Electrophysiological Modifications to Suppress Arrhythmias

Abstract

Described herein are compositions and methods related to the treatment of cardiovascular disease or disorder.Described herein are also cells, stem cells (including embryonic and pluripotent stem cells) and human cardiomyocytes differentiated in vitro, wherein HCN4 (HCN4), Cav3.2 (CACNA1H) and NCX1 (SLC8A1) activity is at least partially inhibited in such cells, and Kir2.1 (KCNJ2) activity is at least partially stimulated.Described herein are also methods for the delivery of such cells and for transplanting cardiomyocytes, for example for the treatment or prevention of disease or disorder. TIFF2024517810000020.tif69170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 292,046, filed December 21, 2021, U.S. Provisional Application No. 63 / 211,843, filed June 17, 2021, and U.S. Provisional Application No. 63 / 183,528, filed May 3, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Technical Field The technology described herein relates to compositions and methods and their uses for treating and preventing electrical disturbances in the heart and promoting engraftment of in vitro differentiated cardiomyocytes. [Background technology]

[0003] background Cardiovascular disease remains the leading cause of death for both men and women worldwide, with a rapidly increasing impact in developing countries. Cardiomyocyte replacement therapy is an area of ​​active development for the treatment of cardiovascular disease and can restore cardiac function after injuries, including but not limited to myocardial infarction. Human stem cells cultured in vitro can serve as starting material for producing human cardiomyocytes for engraftment into the damaged heart. However, the use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) for cardiac regeneration is hampered by the occurrence of transient arrhythmias after transplantation. To improve patient outcomes after cardiomyocyte replacement therapy, compositions and methods for preventing and / or treating cardiac graft-induced arrhythmias are needed. Summary of the Invention

[0004] overview The technology described herein relates to the discovery of compositions and methods for treating and / or preventing electrical disturbances in the heart and promoting the engraftment of in vitro differentiated cardiomyocytes. It has been discovered that by attenuating the impulse-generating activity of cardiomyocytes administered for engraftment into cardiac tissue, transplant-induced disturbances in heart rate and rhythm are eliminated or dramatically reduced. More specifically, it has been discovered that modifying the activity of a set of ion channels that regulate electrophysiological function in graft cardiomyocytes limits or prevents electrical disturbances in heart rate and / or rhythm caused by the transplanted cardiomyocytes. These disturbances, which can last for several weeks after transplantation of cardiomyocytes into cardiac tissue before resolving, can have fatal consequences and are collectively referred to herein as "engraftment arrhythmia" or "EA," and are further described herein below. While individual manipulation of specific ion channels, pairwise manipulation, or triadic manipulation either had no benefit or actually worsened transplant arrhythmias, manipulation of the four-member set of ion channel polypeptides HCN4 (encoded by the gene HCN4), Cav3.2 (encoded by the gene CACNA1H), NCX1 (encoded by the gene SLC8A1), and Kir2.1 (encoded by the gene KCNJ2) was found to dramatically improve or eliminate transplant arrhythmias. More specifically, inhibition or knockout of the activity or expression of HCN4, CACNA1H, and SLC8A1, combined with activation or overexpression of the KCNJ2 (Kir2.1) polypeptide, was found to significantly reduce or eliminate such disturbances.

[0005] Thus, in one aspect, provided herein are in vitro differentiated human cardiomyocytes in which HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited and KCNJ2 activity is at least partially stimulated.

[0006] Another aspect provided herein relates to in vitro differentiated human cardiomyocytes comprising reduced expression of HCN4, CACNA1H, and SLC8A1, and expression of a transgene encoding KCNJ2 regulated by endogenous HCN4 regulatory sequences.

[0007] In one embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited relative to cardiomyocytes or other control cells.

[0008] In another embodiment of this aspect and all other aspects provided herein, KCNJ2 activity is at least partially stimulated relative to cardiomyocytes or other control cells.

[0009] In another embodiment of this aspect and all other aspects provided herein, at least partial inhibition of HCN4, CACNA1H, and SLC8A1 comprises inhibition via contacting the cardiomyocytes with one or more inhibitor drugs and / or comprises genetic manipulation.

[0010] In another embodiment of this aspect and all other aspects provided herein, the at least partial stimulation of KCNJ2 activity comprises contacting the cardiomyocytes with one or more activating drugs and / or comprises genetic manipulation.

[0011] In another embodiment of this aspect and all other aspects provided herein, the reduced expression of HCN4, CACNA1H, or SLC8A1 is due to genetic manipulation.

[0012] In another embodiment of this aspect and all other aspects provided herein, the at least partially stimulated activity of KCNJ2 is due to genetic manipulation.

[0013] In another embodiment of this aspect and all other aspects provided herein, one or more of the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated.

[0014] In another embodiment of this aspect and all other aspects provided herein, each of the genes encoding HCN4, CACNA1H, and SLC8A1 is inactivated.

[0015] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is completely inhibited and KCNJ2 activity is at least partially stimulated relative to control cells.

[0016] In another embodiment of this aspect and all other aspects provided herein, the one or more inhibitor drugs, activator drugs and / or genetic manipulations do not alter expression of HCN4, CACNA1H, SLC8A1 and / or KCNJ2.

[0017] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000%, or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, compared to cardiomyocytes or other control cells that are not manipulated by one or more inhibitor drugs and / or genetic manipulation.

[0018] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000%, or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, relative to cardiomyocytes or other control cells that are not genetically engineered.

[0019] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is completely inhibited by genetic engineering, and KCNJ2 activity is at least partially stimulated, relative to control cells.

[0020] In another embodiment of this aspect and all other aspects provided herein, the cardiomyocyte or other control cell is a wild-type cardiomyocyte, a primary cardiomyocyte, an in vitro differentiated cardiomyocyte derived from a PSC or an ESC, or starting material.

[0021] In another embodiment of this aspect and all other aspects provided herein, the control cells are not manipulated by one or more inhibitor drugs, activator drugs and / or genetic manipulations.

[0022] In another embodiment of this aspect and all other aspects provided herein, the one or more inhibitor drugs, activator drugs and / or genetic manipulations do not alter expression of HCN4, CACNA1H, SLC8A1 and / or KCNJ2.

[0023] In another embodiment of this aspect and all other aspects provided herein, the control cell is an in vitro differentiated cardiomyocyte derived from a PSC or an ESC, and the control cell, the PSC or ESC, comprises one, two, or three, but not all four, edits, and HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited, and KCNJ2 activity is at least partially stimulated.

[0024] In another embodiment of this aspect and all other aspects provided herein, the PSCs are iPSCs.

[0025] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% or 1-fold and KCNJ2 activity is stimulated by at least 10% or 1-fold by genetic engineering compared to control cells that are not genetically engineered.

[0026] In another embodiment of this aspect and all other aspects provided herein, the method of genetic manipulation is gene knockdown, and optionally, the gene knockdown is by RNA silencing or RNAi, optionally selected from the group consisting of siRNA, piRNA, shRNA, and miRNA.

[0027] In another embodiment of this aspect and all other aspects provided herein, the method of genetic engineering is gene knockout, and optionally the gene knockout is by inducing an insertion or deletion in the gene using a gene editing system, wherein the gene editing system is optionally selected from the group consisting of a ZFN, a TALEN, a meganuclease, a transposase, a CRISPR / Cas system, a nickase system, a base editing system, a prime editing system, and a gene writing system.

[0028] In another embodiment of this aspect and all other aspects provided herein, a gene knockout of HCN4, CACNA1H, or SLC8A1 is introduced into one or more alleles of HCN4, CACNA1H, or SLC8A1.

[0029] In another embodiment of this aspect and all other aspects provided herein, the gene knockout of HCN4, CACNA1H, or SLC8A1 is introduced into both alleles of HCN4, CACNA1H, or SLC8A1.

[0030] In another embodiment of this aspect and all other aspects provided herein, the gene knockout of HCN4, CACNA1H, or SLC8A1 results in reduced protein expression and / or reduced gene expression of HCN4, CACNA1H, or SLC8A1.

[0031] In another embodiment of this aspect and all other aspects provided herein, gene inactivation comprises an insertion or deletion of a nucleic acid sequence at the locus encoding HCN4, CACNA1H, and / or SLC8A1.

[0032] In another embodiment of this aspect and all other aspects provided herein, gene inactivation is effected via RNA-guided nuclease, TALEN, or zinc finger nuclease activity.

[0033] In another embodiment of this aspect and all other aspects provided herein, the RNA-guided nuclease comprises a Cas nuclease.

[0034] In another embodiment of this aspect and all other aspects provided herein, gene inactivation or gene knockout is achieved via RNAi, antisense, or RNA-targeted Cas nucleases.

[0035] In another embodiment of this aspect and all other aspects provided herein, gene inactivation or gene knockout is performed using the CRISPR / Cas system.

[0036] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes further comprise at least one exogenous nucleic acid sequence.

[0037] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes express a polypeptide from at least one exogenous nucleic acid sequence.

[0038] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes further comprise reduced expression of at least one additional gene.

[0039] In another embodiment of this aspect and all other aspects provided herein, KCNJ2 is overexpressed from a transgene.

[0040] In another embodiment of this aspect and all other aspects provided herein, the transgene comprises an exogenous KCNJ2 coding sequence driven by an HCN4 or CACNA1H expression regulatory sequence.

[0041] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence replaces the coding sequence for HCN4 or CACNA1H such that the replaced HCN4 or CACNA1H coding sequence is not expressed and the replaced KCNJ2 is expressed under the regulation of the endogenous HCN4 or CACNA1H regulatory sequence.

[0042] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence is replaced using a CRISPR / Cas system.

[0043] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of HCN4 with the KCNJ2 coding sequence.

[0044] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of CACNA1H with the KCNJ2 coding sequence.

[0045] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated and the KCNJ2 polypeptide is overexpressed.

[0046] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SCL8A1 contain an indel in at least one allele.

[0047] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SCL8A1 contain indels in two alleles.

[0048] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated human cardiomyocytes comprise HCN4 インデル / インデル , CACNA1H インデル / インデル , and SCL8A1 インデル / インデル It is a cell.

[0049] In another embodiment of this aspect and all other aspects provided herein, the indels are generated using a CRISPR / Cas system.

[0050] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene and is operably linked to an endogenous HCN4 or CACNA1H regulatory sequence.

