarhspwpmaarsgtrchtlvcsslprrsavsgtatqpraaavhsskkpsstpsstpgpsaqiihpsngrrgrgrppqklraqe Itlqtpakrpllagttctasgpepeplpedgalmdkpvplspglqhpvkasgcswapvp

Claims

CLAIMSWhat is claimed herein is:

1. A method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with: a Janus kinase (JAK) inhibitor; and a genetic construct that directs the expression of a lysine-specific demethylase 4D (KDM4D) polypeptide.

2. The method of claim 1, wherein the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the JAK inhibitor and the genetic construct to the subject.

3. The method of claim 1 or claim 2, wherein the cardiomyocyte is human.

4. The method of any one of claims 1-3, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

5. The method of any one of claims 1-4, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.

6. The method of any one of claims 1-5, wherein the JAK inhibitor is ruxolitinib.

7. The method of any one of claims 1-6, wherein the genetic construct that directs the expression of KDM4D comprises a viral vector.

8. The method of claim 7, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

9. The method of claim 8, wherein the AAV vector is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 andAAV12.

10. The method of any of claims 7-9, wherein the AAV vector is AAV9.

11. The method of any one of claims 2-10, wherein the JAK inhibitor is administered simultaneously with the genetic construct that directs the expression of a KDM4D polypeptide.

12. The method of any one of claims 2-10, wherein the JAK inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of a KDM4D polypeptide.

13. The method of any one of claims 1-12, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.

14. The method of claim 13, wherein the JAK inhibitor is administered at a dosage of 10 pM.

15. The method of any one of claims 1-14, wherein the adeno-associated vims expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.

16. The method of any one of claims 1-15, further comprising contacting the cardiomyocyte with a phosphatidylinositol-3 -kinase (PI3K) inhibitor.

17. The method of claim 16, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

18. The method of claim 16, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.

19. The method of claim 16, wherein the PI3K inhibitor is PHT-427.

20. The method of any one of claims 16-19, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.

21. The method of any one of claims 16-20, wherein the PI3K inhibitor is administered at a dosage of lOpM.

22. The method of any one of claims 16-21, wherein the PI3K inhibitor is administered simultaneously with the genetic construct expressing a KDM4D polypeptide.

23. The method of any one of claims 16-22, wherein the PI3K inhibitor is administered at least 1 hour after the administration of the genetic construct expressing a KDM4D polypeptide.

24. A method for promoting cardiomyocyte proliferation, the method comprising contacting a cardiomyocyte with: a phosphatidylinositol-3 -kinase (PI3K) inhibitor; and a genetic construct that directs the expression of a KDM4D polypeptide.

25. The method of claim 24, wherein the cardiomyocyte is in a subject in vivo, and wherein the contacting comprises administering the PI3K inhibitor and the genetic construct to the subject.

26. The method of claim 24 or claim 25, wherein the cardiomyocyte is human.

27. The method of any one of clams 24-26, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

28. The method of any one of clams 24-26, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.

29. The method of any one of claims 24-26, wherein the PI3K inhibitor is PHT-427.

30. The method of any one of claims 24-29, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.

31. The method of any one of claims 24-30, wherein the PI3K inhibitor is administered at a dosage of lOpM.

32. The method of any one of claims 24-31, wherein the genetic construct that directs the expression of a KDM4D polypeptide comprises a viral vector.

33. The method of claim 32, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

34. The method of claim 33, wherein the AAV vector is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

35. The method of any one of claims 33-34, wherein the AAV vector is AAV9.

36. The method of any one of claims 24-35, wherein the PI3K inhibitor is administered simultaneously with the genetic construct that directs the expression of a KDM4D polypeptide.

37. The method of any one of claims 24-35, wherein the PI3K inhibitor is administered at least 1 hour after the administration of the genetic construct that directs the expression of a KDM4D polypeptide.

38. The method of any one of claims 24-37, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.

39. The method of any one of claims 1-38, wherein the subject has cardiac damage or a cardiac disease or disorder.

40. The method of claim 39, wherein the subject has suffered a myocardial infarction.

41. The method of claim 39, wherein the cardiac disease or disorder is selected from atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.

42. A kit for promoting cardiomyocyte proliferation, the kit comprising: a viral vector expressing a KDM4D polypeptide; and a JAK inhibitor.

43. The kit of claim 42, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

44. The kit of claim 42, further comprising a PI3K inhibitor.

45. A kit for promoting cardiomyocyte proliferation, the kit comprising: a viral vector expressing a KDM4D polypeptide; anda PI3K inhibitor.