[0051] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated using the CRISPR / Cas system, and the KCNJ2 polypeptide is overexpressed under the regulation of endogenous HCN4 regulatory sequences.

[0052] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene operably linked to an endogenous HCN4 regulatory sequence.

[0053] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is overexpressed under the regulation of endogenous HCN4 regulatory sequences at the HCN4 locus.

[0054] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated using the CRISPR / Cas system, and the KCNJ2 polypeptide is overexpressed under the regulation of endogenous CACNA1H regulatory sequences.

[0055] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene operably linked to an endogenous CACNA1H regulatory sequence.

[0056] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is overexpressed under the regulation of endogenous CACNA1H regulatory sequences at the CACNA1H locus.

[0057] In another embodiment of this aspect and all other aspects provided herein, the activity of at least one of HCN4, CACNA1H, SLC8A1, and KCNJ2 is manipulated by contacting the cardiomyocyte with a drug, and the activity of at least one of HCN4, CACNA1H, SLC8A1, and KCNJ2 is genetically manipulated.

[0058] In another embodiment of this aspect and all other aspects provided herein, the cardiomyocytes are differentiated in vitro from pluripotent stem cells.

[0059] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0060] In another embodiment of this aspect and all other aspects provided herein, the cardiomyocytes are differentiated in vitro from iPSCs derived from the subject into which the in vitro differentiated human cardiomyocytes are transplanted.

[0061] In another embodiment of this aspect and all other aspects provided herein, the cardiomyocytes are differentiated in vitro from iPSCs derived from a healthy subject.

[0062] In another embodiment of this aspect and all other aspects provided herein, the cardiomyocytes are differentiated in vitro from a starting material.

[0063] In another embodiment of this aspect and all other aspects provided herein, the starting material includes primary cells collected from a donor.

[0064] In another embodiment of this aspect and all other aspects provided herein, each of the healthy subject, starting material, and / or donor is not derived from the same individual as the subject into whom the in vitro differentiated human cardiomyocytes are transplanted.

[0065] In another embodiment of this aspect and all other aspects provided herein, the primary cells collected from the donor are stem cells.

[0066] In another embodiment of this aspect and all other aspects provided herein, the stem cells are ESCs.

[0067] In another embodiment of this aspect and all other aspects provided herein, the starting material is a stem cell line.

[0068] In another embodiment of this aspect and all other aspects provided herein, the stem cell line is an ESC line or an iPSC line.

[0069] In another embodiment of this aspect and all other aspects provided herein, the stem cell line is an iPSC line.

[0070] In another embodiment of this aspect and all other aspects provided herein, when administered to cardiac tissue of a subject in need thereof, the in vitro differentiated human cardiomyocytes promote reduced arrhythmias compared to a subject administered in vitro differentiated human cardiomyocytes that do not comprise at least partial inhibition of HCN4, CACNA1H, and SLC8A1 activity and at least partial stimulation of KCNJ2 activity.

[0071] In another embodiment of this aspect and all other aspects provided herein, when administered to cardiac tissue of a subject in need thereof, the subject experiences reduced arrhythmias compared to a subject administered in vitro differentiated human cardiomyocytes that do not comprise at least partial inhibition of HCN4, CACNA1H, and SLC8A1 activity and at least partial stimulation of KCNJ2 activity.

[0072] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are admixed with a cryopreservative.

[0073] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are frozen in admixture with a cryopreservative.

[0074] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated human cardiomyocytes express one or more markers selected from the group consisting of NKX2-5, MYH6, MYL7, TBX5, ATP2a2, RYR2, and cTnT.

[0075] In another embodiment of this aspect and all other aspects provided herein, cellular activity and maturity can be determined by a numerical parameter selected from the group consisting of electrical maturity, metabolic maturity, and contractile maturity.

[0076] In another embodiment of this aspect and all other aspects provided herein, the metabolic maturity of in vitro differentiated cardiomyocytes is determined by: Increased activity of mitochondrial function, increased fatty acid metabolism, increased oxygen consumption rate (OCR), increased phosphorylated ACC level or activity, increased level or activity of fatty acid binding protein (FABP), increased level or activity of pyruvate dehydrogenase kinase-4 (PDK4), increased mitochondrial respiratory capacity, increased mitochondrial volume, and increased levels of mitochondrial DNA The markers are determined by one or more of the markers selected from the group consisting of:

[0077] Another aspect provided herein relates to a pluripotent stem cell comprising decreased expression of HCN4, CACNA1H, and SLC8A1 and increased expression of KCNJ2.

[0078] In one embodiment of this aspect and all other aspects provided herein, decreased expression of HCN4, CACNA1H, and SLC8A1 includes decreased protein expression and / or decreased gene expression for each of HCN4, CACNA1H, and SLC8A1.

[0079] In another embodiment of this aspect and all other aspects provided herein, increased expression of KCNJ2 includes increased protein expression and / or increased gene expression.

[0080] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells comprise reduced expression of HCN4, CACNA1H, and SLC8A1, and expression of a transgene encoding KCNJ2 that is regulated by endogenous HCN4 regulatory sequences.

[0081] In another embodiment of this aspect and all other aspects provided herein, inhibition of HCN4, CACNA1H, and SLC8A1 comprises inhibition via contacting cardiomyocytes with one or more inhibitor drugs and / or comprises genetic manipulation.

[0082] In another embodiment of this aspect and all other aspects provided herein, stimulating KCNJ2 activity comprises contacting the pluripotent stem cells with one or more activating drugs and / or comprises genetic manipulation.

[0083] In another embodiment of this aspect and all other aspects provided herein, the reduced expression of HCN4, CACNA1H, or SLC8A1 is due to genetic manipulation.

[0084] In another embodiment of this aspect and all other aspects provided herein, one or more of the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated in the pluripotent stem cells.

[0085] In another embodiment of this aspect and all other aspects provided herein, each of the genes encoding HCN4, CACNA1H, and SLC8A1 is inactivated in the pluripotent stem cells.

[0086] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is completely inhibited and KCNJ2 activity is at least partially stimulated compared to control cells that are not manipulated by one or more inhibitor drugs and / or genetic manipulation.

[0087] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000%, or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, relative to control cells that are not manipulated by one or more inhibitor drugs and / or genetic manipulation.

[0088] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000%, or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, relative to control cells that are not genetically engineered.

[0089] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is completely inhibited by genetic engineering, and KCNJ2 activity is at least partially stimulated, relative to control cells that are not genetically engineered.

[0090] In another embodiment of this aspect and all other aspects provided herein, the control cells are wild-type cardiomyocytes, primary cardiomyocytes, in vitro differentiated cardiomyocytes derived from PSCs or ESCs, or starting material.

[0091] In another embodiment of this aspect and all other aspects provided herein, the control cell is an in vitro differentiated cardiomyocyte derived from a PSC or an ESC, and the control cell, PSC, ESC, comprises one, two, or three, but not all four, edits, and HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited, and KCNJ2 activity is at least partially stimulated.

[0092] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% or 1-fold and KCNJ2 activity is stimulated by at least 10% or 1-fold by genetic engineering compared to control cells that are not genetically engineered.

[0093] In another embodiment of this aspect and all other aspects provided herein, the method of genetic manipulation is gene knockdown, and optionally, the gene knockdown is by RNA silencing or RNAi, optionally selected from the group consisting of siRNA, piRNA, shRNA, and miRNA.

[0094] In another embodiment of this aspect and all other aspects provided herein, the method of genetic engineering is gene knockout, and optionally the gene knockout is by inducing an insertion or deletion in the gene using a gene editing system, wherein the gene editing system is optionally selected from the group consisting of a ZFN, a TALEN, a meganuclease, a transposase, a CRISPR / Cas system, a nickase system, a base editing system, a prime editing system, and a gene writing system.

[0095] In another embodiment of this aspect and all other aspects provided herein, a gene knockout of HCN4, CACNA1H, or SLC8A1 is introduced into one or more alleles of HCN4, CACNA1H, or SLC8A1.

[0096] In another embodiment of this aspect and all other aspects provided herein, the gene knockout of HCN4, CACNA1H, or SLC8A1 is introduced into both alleles of HCN4, CACNA1H, or SLC8A1.

[0097] In another embodiment of this aspect and all other aspects provided herein, the gene knockout of HCN4, CACNA1H, or SLC8A1 results in reduced protein expression and / or reduced gene expression of HCN4, CACNA1H, or SLC8A1.

[0098] In another embodiment of this aspect and all other aspects provided herein, gene inactivation comprises an insertion or deletion of a nucleic acid sequence at the locus encoding HCN4, CACNA1H, and / or SLC8A1.

[0099] In another embodiment of this aspect and all other aspects provided herein, gene inactivation is effected via RNA-guided nuclease, TALEN, or zinc finger nuclease activity.

[0100] In another embodiment of this aspect and all other aspects provided herein, the RNA-guided nuclease comprises a Cas nuclease.

[0101] In another embodiment of this aspect and all other aspects provided herein, gene inactivation or gene knockout is achieved via RNAi, antisense, or RNA-targeted Cas nucleases.

[0102] In another embodiment of this aspect and all other aspects provided herein, gene inactivation or gene knockout is performed using the CRISPR / Cas system.

[0103] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells further comprise at least one exogenous nucleic acid sequence.

[0104] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells express a polypeptide from at least one exogenous nucleic acid sequence.

[0105] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cell further comprises reduced expression of at least one additional gene.

[0106] In another embodiment of this aspect and all other aspects provided herein, KCNJ2 is overexpressed from a transgene.

[0107] In another embodiment of this aspect and all other aspects provided herein, the transgene comprises an exogenous KCNJ2 coding sequence driven by an HCN4 or CACNA1H expression regulatory sequence.

[0108] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence replaces the coding sequence for HCN4 or CACNA1H such that the replaced HCN4 or CACNA1H coding sequence is not expressed and the replaced KCNJ2 is expressed under the regulation of the endogenous HCN4 or CACNA1H regulatory sequence.

[0109] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence is replaced using a CRISPR / Cas system.

[0110] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of HCN4 with the KCNJ2 coding sequence.

[0111] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of CACNA1H with the KCNJ2 coding sequence.

[0112] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated and the KCNJ2 polypeptide is overexpressed.

[0113] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SCL8A1 contain an indel in at least one allele.

[0114] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SCL8A1 contain indels in two alleles.

[0115] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are HCN4 インデル / インデル , CACNA1H インデル / インデル , and SCL8A インデル / インデル It is a cell.

[0116] In another embodiment of this aspect and all other aspects provided herein, the indels are generated using a CRISPR / Cas system.

[0117] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene and is operably linked to an endogenous HCN4 or CACNA1H regulatory sequence.

[0118] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated using the CRISPR / Cas system, and the KCNJ2 polypeptide is overexpressed under the regulation of endogenous HCN4 regulatory sequences.

[0119] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene operably linked to an endogenous HCN4 regulatory sequence.

[0120] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is overexpressed under the regulation of endogenous HCN4 regulatory sequences at the HCN4 locus.

[0121] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated using the CRISPR / Cas system, and the KCNJ2 polypeptide is overexpressed under the regulation of endogenous CACNA1H regulatory sequences.

[0122] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene operably linked to an endogenous CACNA1H regulatory sequence.

[0123] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is overexpressed under the regulation of endogenous CACNA1H regulatory sequences at the CACNA1H locus.

[0124] In another embodiment of this aspect and all other aspects provided herein, the activity of at least one of HCN4, CACNA1H, SLC8A1, and KCNJ2 is manipulated by contacting the cardiomyocyte with a drug, and the activity of at least one of HCN4, CACNA1H, SLC8A1, and KCNJ2 is genetically manipulated.

[0125] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are embryonic stem cells (ESCs).

[0126] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0127] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are derived from iPSCs derived from the subject into which the pluripotent stem cells are to be transplanted.

[0128] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are derived from iPSCs derived from a healthy subject.

[0129] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are differentiated in vitro from a starting material.

[0130] In another embodiment of this aspect and all other aspects provided herein, the starting material includes primary cells collected from a donor.

[0131] In another embodiment of this aspect and all other aspects provided herein, each of the healthy subject, starting material, and / or donor is not derived from the same individual as the subject into whom the pluripotent stem cells are transplanted.

[0132] In another embodiment of this aspect and all other aspects provided herein, the primary cells collected from the donor are stem cells.

[0133] In another embodiment of this aspect and all other aspects provided herein, the stem cells are ESCs.

[0134] In another embodiment of this aspect and all other aspects provided herein, the starting material is a stem cell line.

[0135] In another embodiment of this aspect and all other aspects provided herein, the stem cell line is an ESC line or an iPSC line.

[0136] In another embodiment of this aspect and all other aspects provided herein, the stem cell line is an iPSC line.

[0137] In another embodiment of this aspect and all other aspects provided herein, when administered to cardiac tissue of a subject in need thereof, the pluripotent stem cells promote reduced arrhythmias compared to a subject administered pluripotent stem cells that do not include inhibition of HCN4, CACNA1H, and SLC8A1 activity and stimulation of KCNJ2 activity.

[0138] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are admixed with a cryopreservation agent.

[0139] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are frozen in admixture with a cryopreservative.

[0140] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated human cardiomyocytes derived from pluripotent stem cells express one or more markers selected from the group consisting of NKX2-5, MYH6, MYL7, TBX5, ATP2a2, RYR2, and cTnT.

[0141] In another embodiment of this aspect and all other aspects provided herein, the cellular activity and maturity of in vitro differentiated human cardiomyocytes derived from pluripotent stem cells can be determined by a numerical parameter selected from the group consisting of electrical maturity, metabolic maturity, and contractile maturity.

[0142]

[0013] In another embodiment of this aspect and all other aspects provided herein, the metabolic maturity of in vitro differentiated human cardiomyocytes derived from pluripotent stem cells is increased compared to a baseline level by: Increased activity of mitochondrial function, increased fatty acid metabolism, increased oxygen consumption rate (OCR), increased phosphorylated ACC level or activity, increased level or activity of fatty acid binding protein (FABP), increased level or activity of pyruvate dehydrogenase kinase-4 (PDK4), increased mitochondrial respiratory capacity, increased mitochondrial volume, and increased levels of mitochondrial DNA The markers are determined by one or more of the markers selected from the group consisting of:

[0143] Another aspect provided herein relates to a cell bank comprising the pluripotent stem cells described herein in any embodiment.

[0144] Another aspect provided herein relates to in vitro differentiated cardiomyocytes derived from the pluripotent stem cells described herein in any embodiment.

[0145] In another aspect, also provided herein are cells that comprise decreased expression of HCN4, CACNA1H, and SLC8A1 and increased expression of KCNJ2 compared to the starting material.

[0146] In one embodiment of this aspect and all other aspects provided herein, the starting material comprises primary cells collected from a donor.

[0147] In another embodiment of this aspect and all other aspects provided herein, decreased protein expression of HCN4, CACNA1H, and SLC8A1 comprises decreased protein expression and / or decreased gene expression for each of HCN4, CACNA1H, and SLC8A1.

[0148] In another embodiment of this aspect and all other aspects provided herein, increased expression of KCNJ2 includes increased protein expression and / or increased gene expression.

[0149] In another embodiment of this aspect and all other aspects provided herein, the cell comprises reduced expression of HCN4, CACNA1H, and SLC8A1, and expression of a transgene encoding KCNJ2 that is regulated by endogenous HCN4 regulatory sequences.

[0150] In another embodiment of this aspect and all other aspects provided herein, inhibition of HCN4, CACNA1H, and SLC8A1 comprises inhibition via contacting the cell with one or more inhibitor drugs and / or comprises genetic manipulation.

[0151] In another embodiment of this aspect and all other aspects provided herein, stimulating KCNJ2 activity comprises contacting the cells with one or more activating drugs and / or comprises genetic manipulation.

[0152] In another embodiment of this aspect and all other aspects provided herein, the reduced expression of HCN4, CACNA1H, or SLC8A1 is due to genetic manipulation.

[0153] In another embodiment of this aspect and all other aspects provided herein, one or more of the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated in the cell.

[0154] In another embodiment of this aspect and all other aspects provided herein, each of the genes encoding HCN4, CACNA1H, and SLC8A1 is inactivated in the cell.

[0155] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is completely inhibited and KCNJ2 activity is at least partially stimulated compared to control cells that are not manipulated by one or more inhibitor drugs and / or genetic manipulation.

[0156] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000%, or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, relative to control cells that are not manipulated by one or more inhibitor drugs and / or genetic manipulation.

[0157] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000%, or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, relative to control cells that are not genetically engineered.

[0158] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is completely inhibited by genetic engineering, and KCNJ2 activity is at least partially stimulated, relative to control cells that are not genetically engineered.

[0159] In another embodiment of this aspect and all other aspects provided herein, the control cells are wild-type cardiomyocytes, primary cardiomyocytes, in vitro differentiated cardiomyocytes derived from PSCs or ESCs, or starting material.

[0160] In another embodiment of this aspect and all other aspects provided herein, the control cell is an in vitro differentiated cardiomyocyte derived from a PSC or an ESC, and the control cell, PSC, ESC, comprises one, two, or three, but not all four, edits, and HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited, and KCNJ2 activity is at least partially stimulated.

[0161] In another embodiment of this aspect and all other aspects provided herein, HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% or 1-fold and KCNJ2 activity is stimulated by at least 10% or 1-fold by genetic engineering compared to control cells that are not genetically engineered.

[0162] In another embodiment of this aspect and all other aspects provided herein, the method of genetic manipulation is gene knockdown, and optionally, the gene knockdown is by RNA silencing or RNAi, optionally selected from the group consisting of siRNA, piRNA, shRNA, and miRNA.

[0163] In another embodiment of this aspect and all other aspects provided herein, the method of genetic engineering is gene knockout, and optionally the gene knockout is by inducing an insertion or deletion in the gene using a gene editing system, wherein the gene editing system is optionally selected from the group consisting of a ZFN, a TALEN, a meganuclease, a transposase, a CRISPR / Cas system, a nickase system, a base editing system, a prime editing system, and a gene writing system.

[0164] In another embodiment of this aspect and all other aspects provided herein, a gene knockout of HCN4, CACNA1H, or SLC8A1 is introduced into one or more alleles of HCN4, CACNA1H, or SLC8A1.

[0165] In another embodiment of this aspect and all other aspects provided herein, the gene knockout of HCN4, CACNA1H, or SLC8A1 is introduced into both alleles of HCN4, CACNA1H, or SLC8A1.

[0166] In another embodiment of this aspect and all other aspects provided herein, the gene knockout of HCN4, CACNA1H, or SLC8A1 results in reduced protein expression and / or reduced gene expression of HCN4, CACNA1H, or SLC8A1.

[0167] In another embodiment of this aspect and all other aspects provided herein, gene inactivation comprises an insertion or deletion of a nucleic acid sequence at the locus encoding HCN4, CACNA1H, and / or SLC8A1.

[0168] In another embodiment of this aspect and all other aspects provided herein, gene inactivation is effected via RNA-guided nuclease, TALEN, or zinc finger nuclease activity.

[0169] In another embodiment of this aspect and all other aspects provided herein, the RNA-guided nuclease comprises a Cas nuclease.

[0170] In another embodiment of this aspect and all other aspects provided herein, gene inactivation or gene knockout is achieved via RNAi, antisense, or RNA-targeted Cas nucleases.

[0171] In another embodiment of this aspect and all other aspects provided herein, gene inactivation or gene knockout is performed using the CRISPR / Cas system.

[0172] In another embodiment of this aspect and all other aspects provided herein, the cell further comprises at least one exogenous nucleic acid sequence.

[0173] In another embodiment of this aspect and all other aspects provided herein, the cell expresses a polypeptide from at least one exogenous nucleic acid sequence.

[0174] In another embodiment of this aspect and all other aspects provided herein, the cell further comprises reduced expression of at least one additional gene.

[0175] In another embodiment of this aspect and all other aspects provided herein, KCNJ2 is overexpressed from a transgene.

[0176] In another embodiment of this aspect and all other aspects provided herein, the transgene comprises an exogenous KCNJ2 coding sequence driven by an HCN4 or CACNA1H expression regulatory sequence.

[0177] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence replaces the coding sequence for HCN4 or CACNA1H such that the replaced HCN4 or CACNA1H coding sequence is not expressed and the replaced KCNJ2 is expressed under the regulation of the endogenous HCN4 or CACNA1H regulatory sequence.

[0178] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence is replaced using a CRISPR / Cas system.