46. The kit of claim 45, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

47. The kit of claim 45, further comprising a JAK inhibitor.

48. The kit of any one of claims 42-47, further comprising instructions for use.

49. The kit of any one of claims 42-48, for use in treating a cardiac disease or disorder.

50. The kit of any one of claims 42-49, for use in treating a cardiovascular disease or disorder.

51. The kit of any one of claims 42-50, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

52. The kit of any one of claims 42-50, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.

53. The kit of any one of claims 42-50, wherein the JAK inhibitor is ruxolitinib.

54. The kit of any one of claims 42-53, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

55. The kit of any one of claims 42-53, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.

56. The kit of any one of claims 42-53, wherein the PI3K inhibitor is PHT-427.

57. The kit of any one of claims 42-56, wherein the adeno-associated virus expressing a KDM4D polypeptide is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

58. The kit of any one of claims 42-57, wherein the adeno-associated virus expressing a KDM4D polypeptide is AAV9.

59. A method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof:an amount of a JAK inhibitor; and an amount of a genetic construct that directs the expression of a KDM4D polypeptide; sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.

60. The method of claim 59, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

61. The method of claim 59, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.

62. The method of claim 59, wherein the JAK inhibitor is ruxolitinib.

63. The method of any one of claims 59-62, wherein the subject in need thereof is human.

64. The method of any one of claims 59-63, wherein the genetic construct that directs expression of a KDM4D polypeptide comprises a viral vector.

65. The method of claim 64, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

66. The method of claims 64 or 65, wherein the AAV is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

67. The method of any one of claims 59-66, wherein the AAV vector is AAV9.

68. The method of any one of claims 59-67, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.

69. The method of any one of claims 59-68, wherein the JAK inhibitor is administered at a dosage lOpM.

70. The method of any one of claims 59-69, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.

71. The method of any one of claims 59-70, further comprising administering a PI3K inhibitor to the subject.

72. The method of claim 61, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

73. The method of claim 61, wherein the PI3K inhibitor is selected from the group consisting of:cx-4945, PHT-427, A-769662, and XL765.

74. The method of claim 61, wherein the PI3K inhibitor is PHT-427.

75. A method of treating cardiac damage or a cardiac disease or disorder, the method comprising administering to a subject in need thereof: an amount of a PI3K inhibitor; and an amount of a genetic construct that directs the expression of a KDM4D polypeptide; sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or a cardiac disease or disorder is treated.

76. The method of claim 75, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

77. The method of claim 75, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.

78. The method of claim 75, wherein the PI3K inhibitor is PHT-427.

79. The method of any one of claims 75-78, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.

80. The method of any one of claims 75-79, wherein the PI3K inhibitor is administered at a dosage of lOpM.

81. The method of any one of claims 75-80, wherein the genetic construct that directs expression of a KDM4D polypeptide comprises a viral vector.

82. The method of claim 81, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

83. The method of claim 82, wherein the AAV is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

84. The method of any one of claims 82-83, wherein the AAV vector is AAV9.

85. The method of any one of claims 82-84, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.

86. The method of any one of claims 75-85, wherein the subject in need thereof is human.

87. The method of any one of claims 75-86, further comprising administering a JAK inhibitor to the subject.

88. The method of claim 87, wherein the JAK inhibitor is selected from the group consisting of:ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

89. The method of claim 87, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.

90. The method of claim 87, wherein the JAK inhibitor is ruxolitinib.

91. The method of any one of claims 87-90, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.

92. The method of any one of claims 87-90, wherein the JAK inhibitor is administered at a dosage of 10 pM.

93. A method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject: an amount of a JAK inhibitor; and an amount of a genetic construct directing the expression of a KDM4D polypeptide; sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.

94. The method of claim 93, wherein the subject has suffered ischemic injury to cardiac tissue.

95. The method of claim 93 or 94, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

96. The method of claim 93 or 94, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.

97. The method of any one of claims 93 or 94, wherein the JAK inhibitor is ruxolitinib.

98. The method of any one of claims 93-97, wherein the subject in need thereof is human.

99. The method of any one of claims 93-98, wherein the genetic construct that directs the expression of a KDM4D polypeptide comprises a viral vector.

100. The method of claim 99, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

101. The method of claim 100, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

102. The method of any one of claims 100-101, wherein the AAV vector is AAV9.

103. The method of any one of claims 93-102, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.

104. The method of any one of claims 93-103, wherein the JAK inhibitor is administered at a dosage of lOpM.

105. The method of any one of claims 100-104, wherein the adeno-associated virus expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.

106. The method of any one of claims 93-105, further comprising administering a PI3K inhibitor to the subject.

107. The method of claim 106, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

108. The method of claim 106, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.

109. The method of claim 106, wherein the PI3K inhibitor is PHT-427.

110. The method of any one of claims 106-109, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.

111. The method of any one of claims 106-110, wherein the PI3K inhibitor is administered at a dosage of lOpM.

112. A method of reducing cardiac damage or injury in a subject in need thereof, the method comprising administering to the subject an amount of a PI3K inhibitor and an amount of a genetic construct directing the expression of a KDM4D polypeptide sufficient to promote cardiomyocyte proliferation in the subject, whereby cardiac damage or injury is reduced.