[0179] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of HCN4 with the KCNJ2 coding sequence.

[0180] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of CACNA1H with the KCNJ2 coding sequence.

[0181] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated and the KCNJ2 polypeptide is overexpressed.

[0182] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SCL8A1 contain an indel in at least one allele.

[0183] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SCL8A1 contain indels in two alleles.

[0184] In another embodiment of this aspect and all other aspects provided herein, the cells are HCN4 インデル / インデル , CACNA1H インデル / インデル , and SCL8A1 インデル / インデル It is a cell.

[0185] In another embodiment of this aspect and all other aspects provided herein, the indels are generated using a CRISPR / Cas system.

[0186] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene and is operably linked to an endogenous HCN4 or CACNA1H regulatory sequence.

[0187] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated using the CRISPR / Cas system, and the KCNJ2 polypeptide is overexpressed under the regulation of endogenous HCN4 regulatory sequences.

[0188] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene operably linked to an endogenous HCN4 regulatory sequence.

[0189] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is overexpressed under the regulation of endogenous HCN4 regulatory sequences at the HCN4 locus.

[0190] In another embodiment of this aspect and all other aspects provided herein, the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated using the CRISPR / Cas system, and the KCNJ2 polypeptide is overexpressed under the regulation of endogenous CACNA1H regulatory sequences.

[0191] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is encoded by a transgene operably linked to an endogenous CACNA1H regulatory sequence.

[0192] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 polypeptide is overexpressed under the regulation of endogenous CACNA1H regulatory sequences at the CACNA1H locus.

[0193] In another embodiment of this aspect and all other aspects provided herein, the activity of at least one of HCN4, CACNA1H, SLC8A1, and KCNJ2 is manipulated by contacting the cell with a drug, and the activity of at least one of HCN4, CACNA1H, SLC8A1, and KCNJ2 is genetically manipulated.

[0194] In another embodiment of this aspect and all other aspects provided herein, the cells are differentiated in vitro from pluripotent stem cells.

[0195] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0196] In another embodiment of this aspect and all other aspects provided herein, the cells are differentiated in vitro from iPSCs derived from the subject into which the in vitro differentiated human cardiomyocytes are transplanted.

[0197] In another embodiment of this aspect and all other aspects provided herein, the cells are differentiated in vitro from iPSCs derived from a healthy subject.

[0198] In another embodiment of this aspect and all other aspects provided herein, the cells are differentiated in vitro from a starting material.

[0199] In another embodiment of this aspect and all other aspects provided herein, the starting material includes primary cells collected from a donor.

[0200] In another embodiment of this aspect and all other aspects provided herein, each of the healthy subject, starting material, and / or donor is not derived from the same individual as the subject into whom the cells are to be transplanted.

[0201] In another embodiment of this aspect and all other aspects provided herein, the primary cells collected from the donor are stem cells.

[0202] In another embodiment of this aspect and all other aspects provided herein, the stem cells are ESCs.

[0203] In another embodiment of this aspect and all other aspects provided herein, the starting material is a stem cell line.

[0204] In another embodiment of this aspect and all other aspects provided herein, the stem cell line is an ESC line or an iPSC line.

[0205] In another embodiment of this aspect and all other aspects provided herein, the stem cell line is an iPSC line.

[0206] In another embodiment of this aspect and all other aspects provided herein, when administered to cardiac tissue of a subject in need thereof, the cells promote reduced arrhythmias compared to a subject administered cells that do not include inhibition of HCN4, CACNA1H, and SLC8A1 activity and stimulation of KCNJ2 activity.

[0207] In another embodiment of this aspect and all other aspects provided herein, the cells are admixed with a cryopreservative.

[0208] In another embodiment of this aspect and all other aspects provided herein, the cells are frozen in admixture with a cryopreservative.

[0209] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated human cardiomyocytes derived from pluripotent stem cells express one or more markers selected from the group consisting of NKX2-5, MYH6, MYL7, TBX5, ATP2a2, RYR2, and cTnT.

[0210] In another embodiment of this aspect and all other aspects provided herein, the cellular activity and maturity of in vitro differentiated human cardiomyocytes derived from pluripotent stem cells can be determined by a numerical parameter selected from the group consisting of electrical maturity, metabolic maturity, and contractile maturity.

[0211]

[0013] In another embodiment of this aspect and all other aspects provided herein, the metabolic maturity of in vitro differentiated human cardiomyocytes derived from the cells is increased, compared to a baseline level, by: Increased activity of mitochondrial function, increased fatty acid metabolism, increased oxygen consumption rate (OCR), increased phosphorylated ACC level or activity, increased level or activity of fatty acid binding protein (FABP), increased level or activity of pyruvate dehydrogenase kinase-4 (PDK4), increased mitochondrial respiratory capacity, increased mitochondrial volume, and increased levels of mitochondrial DNA The markers are determined by one or more of the markers selected from the group consisting of:

[0212] Another aspect provided herein relates to a cell bank comprising the cells described herein in any embodiment.

[0213] In another aspect, also provided herein are in vitro differentiated cardiomyocytes derived from the cells described herein in any embodiment.

[0214] Another aspect provided herein relates to in vitro differentiated cardiomyocytes derived from the starting material cells described herein in any embodiment.

[0215] Another aspect provided herein relates to a pharmaceutical composition comprising the in vitro differentiated human cardiomyocytes described herein and a pharmaceutically acceptable carrier.

[0216] In one embodiment of this aspect and all other aspects provided herein, the pharmaceutical composition comprises an extracellular matrix or scaffold composition.

[0217] In another embodiment of this aspect and all other aspects provided herein, the pharmaceutical composition further comprises at least one additional cell type.

[0218] Another aspect provided herein relates to a transplant composition comprising any one of the cells described herein or in vitro differentiated human cardiomyocytes derived from any one of the cells described herein or a pharmaceutical composition described herein.

[0219] Another aspect provided herein relates to a cardiac delivery device or system comprising a pharmaceutical or implant composition described herein.

[0220] In one embodiment of this aspect and all other aspects provided herein, the cardiac delivery device comprises a syringe containing the pharmaceutical or implant composition.

[0221] In another embodiment of this aspect and all other aspects provided herein, the cardiac delivery device comprises a needle with a lumen sufficient for passage of the pharmaceutical or implant composition.

[0222] In another embodiment of this aspect and all other aspects provided herein, the needle is in fluid communication with the syringe.

[0223] In another embodiment of this aspect and all other aspects provided herein, the cardiac delivery device further comprises a cardiac catheter.

[0224] Another aspect provided herein relates to a method of preparing a pharmaceutical composition, the method comprising inhibiting the activity of HCN4, CACNA1H, and SLC8A1 and stimulating the activity of KCNJ2 in an isolated population of cardiomyocytes.

[0225] Another aspect provided herein relates to a method of preparing a pharmaceutical composition, the method comprising inhibiting the activity of HCN4, CACNA1H, and SLC8A1 and stimulating the activity of KCNJ2 in a population of PSCs, and differentiating the population of PSCs into cardiomyocytes in vitro.

[0226] In one embodiment of this aspect and all other aspects provided herein, the PSC is modified according to any embodiment described herein.

[0227] In another embodiment of this aspect and all other aspects provided herein, the method further comprises admixing the population of cardiomyocytes with a pharmaceutically acceptable carrier.

[0228] In another embodiment of this aspect and all other aspects provided herein, one or more of HCN4, CACNA1H, and SCL8A1 are inhibited by contacting the cardiomyocytes with one or more inhibitor drugs and / or by genetic manipulation.

[0229] In another embodiment of this aspect and all other aspects provided herein, KCNJ2 is stimulated by contacting the cardiomyocytes with one or more activating drugs and / or by genetic manipulation.

[0230] In another embodiment of this aspect and all other aspects provided herein, one or more of the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated.

[0231] In another embodiment of this aspect and all other aspects provided herein, gene inactivation comprises an insertion or deletion of a nucleic acid sequence at the locus encoding HCN4, CACNA1H, and / or SLC8A1.

[0232] In another embodiment of this aspect and all other aspects provided herein, gene inactivation is effected via RNA-guided nuclease, TALEN, or zinc finger nuclease activity.

[0233] In another embodiment of this aspect and all other aspects provided herein, the RNA-guided nuclease comprises a Cas nuclease.

[0234] In another embodiment of this aspect and all other aspects provided herein, gene inactivation is achieved via RNAi, antisense, or RNA-targeted Cas nucleases.

[0235] Another aspect provided herein relates to a method of transplanting in vitro differentiated cardiomyocytes, the method comprising contacting the in vitro differentiated cardiomyocytes of or derived from any embodiment described herein, a pharmaceutical composition described herein, a transplant composition described herein, or a cardiac delivery device or system described herein with cardiac tissue of a subject in need thereof.

[0236] Another aspect provided herein relates to a method of transplanting in vitro differentiated cardiomyocytes, comprising delivering any of the cells described herein or in vitro differentiated cardiomyocytes derived from any of the cells described herein, a pharmaceutical composition described herein, a transplant composition described herein, or a cardiac delivery device or system described herein to cardiac tissue of a subject in need thereof.

[0237] In another embodiment of this aspect and all other aspects provided herein, transplanting results in reduced transplant arrhythmias compared to transplantation of in vitro differentiated cardiomyocytes lacking inhibition of HCN4, CACNA1H, and SLC8A1 and lacking stimulation of KCNJ2.

[0238] Another aspect provided herein relates to a method of treating a disease or disorder involving damage or dysfunction of cardiac tissue in a subject in need thereof, comprising contacting cardiac tissue of the subject with a cell described herein, a pharmaceutical composition described herein, a transplant composition described herein, or a cardiac delivery device or system described herein.

[0239] Another aspect provided herein relates to a method of treating a disease or disorder involving damage or dysfunction of cardiac tissue in a subject in need thereof, comprising delivering to cardiac tissue of a subject in need thereof any of the cells described herein or in vitro differentiated cardiomyocytes derived from any of the cells described herein, a pharmaceutical composition described herein, a transplant composition described herein, or a cardiac delivery device or system described herein.

[0240] In one embodiment of this aspect and all other aspects provided herein, the contacting or delivering results in reduced transplant arrhythmias compared to transplantation of in vitro differentiated cardiomyocytes lacking inhibition of HCN4, CACNA1H, and SLC8A1 and lacking stimulation of KCNJ2.

[0241] In another embodiment of this aspect and all other aspects provided herein, the method further comprises administering amiodarone and ivabradine to the subject.

[0242] Another aspect provided herein relates to a composition comprising an inhibitor of two or more of HCN4, CACNA1H, and SLC8A1.

[0243] In another embodiment of this aspect and all other aspects provided herein, the composition is admixed with a population of in vitro differentiated cardiomyocytes.

[0244] In another embodiment of this aspect and all other aspects provided herein, the composition further comprises an activator for KCNJ2.

[0245] In another embodiment of this aspect and all other aspects provided herein, the composition comprises an inhibitor for each of HCN4, CACNA1H, and SLC8A1.

[0246] In another embodiment of this aspect and all other aspects provided herein, the composition comprises an inhibitor for each of HCN4, CACNA1H, and SLC8A1 and an activator for KCNJ2.

[0247] Another aspect provided herein relates to isolated human cardiomyocytes in which expression of the HCN4, CACNA1H, and SLC8A1 genes is partially or completely inactivated by deleterious mutations or by insertions, and expression of the KCNJ2 gene is at least partially increased.

[0248] In another embodiment of this aspect and all other aspects provided herein, the inactivation comprises an insertion or deletion of a nucleic acid sequence at the locus encoding HCN4, CACNA1H, and / or SLC8A1.

[0249] In another embodiment of this aspect and all other aspects provided herein, the inactivation is effected via RNA-guided nuclease, RNAi, antisense, TALEN, or zinc finger nuclease activity.

[0250] In another embodiment of this aspect and all other aspects provided herein, the RNA-guided nuclease comprises a Cas nuclease.

[0251] In another embodiment of this aspect and all other aspects provided herein, the isolated human cardiomyocyte further comprises at least one exogenous nucleic acid sequence.

[0252] In another embodiment of this aspect and all other aspects provided herein, the polypeptide is expressed from at least one exogenous nucleic acid sequence.

[0253] In another embodiment of this aspect and all other aspects provided herein, the isolated human cardiomyocyte further comprises reduced expression of at least one additional gene.

[0254] In another embodiment of this aspect and all other aspects provided herein, KCNJ2 is overexpressed from a transgene.

[0255] In another embodiment of this aspect and all other aspects provided herein, the transgene comprises an exogenous KCNJ2 coding sequence driven by an HCN4 or CACNA1H expression regulatory sequence.

[0256] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence replaces the coding sequence for HCN4 or CACNA1H such that the replaced HCN4 or CACNA1H coding sequence is not expressed and KCNJ2 is expressed under the regulation of the endogenous HCN4 or CACNA1H regulatory sequence.

[0257] In another embodiment of this aspect and all other aspects provided herein, the KCNJ2 coding sequence is replaced using a CRISPR / Cas system.

[0258] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of HCN4 with the KCNJ2 coding sequence.

[0259] In another embodiment of this aspect and all other aspects provided herein, the CRISPR / Cas system is used to replace the coding sequence of CACNA1H with the KCNJ2 coding sequence.

[0260] In another embodiment of this aspect and all other aspects provided herein, the cardiomyocytes are differentiated in vitro from pluripotent stem cells.

[0261] In another embodiment of this aspect and all other aspects provided herein, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0262] In another embodiment of this aspect and all other aspects provided herein, the cardiomyocytes are differentiated in vitro from iPSCs derived from a first subject that is different from a second subject into which the in vitro differentiated human cardiomyocytes are transplanted.

[0263] In another embodiment of this aspect and all other aspects provided herein, when administered to cardiac tissue of a subject in need thereof, the isolated human cardiomyocytes promote reduced arrhythmias compared to a subject administered isolated human cardiomyocytes that do not comprise partial or complete inactivation of HCN4, CACNA1H, and SLC8A1 gene expression and at least partially increased expression of the KCNJ2 gene.

[0264] Another aspect provided herein relates to a composition for use in treating a disease or disorder involving cardiac tissue damage or dysfunction in a subject, the composition comprising a cell described herein or an in vitro differentiated cardiomyocyte derived from a cell described herein, a pharmaceutical composition described herein, a transplant composition described herein, or a cardiac delivery device or system of any embodiment described herein, for delivery to cardiac tissue of a subject in need thereof.

[0265] In another embodiment of this aspect and all other aspects provided herein, delivery of the composition results in reduced transplant arrhythmias compared to transplantation of in vitro differentiated cardiomyocytes lacking inhibition of HCN4, CACNA1H, and SLC8A1 and lacking stimulation of KCNJ2.

[0266] In another aspect, also provided herein is a composition comprising an inhibitor of two or more of HCN4, CACNA1H, and SLC8A1 for use in a method of treating or preventing cardiac transplant arrhythmia in a subject.

[0267] In another embodiment of this aspect and all other aspects provided herein, the composition is admixed with a population of in vitro differentiated cardiomyocytes.

[0268] In another embodiment of this aspect and all other aspects provided herein, the composition further comprises an activator for KCNJ2.

[0269] In another embodiment of this aspect and all other aspects provided herein, the composition comprises an inhibitor for each of HCN4, CACNA1H, and SLC8A1.

[0270] In another embodiment of this aspect and all other aspects provided herein, the composition comprises an inhibitor for each of HCN4, CACNA1H, and SLC8A1 and an activator for KCNJ2. [Brief explanation of the drawings]