113. The method of claim 112, wherein the subject has suffered ischemic injury to cardiac tissue.

114. The method of claim 112 or 113, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

115. The method of claim 112 or 113, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.

116. The method of claim 112 or 113, wherein the PI3K inhibitor is PHT-427.

117. The method of any one of claims 112-116, wherein the subject in need thereof is human.

118. The method of any one of claims 112-117, wherein the genetic construct directing the expression of a KDM4D polypeptide comprises a viral vector.

119. The method of claim 118, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

120. The method of claim 119, wherein the AAV is selected from the group consisting of AAV 1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

121. The method of claim 119, wherein the AAV is AAV9.

122. The method of any one of claims 112-121, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.

123. The method of any one of claims 112-122, wherein the PI3K inhibitor is administered at a dosage of lOpM.

124. The method of any one of claims 119-123, wherein the AAV expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.

125. The method of any one of claims 112-124, further comprising administering a JAK inhibitor.

126. The method of claim 125, wherein the JAK inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

127. The method of claim 125, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.

128. The method of claim 125, wherein the JAK inhibitor is ruxolitinib.

129. The method of any one of claims 125-128, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.

130. The method of any one of claims 125-129, wherein the JAK inhibitor is administered at a dosage of lOpM.

131. A combination of: a) at least one of: a JAK inhibitor and PI3K inhibitor; and b) a genetic construct that directs the expression of a KDM4D polypeptide for use in a method of reducing cardiac damage or injury in a subject in need thereof.

132. The combination of claim 131, comprising a JAK inhibitor.

133. The combination of claim 131, comprising a PI3K inhibitor.

134. The combination of any one of claims 131-133, comprising a JAK inhibitor and a PI3K inhibitor.

135. The combination of claims 131, 132, or 134, wherein the JAK inhibitor is selected from the group consisting of:ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, fdgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, nilotinib, cucurbitacin I, AG-490, cx-4945, CHZ868, LS104, ON044580, NVP-BBT594, and NVP-CHZ868.

136. The combination of claims 131, 132, or 134, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib citrate, ruxolitinib, nilotinib, AG-490, and cx-4945.

137. The combination of claims 131, 132, or 134, wherein the JAK inhibitor is ruxolitinib.

138. The combination of any one of claims 131, 132, or 134-137, wherein the JAK inhibitor is administered at a dosage of InM to lOOpM, inclusive.

139. The combination of any one of claims 131, 132, or 134-138, wherein the JAK inhibitor is administered at a dosage lOpM.

140. The combination of any one of claims 131, or 133-139, wherein the PI3K inhibitor is selected from the group consisting of: idelalisib, alpelisib, leniolisib, duvelisib, copanlisib, umbralisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, boxtalisib, AMG 319, IPI-549, AZD8186, GSK2636771, SF1126, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, cx-4945, PHT-427, A-769662, and XL765.

141. The combination of any one of claims 131, or 133-139, wherein the PI3K inhibitor is selected from the group consisting of: cx-4945, PHT-427, A-769662, and XL765.

142. The combination of any one of claims 131, or 133-139, wherein the PI3K inhibitor is PHT-427.

143. The combination of any one of claims 131 or 133-142, wherein the PI3K inhibitor is administered at a dosage of InM to lOOpM, inclusive.

144. The combination of any one of claims 131 or 133-143, wherein the PI3K inhibitor is administered at a dosage 1 OpM.

145. The combination of any one of claims 131-144, wherein the genetic construct that directs expression of a KDM4D polypeptide comprises a viral vector.

146. The combination of claim 145, wherein the viral vector is an adenoviral vector or an adeno- associated virus (AAV) vector.

147. The combination of claim 146, wherein the AAV is selected from the group consisting of:AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 andAAV12.

148. The combination of any one of claims 146-147, wherein the AAV vector is AAV9.

149. The combination of any one of claims 146-148, wherein the adeno-associated vims expressing a KDM4D polypeptide is administered at a total dose within the range of lxl09vg to about lxl016vg.

150. The combination of any one of claims 131-149, wherein the subject in need thereof is human.

151. The combination of any one of claims 131-150, wherein the subject has suffered ischemic injury to cardiac tissue.

152. The combination of any one of claims 131-151, the subject has cardiac damage or a cardiac disease or disorder.

153. The combination of claim 152, the cardiac disease or disorder is selected from atherosclerotic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, myocardial infarction, heart failure, cardiac hypertrophy, valvular stenosis, regurgitation, ischemia, fibrillation, and polymorphic ventricular tachycardia.

154. The combination of any one of claims 131-152, the subject has suffered a myocardial infarction.

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