[0271] [Figure 1A]Figures 1A-1C demonstrate the expression of ion channels involved in hPSC-CM automaticity. (Figure 1A) Action potential traces of hPSC-CM and adult CM with relative phase numbers. The dotted line indicates phase 4 only for adult CM from the Purkinje system. (Figure 1B) RNA-seq analysis of ion channel gene expression during in vivo transplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) maturation in a rat heart. The solid line represents the average of two independent experiments. The red-shaded area represents the onset of transplant arrhythmia (EA). (Figure 1C) Gene editing manipulation of human embryonic stem cell-derived cardiomyocyte (hESC-CM) automaticity. KO, knockout; OE, overexpression. Each round of gene editing is depicted in a different color. Symbols indicate cell lines tested in vivo that either caused EA (red star) or were well-integrated with sinus rhythm (green circle). The absence of a symbol indicates cells that have not yet been tested in vivo. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A] Figures 2A-2B. MEDUSA-CM in vitro electrical activity. MEDUSA stands for "Modulation of Electrophysiological DNA to Understand and Suppress Arrhythmias." Here, cells contain a quadruple genome edit involving knockout of HCN4 / SLC8A1 / CACNA1H and overexpression of KCNJ2 from the endogenous HCN4 locus. Pluripotent MEDUSA cells (prior to differentiation into CM) were generated from the RUES2 hESC parental cell line. (Figure 2A) Onset of automaticity during cardiac differentiation: Left panel, quantification of beat rate in beats / min; right panel, percentage of beating wells out of a total of 12 wells per cell line. Data are shown as the average of one batch of differentiation. ES = embryonic state, MS = mesoderm state, CP = cardiac progenitor cell, CM = cardiomyocyte state. (Figure 2B) Representative MEA analysis of MEDUSA CM. MEDUSA CM frequencies are shown as the mean ± SEM of 8 wells / cell line. [Figure 2B] See legend to Figure 2A. [Figure 3A-1] Figures 3A-3C. Transplantation of gene-edited hESC-CM and EA burden in pigs. (Figures 3A, 3B) Heart rate (right panel) and arrhythmia burden (left panel) of recipients of different gene-edited hESC-CM (Figure 3A) and MEDUSA-CM (Figure 3B) compared to controls. Data are shown as mean ± SEM or mean only. The number of recipients is indicated within the graph. (Figure 3C) MEDUSA-CM grafts detected by slow skeletal troponin I staining. Scale bar = 200 µm. [Figure 3A-2] See the description of Figure 3A-1. [Figure 3A-3] See the description of Figure 3A-1. [Figure 3B] See the description of Figure 3A-1. [Figure 3C] See the description of Figure 3A-1. [Figure 4A] Figures 4A-4C. Electrical pacing studies of HCN4 / CACNA1H / SLC8A1-3KO+KCNJ2-OE MEDUSA-CMs. (Figure 4A) Calcium imaging studies demonstrating that MEDUSA cardiomyocytes exhibit calcium transients in response to electrical field stimulation identical to wild-type cardiomyocytes. (Figure 4B) Calcium imaging kymograms show identical calcium transients in unedited hESC-CMs or another source of wild-type CMs and MEDUSA-CMs. (Figure 4C) Quantitative analysis demonstrates similar frequency responses to electrical field stimulation in MEDUSA-CMs and wild-type cells. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A]Figures 5A-5F. Gene expression analysis of hiPSC-CMs during in vivo transplantation compared to 2D culture. (Figure 5A) Representative action potential traces from hESC-CMs compared to adult CMs. (Figure 5B) Experimental layout for an RNA-seq experiment using hiPSC-CMs. (Figure 5C) Representative histological analysis of a rat heart transplanted with hiPSC-CMs at 84 days post-injection, before and after laser capture microdissection (LCM). H&E = hematoxylin and eosin staining. Scale bar = 200 µM for a, d, 50 µM for b, c. (Figure 5D) Principal component analysis (PCA) of the RNA-seq dataset described in Figure 5B. The percentage of gene expression variance represented by each PCA is shown. (Figure 5E) Heatmap of maturation-related genes in hiPSC-CMs. (Figure 5F) Time course analysis of ion channel genes during in vivo transplantation of hiPSC-CMs. Shading indicates the approximate window of transplantation arrhythmias. [Figure 5B] See the legend to Figure 5A. [Figure 5C] See the legend to Figure 5A. [Figure 5D] See the legend to Figure 5A. [Figure 5E] See the legend to Figure 5A. [Figure 5F] See the legend to Figure 5A. [Figure 6A]Figures 6A-6F. Pharmacological inhibition of RUES2 hESC-CM wild-type motility in vitro. (Figures 6A, 6C, 6E) Dose-response curves for ivabradine (If, HCN4 inhibitor, Figure 6A), ML-218 (ICaT inhibitor, Figure 6C), and SEA0400 / KB-R7943 (INCX1 inhibitor, Figure 6E) in the MEA system. Data are shown as the mean ± SEM of two independent experiments with six technical replicates (wells) each, normalized to the experimental baseline, and expressed as % of the DMSO control. The line represents a nonlinear regression of the normalized response Y = 100 / (1 + 10^((LogIC50-X) * Hill slope)). (Figures 6B, 6D, 6F) Representative MEA recordings from RUES2 hESC-CM wild-type treated with ivabradine (Figure 6B), ML-218 (Figure 6D), and SEA0400 / KB-R7943 (Figure 6F) compared to the corresponding DMSO-treated controls of the experiment. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 7A]Figures 7A-7E. Cleavage of HCN4 and CACNA1H is not sufficient to disrupt hESC-CM motility. (Figure 7A) Experimental layout for the generation of gene-edited cell lines, cardiac differentiation, and in vitro / in vivo characterization. SpCas9 = Streptococcus pyogenes Cas9, sgRNA = single guide RNA. (Figure 7B) Patch clamp analysis of funny currents (If) from HCN4 knockout and RUES2 hESC-CM wild-type, demonstrating the lack of hyperpolarization-induced If after KO. A representative current trace from clone 1 is shown. See Figure 14C for quantification. (Figures 7C, 7D) Spontaneous activity and spike amplitude of gene-edited RUES2 hESC-CMs in the MEA system. Data are shown as the mean ± SEM of two to three independent experiments with eight technical replicates each and normalized to the RUES2 hESC-CM wild-type frequency. Statistical differences are reported compared to RUES2 hESC-CM wild-type by two-way ANOVA with Sidak correction for multiple comparisons (*p<0.05, **p<0.01, and ***p<0.001). (Figure 7E) Quantification of transplant arrhythmia burden (% of time on each day) and heart rate after HCN4 KO RUES2 hESC-CM transplantation compared to RUES2 hESC-CM wild-type. Data are shown as mean ± SEM of N=3 for RUES2 hESC-CM wild-type and N=2 for HCN4 KO. Black symbols represent euthanized animals. The right panel shows a representative ECG trace from an animal receiving HCN4 KO during EA. [Figure 7B] See the legend to Figure 7A. [Figure 7C] See the legend to Figure 7A. [Figure 7D] See the legend to Figure 7A. [Figure 7E] See the legend to Figure 7A. [Figure 8A]Figures 8A-8E. Perturbation of HCN4 and KCNJ2 is not sufficient to disrupt hESC-CM motility. (Figure 8A) Gene editing approach to knock in KCNJ2 under the transcriptional regulation of the HCN4 promoter in RUES2 hESCs. Genotyping PCR strategies for on-target and off-target insertions are shown. See Supplemental Figure 5A. (Figure 8B) Time course analysis of HCN4, KCNJ2, and TNNT2 expression during cardiac differentiation of RUES2 hESC-CM wild-type and gene-edited hESCs. (Figure 8C) Representative quantification of spontaneous beating during hESC-CM differentiation from HCN4 KO / KCNJ2 KI clones compared to wild-type. (Figure 8D) Spontaneous activity and representative traces of HCN4 KO / KCNJ2 KI clones quantified by MEA. Given the pronounced irregularity of automaticity in these strains, data are reported as the average beat rate (left panel) and corresponding % beat irregularity (right panel) for a 5-minute recording. % beat irregularity is calculated as the standard deviation of the beat interval recording at 100 seconds divided by the average beat interval over that same period. Data are plotted as the mean ± SEM of three independent experiments, each with eight technical replicates. (Figure 8E) In vivo data showing EA load, left panel, and heart rate of animals transplanted with RUES2 hESC-CM compared to wild-type RUES2 hESC-CM. On the right is a representative ECG trace. Data are presented as described in Figure 7E. [Figure 8B] See the legend to Figure 8A. [Figure 8C] See the legend to Figure 8A. [Figure 8D] See the legend to Figure 8A. [Figure 8E] See the legend to Figure 8A. [Figure 9A]Figures 9A-9I. The triple gene editing combination reduces the motility of RUES2 hESC-CM but does not prevent EA. (Figure 9A) RT-qPCR gene expression analysis of HCN, T-type ion channel genes, and KCNJ2 in HCN4 / CACNA1H 2KO / KCNJ2 KI compared to RUES2 hESC-CM wild-type at day 14 of differentiation. Data are shown as the mean ± SEM of three independent experiments normalized to WT. Differences from WT (***p<0.001) by two-way ANOVA with Sidak correction for multiple comparisons. (Figure 9B) Representative beating initiation during cardiac differentiation in HCN4 / CACNA1H 2KO / KCNJ2 KI CM. (Figure 9C) MEA analysis of HCN4 / CACNA1H 2KO / KCNJ2 KI clones compared to RUES2 hESC-CM wild-type. Data are shown as the mean beats / min ± SEM recorded at 5 min from two independent experiments with eight replicates each. See also Figure 15F. (Figure 9D) Arrhythmia burden and heart rate of one pig transplanted with HCN4 / CACNA1H 2KO / KCNJ2 KI hESC-CM compared to RUES2 hESC-CM wild-type (n=3), as well as a representative EKG trace during EA. Data are presented as described in Figures 3E and 3F. (Figure 9E) Western blot of NCX1 KO clone compared to RUES2 hESC-CM wild-type. cTnT = cardiac troponin T. (Figure 9F) Representative time course analysis of the initiation of beating during cardiac differentiation of SLC8A1 KO clone compared to WT. (Figure 9G) Representative field potential traces and quantification of beating behavior of SLC8A1 KO clone at different time points of a 2-week culture on MEA plates. Data are shown as the mean beats / min ± SEM recorded over 5 min from three independent experiments (N = 3, n = 4). (Figure 9H) EA load and heart rate of pigs (n = 3) transplanted with HCN4 / NCX1 2KO / KCNJ2 KI hESC-CM compared to WT (n = 3). Data are shown as the mean ± SEM for individual animals from WT and HCN4 / SLC8A1 2KO_KCNJ2 KI.(Figure 9I) Representative histological analysis of HCN4 / NCX1 2KO / KCNJ2 KI grafts 4 weeks after injection. Scale bar = 200 μm. [Figure 9B] See the legend to Figure 9A. [Figure 9C] See the legend to Figure 9A. [Figure 9D] See the legend to Figure 9A. [Figure 9E] See the legend to Figure 9A. [Figure 9F] See the legend to Figure 9A. [Figure 9G] See the legend to Figure 9A. [Figure 9H] See the legend to Figure 9A. [Figure 9I] See the legend to Figure 9A. [Figure 10A] Figures 10A-10C. In vitro characterization of MEDUSA-CM. (Figure 10A) qRT-PCR of genes responsible for If, ICaT, and INCX in MEDUSA-CM compared to hESC-CM wild-type controls. Data are shown as the mean ± SEM of two independent experiments. Statistical differences are reported compared to hESC-CM wild-type controls by two-way ANOVA with Sidak correction for multiple comparisons (*p<0.05 and **p<0.01). (Figure 10B) Beat onset and beat rate during cardiac differentiation of MEDUSA-CM. Data are shown as the mean ± SEM of two independent batches of differentiation. (Figure 10C) Representative calcium transient analysis of single-cell MEDUSA-CM compared to hESC-CM wild-type controls using Fluo-4. Data are shown as the mean ± SEM of 12 single cells for both hESC-CM wild-type control cells and MEDUSA-CM. [Figure 10B] See the legend to Figure 10A. [Figure 10C] See the legend to Figure 10A. [Figure 11A]Figures 11A-11F. In vivo characterization of MEDUSA-CM. (Figure 11A) Representative electrocardiogram traces from Yucatan minipigs injected with hESC-CM wild-type control cells during EA and from Yucatan minipigs injected with MEDUSA-CM in normal sinus rhythm at the same time points after transplantation. (Figure 11B) EA load and heart rate of MEDUSA-CM after in vivo transplantation. Data are shown as described in Figure 6E. (Figure 11C) Representative image of a MEDUSA-CM graft 4 weeks after injection. Scale bar = 200 μM. (Figure 11D) Immunofluorescence image of MLC2v / MLC2a and ssTnI / cTnI in a MEDUSA-CM graft. Scale bar = 10 μM. See also Figure 17F. (Figures 11E, 11F) Representative immunofluorescence images of a MEDUSA-CM graft integrated with the host. Scale bar = 10 μM. [Figure 11B] See the legend to Figure 11A. [Figure 11C] See the legend to Figure 11A. [Figure 11D] See the legend to Figure 11A. [Figure 11E] See the legend to Figure 11A. [Figure 11F] See the legend to Figure 11A. [Figure 12A] Figures 12A-12D. RNAseq analysis of in vivo transplanted hiPSC-CMs. (Figure 12A) Percentage of human / rat reads in in vivo samples after LCM. (Figure 12B) GO term analysis of selected up- and down-regulated pathways at 3 months after hiPSC-CM transplantation. See also Tables 4A-4B. Expression kinetics of (Figure 12C) HCN and (Figure 12D) T-type ion channel isoforms during in vivo maturation of hiPSC-CMs. [Figure 12B] See the legend to Figure 12A. [Figure 12C] See the legend to Figure 12A. [Figure 12D] See the legend to Figure 12A. [Figure 13]Figures 13A-13C. Pharmacological inhibition of in vitro motility of RUES2 hESC-CM wild-type control cells. (Figures 13A, 13B, 13C) Dose-response curves for mibefradil (ICaT, Figure 13A), zacopride (IK1 agonist, Figure 13B), and verapamil (L-type calcium channel inhibitor, Figure 13C) in the MEA system. Data are shown as the mean ± SEM of two independent experiments, normalized to baseline, and expressed as % of DMSO control (N = 2, n = 6). The line represents a nonlinear regression of the normalized response Y = 100 / (1 + 10^((LogIC50-X) * Hill slope)). [Figure 14A] Figures 14A-14E. Characterization of gene-edited cell lines targeting phase 4 of the action potential. (Figure 14A) Gene editing approaches for the generation of different cell lines are described in the MEDUSA-CM project. (Figure 14B) Sanger sequencing of HCN4 KO clones (HCN4 KO cl.1 = -1 bp homozygous deletion; HCN4 KO cl.2 = -5 bp homozygous deletion), CACNA1H KO clones (CACNA1H KO cl.1 = -20 bp deletion, CACNA1H KO cl.2 = 2 bp heterozygous insertion on different alleles), and double-edited cells (HCN4 / CACNA1H KO cl.1 = +1 bp homozygous insertion, HCN4 / CACNA1H KO cl.2 = -1 bp homozygous deletion). (Figure 14C) Voltage / current plots of If in hESC-CM wild-type control cells and HCN4 KO hESC-CM. Data are shown as mean ± standard error for n = 3 cells (WT), n = 6 for HCN4 KO cl.1, and n = 4 for HCN4 KO cl.2. (Figures 14D, 14E) Gene expression analysis by RTqPCR of HCN and T-type isoforms in HCN4 KO clones (Figure 14D) and CACNA1H KO and HCN / CACNA1H 2KO (Figure 14S) compared to hESC-CM wild-type control cells. Differences quantified by two-way ANOVA with Sidak correction for multiple comparisons (***p<0.001). [Figure 14B] See the legend to Figure 14A. [Figure 14C]See the legend to Figure 14A. [Figure 14D] See the legend to Figure 14A. [Figure 14E] See the legend to Figure 14A. [Figure 15A] Figures 15A-15F. Characterization of PIEZO1 KO cell lines. (Figure 15A) The most differentially regulated ion channel genes (left panel) in vivo compared with day 0 and day 7 and the corresponding expression kinetics after in vivo transplantation. (Figure 15B) PIEZO1 gene expression in vivo and in vitro at different time points. (Figure 15C) Sanger sequencing of the PIEZO1 KO hESC-RUES2 cell line. (PIEZO1 KO cl.1 homozygous deletion of 5 bp, PIEZO1 KO cl.2 heterozygous deletion of -5 bp and -1 bp on two alleles). (Figure 15D) Western blotting analysis of PIEZO1 KO CM compared with hESC-CM wild-type control cells. (Figure 15E) MEA analysis of spontaneous electrical activity of PIEZO1 KO CM. Data are shown as the mean ± SEM of two independent experiments (N = 2, n = 8) and the normalized beating rate to that of hESC-CM wild-type control cells (Figure 15F). EA load and heart rate of pigs transplanted with PIEZO1 KO CM and representative staining with β-myosin heavy chain-positive grafts. [Figure 15B] See the legend to Figure 15A. [Figure 15C] See the legend to Figure 15A. [Figure 15D] See the legend to Figure 15A. [Figure 15E] See the legend to Figure 15A. [Figure 15F] See the legend to Figure 15A. [Figure 16A]Figures 16A-16H. Genotyping and characterization of triple-edited cell lines. (Figure 16A) Genotyping of HCN4 KO / KCNJ2 KI clones generated by CRISPR / Cas9 homology-directed repair using the plasmid described in Figure 4A. (Figure 16B) Representative flow cytometry data of HCN4 KO / KCNJ2 KI clones at day 14 of differentiation stained for Nkx2.5 (ventricular CM marker) and cardiac troponin T (pan-CM marker). (Figure 16C) Genotyping of HCN4 / CACNA1H 2KO / KCNJ2 KI clones generated with the same vector shown in Figure 4A. See also Figure 15A for gene editing approaches. (Figure 16D) Representative flow cytometry analysis of HCN4 / CACNA1H 2KO / KCNJ2 KI CMs at day 14 of differentiation. (Figure 16E) Karyotype analysis of HCN4 / CACNA1H 2KO / KCNJ2 KI clones. (Figure 16F) Beat interval irregularity was quantified by dividing the standard deviation of the beat interval recording at 100 seconds by the mean beat interval over that same period. Data are shown as the mean ± standard error of two independent experiments (N=1 for clones gene-edited for lack of spontaneous activity). (Figure 16G) Genotype of SLC8A1 KO clones generated via three gRNA combinations. The left panel shows the Sanger sequencing alignment of SLC8A1 KO cl.1 and 2 on the SLC8A1 exon 2 sequence. (Figure 16H) Genotype of HCN4 / SLC8A1 2KO / KCNJ2 KI clones generated using the same approach described in Figure 8A. [Figure 16B] See the legend to Figure 16A. [Figure 16C] See the legend to Figure 16A. [Figure 16D] See the legend to Figure 16A. [Figure 16E] See the legend to Figure 16A. [Figure 16F] See the legend to Figure 16A. [Figure 16G] See the legend to Figure 16A. [Figure 16H] See the legend to Figure 16A. [Figure 17A] Figures 17A-17E. Characterization of MEDUSA cell lines. (Figure 17A) Karyotype analysis of MEDUSA hESCs. (Figure 17B) Representative flow cytometry analysis of MEDUSA hESCs and hESC-CM wild-type control cells stained with Oct3 / 4 and SSEA4 as pluripotency markers. (Figure 17C) Western blotting analysis of NCX1 in MEDUSA-CM. (Figure 17D) Representative flow cytometry analysis of differentiation markers during cardiac differentiation in hESC-CM wild-type control cells and MEDUSA hESCs from the same batch. (Figure 17E) Single channel images of MLC2v / 2a and ssTnI / cTnI MEDUSA explants. [Figure 17B] See the legend to Figure 17A. [Figure 17C] See the legend to Figure 17A. [Figure 17D] See the legend to Figure 17A. [Figure 17E] See the legend to Figure 17A. [Figure 18] Figure 18. ML-218 treatment of HCN4 KO hESC-CM. Data are shown as the mean ± SEM of spontaneous frequency recorded with the MEA system in two independent experiments. The solid line represents a four-parameter logistic curve (Y = lower limit + (upper limit - lower limit) / (1 + 10^((LogIC50 - X) * Hill slope)); variables are listed in the table below. In this experiment, hESCs were genetically modified to prevent HCN4 expression. HCN4 hESC KO clones and hESC wild-type control cells were then differentiated into cardiomyocytes in vitro (hESC-CM). Then, on day 14, hPSC-CM were treated with increasing doses of ML-218, which targets the T-type ion channel (Cav3.2) responsible for ICaT current. ML-218 effects were evaluated with the MEA system. DETAILED DESCRIPTION OF THE INVENTION

[0272] Detailed Description The compositions and methods described herein relate, in part, to the discovery of a series of ion channels whose function can be regulated to reduce or prevent arrhythmias resulting from the engraftment of cardiomyocytes, including, but not limited to, stem cell-derived cardiomyocytes. Thus, compositions and methods for the treatment and / or prevention of cardiac diseases that can benefit from cardiomyocyte transplantation are described herein. The following provides considerations for the preparation of cardiomyocyte transplant or graft preparations, their use in cardiomyocyte transplantation or engraftment, and additional approaches and considerations for reducing or eliminating arrhythmias that commonly result from such transplantation.

[0273] Exogenous cardiomyocytes, including but not limited to cardiomyocytes (CMs) derived from stem cells via in vitro differentiation, promote arrhythmias when administered to cardiac tissue for engraftment. Without wishing to be bound by theory, it is possible to consider that the introduced cells disrupt normal electrophysiological control of the heart by, in effect, behaving as an autonomous impulse-generating center distinct from the endogenous cardiac pacemaker mechanism or cellular homeostasis. In this scenario, reducing this autonomous impulse generation while maintaining the cardiomyocyte phenotype may allow the engrafted cells to integrate with cardiac tissue with less disruption of the endogenous electrophysiological program.

[0274] Specifically, cardiomyocytes prepared by in vitro differentiation from ESCs and iPS cells (iPSCs) promote transplant arrhythmias. Therefore, the approaches described herein are directly applicable to cardiomyocytes derived from either of these stem cell types. Where iPSCs can be derived from the subject to whom the iPSC-differentiated cells are administered—i.e., autologous stem cell—iPS approaches have the advantage of avoiding or minimizing the risk of immune graft rejection. While ESCs and iPSCs have clear advantages, it is also envisioned that cardiomyocytes generated from other exogenous or endogenous sources would also benefit from the electrophysiological manipulations described herein to avoid or limit transplant arrhythmias (e.g., insofar as newly generated cardiomyocytes generated without the transplantation of exogenous cells can also cause arrhythmias, the term "regenerative arrhythmia" can also be used). For example, cardiomyocytes prepared via transdifferentiation from another somatic cell type and expressing all or part of the set of electrophysiological genes described herein (e.g., HCN4, CACNA1H, and SLC8A1 expression, with or without low or no expression of KCNJ2) are also expected to benefit from the manipulations / approaches described herein. Indeed, it is particularly envisioned that endogenous cardiomyocytes that are arrhythmogenic by activating the cell cycle, or that dedifferentiate in response to injury, e.g., myocardial infarction, viral myocarditis, etc., such that this set of genes is expressed, could benefit from manipulation of the expression or activity of these genes or their encoded proteins. That is, the approaches described herein may also be applicable to treating, reducing, or limiting arrhythmias caused by processes or pathologies that do not involve in vitro differentiated cells.

[0275] Since it has been discovered that genetically knocking out the expression of ion channels encoded by HCN4, CACNA1H, and SLC8A1 and genetically enhancing the expression of ion channels encoded by KCNJ2 in transplanted cells can suppress automaticity and transplant arrhythmia, it is believed that different approaches, such as reducing the expression or activity of HCN4, CACNA1H, and SLC8A1 and increasing the expression or activity of KCNJ2 in cells for engraftment, can provide therapeutic benefits.Therefore, for example, any combination of genetic and / or pharmacological manipulation of target ion channels can suppress or prevent transplant arrhythmia. Thus, as a non-limiting example, pharmacological inhibition of HCN4 channels in combination with genetic knockout of CACNA1H and SLC8A1 and genetic enhancement of KCNJ2 expression in in vitro differentiated cardiomyocytes for engraftment can suppress transplant arrhythmias, and it is particularly envisioned that any other combination of HCN4, CACNA1H, and SLC81 inhibition with KCNJ2 activation may also be beneficial in suppressing transplant arrhythmias. Pharmacological agents useful for manipulating various channels, as well as gene knockdown, knockout, and knockin approaches as needed, and considerations for using each (e.g., appropriate timing and / or amount for knockout or overexpression) are further described herein below.

[0276] definition For convenience, the meanings of some terms and phrases used in this specification, examples and the appended claims are provided below.Unless otherwise specified or implied by the context, the following terms and phrases include the meanings provided below.The definitions are provided to help describe specific embodiments and are not intended to limit the claimed technology, since 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 those of ordinary skill in the art to which this technology belongs.If there is an obvious discrepancy between the usage of a term in this field and its definition provided herein, the definition provided herein shall prevail.

[0277] Definitions of common terms in cell biology and molecular biology 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 Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, published by Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, 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, NY, 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 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 all of which are incorporated herein by reference in their entireties.

[0278] As used herein, the term "cardiomyocyte" or "CM" refers to a cardiac muscle cell. Cardiomyocytes (CM) generally comprise phenotypic and / or structural characteristics associated with cardiac muscle (e.g., electrical phenotype, sarcomere, actin, myosin, and cardiac troponin T expression, etc.). Cardiomyocytes can be native cardiomyocytes isolated from an organism or cardiomyocytes differentiated from stem cells or cardiac progenitors (e.g., cardiomyocytes differentiated in vitro).

[0279] As used herein, the term "human stem cell" refers to a human cell that can self-renew and differentiate into at least one cell type. The term "human stem cell" encompasses human stem cell lines, human-derived induced pluripotent stem (iPS) cells (or iPSCs), human embryonic stem cells, human pluripotent cells, human multipotent stem cells, amniotic stem cells, placental stem cells, or human adult stem cells.

[0280] As used herein, "in vitro differentiated cardiomyocytes" refer to cardiomyocytes generated in culture, typically, but not necessarily, through stepwise differentiation from stem cells or progenitor cells, such as pluripotent stem cells. Examples of stem cell types include, but are not limited to, human embryonic stem cells, induced pluripotent stem cells, early mesoderm cells, lateral plate mesoderm cells, or cardiac progenitor cells. Thus, although in vivo cardiomyocytes ultimately originate from stem cells, i.e., during the development of a tissue or organism, the stem cell-derived cardiomyocytes described herein are produced by in vitro differentiation from stem cells. As used herein, cells differentiated in vitro from stem cells, e.g., induced pluripotent stem (iPS) cells or embryonic stem cells ("ES cells" or "ESCs"), are "stem cell-derived cardiomyocytes" or "in vitro differentiated cardiomyocytes" if they express cardiac troponin T (cTnT). Regardless of the differentiation approach used to generate cardiomyocytes, if electrophysiological disturbances of transplant arrhythmias are expected to occur, cardiac progenitor cells that have been genetically modified as described herein and are capable of in vitro differentiation into a cTnT-expressing cardiomyocyte phenotype are particularly contemplated. Methods for differentiating stem cells into cardiomyocytes in vitro are known in the art and described elsewhere herein.

[0281] The term "pluripotent" as used herein refers to cells that have the ability to differentiate into cell types characteristic of all three germ cell layers (endoderm, mesoderm, and ectoderm) under different conditions. Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, but the preferred test for pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. In some embodiments, pluripotent stem cells are produced or generated from cells that are not pluripotent, and are referred to herein as "induced pluripotent stem cells."

[0282] As used herein, the terms "induced pluripotent stem cells," "iPS cells," "iPSCs," "hiPSCs," and "human induced pluripotent stem cells" are used interchangeably herein and refer to pluripotent cells artificially derived from parent cells, such as differentiated somatic cells. iPSCs are capable of self-renewal and differentiation into committed stem cells, including various types of mature cells, as well as cells of the cardiac lineage. Examples of parent cells useful for generating induced pluripotent stem cells include, but are not limited to, somatic cells such as fibroblasts, cardiac progenitor cells, and skeletal muscle cells. Such "iPS" or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the derivation of iPSCs are known in the art and are further described below (see, e.g., Zhou et al., Stem Cells, 27(11):2667-74(2009); Huangfu et al., Nature Biotechnol. 26(7):795(2008); Woltjen et al., Nature 458(7239):766-770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009); each of which is incorporated herein by reference in its entirety.) As used herein, "hiPSCs" are human induced pluripotent stem cells. Parental cells can include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means.

[0283] The term "derived" when used with reference to stem cells means that the stem cell was generated by reprogramming a differentiated cell to a stem cell phenotype. The term "derived" when used with reference to a differentiated cell means that the cell is the result of differentiation of a stem cell, for example, in vitro differentiation. As used herein, "iPSC-CM" or "induced pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from induced pluripotent stem cells. As used herein, the term "derived" when referring to a cell can encompass the initial cell generated and any subsequent progeny thereof.

[0284] As used herein, the term "progeny" encompasses, for example, the progeny of the first generation, i.e., the progeny are directly derived from, obtained directly from, obtainable directly from, or derivable directly from the original cell, e.g., by traditional propagation methods. The term "progeny" also encompasses further generations, such as the second, third, fourth, fifth, sixth, seventh, or even later generations, i.e., generations of cells derived from, obtained from, obtainable from, or derivable from the previous generation, e.g., by traditional propagation methods. The term "progeny" also encompasses modified cells resulting from the modification or alteration of the original cell or its progeny. In some embodiments, wild-type or control cells are the starting material. In some embodiments, the starting material is a stem cell, such as a pluripotent stem cell (PSC), including, but not limited to, an embryonic stem cell (ESC) and an induced pluripotent stem cell (iPSC). For example, unmodified stem cells obtained from a donor are considered the starting material for wild-type or control cells contemplated herein. In another example, stem cell line starting materials, such as ESC lines, PSC lines, or iPSC lines, are considered wild-type or control cells as contemplated herein. In some embodiments, the starting materials are otherwise modified or engineered to alter the expression of one or more genes to generate engineered cells. For example, the one or more genes are selected from the group of genes consisting of HCN4, CACNA1H, KCNJ2, and SLC8A1. As will be understood by those skilled in the art, there are various different methods for generating iPCS. Initial induction was performed from mouse embryonic fibroblasts or adult fibroblasts using viral transfer of four transcription factors: Oct3 / 4, Sox2, c-Myc, and Klf4. See Takahashi and Yamanaka Cell 126:663-676 (2006), which is incorporated herein by reference in its entirety, particularly for the techniques outlined therein. Since then, numerous methods have been developed.For reviews, see Seki et al., World J. Stem Cells 7(1):116-125 (2015) and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013. Both of these are expressly incorporated herein by reference in their entirety, particularly for methods of generating hiPSCs (see, e.g., Chapter 3 of the latter reference). Generally, iPSCs are generated by transient expression of one or more reprogramming factors in host cells, usually introduced using an episomal vector. Under these conditions, a small number of cells are induced to become iPSCs (generally, the efficiency of this process is low because no selection markers are used). Once cells are "reprogrammed" and become pluripotent, they lose the episomal vector and use endogenous genes to produce these factors. As will also be understood by those skilled in the art, the number of reprogramming factors that can be or are used can vary. Generally, the use of fewer reprogramming factors reduces the efficiency of transformation of cells into a pluripotent state and also reduces "pluripotency", e.g., fewer reprogramming factors may result in cells that are not fully pluripotent but may only be capable of differentiating into fewer cell types.

[0285] As used herein, the term "isolated cell" refers to a cell removed from the organism in which the cell was originally found, or the progeny of such a cell. Optionally, the cell is cultured in vitro, for example, in the presence of other cells. Optionally, the cell is subsequently introduced into a second organism, or reintroduced into the organism from which the cell (or a cell that is the ancestor of the cell) was isolated.

[0286] As used herein, the term "stimulated activity" with respect to ion channel activity means that the expression of an ion channel is increased compared to cardiomyocytes (e.g., cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), human pluripotent stem cells (hPSC-CMs), or human embryonic stem cells (hESC-CMs), isolated primary cardiomyocytes, etc.) that have not been manipulated by pharmacological or genetic means to affect the activity of the ion channel. Alternatively or additionally, "stimulated activity" can refer to an increase in ion channel activity induced by pharmacological or genetic means, as measured, for example, by patch clamp assay. As used herein, "stimulated" includes any stimulation that causes an increase compared to cardiomyocytes that have not been manipulated by pharmacological or genetic means to affect the activity of the ion channel, including partial stimulation. For example, an increase can include an increase in activity of at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000% or more, as compared to cardiomyocytes that have not been manipulated by pharmacological or genetic means to affect the activity of an ion channel.The increase can also include an increase in activity of at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold compared to cardiomyocytes that have not been manipulated by pharmacological or genetic means to affect ion channel activity.

[0287] As used herein, the term "inhibited activity" with respect to ion channel activity means that the expression of an ion channel is reduced compared to cardiomyocytes (e.g., cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), human pluripotent stem cells (hPSC-CMs), or human embryonic stem cells (hESC-CMs), isolated primary cardiomyocytes, etc.) that have not been manipulated by pharmacological or genetic means to affect the activity of the ion channel. Alternatively or additionally, "inhibited activity" can refer to a decrease in ion channel activity induced by pharmacological or genetic means, e.g., as measured by whole-cell patch clamp. As used herein, "inhibited" includes any inhibition that results in a decrease compared to cardiomyocytes that have not been manipulated by pharmacological or genetic means to affect the activity of the ion channel, and can include partial inhibition. For example, the decrease can include at least a 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000% or more decrease in activity compared to cardiomyocytes that have not been manipulated by pharmacological or genetic means to affect ion channel activity.The reduction can also include at least a 1-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 6-fold, at least a 7-fold, at least a 8-fold, at least a 9-fold, at least a 10-fold, at least a 20-fold, at least a 30-fold, at least a 40-fold, at least a 50-fold, at least a 60-fold, at least a 70-fold, at least a 80-fold, at least a 90-fold, at least a 100-fold decrease in activity compared to cardiomyocytes that have not been manipulated by pharmacological or genetic means to affect the activity of ion channels.

[0288] As used herein, "genetic manipulation" or "genetic modification" are used interchangeably to refer to changes in the genetic and / or epigenetic makeup of cells introduced by human hands, including, for example, gene editing that changes the chromosomal DNA of a cell, such as the introduction of one or more transgenes via a nucleic acid construct or vector (whether integrated or episomal), or a combination thereof. Any site-specific mutagenesis approach for modifying chromosomal DNA can be used, but non-limiting examples of gene editing include CRISPR-Cas-mediated chromosome cleavage with or without the use of a homologous recombination template, heritable epigenetic silencing (also known as "CRISPRoff"), base editing, prime editing, and zinc finger nuclease or TALEN-mediated cleavage of one or more target sequences, also with or without the use of a homologous recombination or replacement template, as well as other gene editing systems described herein. The term "genetic manipulation" also includes approaches that transiently modify the expression of one or more genes in a cell at either the RNA or protein level. These include, for example, RNA interference (RNAi), a class of gene regulation approaches involving double-stranded or single-stranded RNA, including but not limited to siRNA, shRNA, and miRNA, which function through RNA-induced silencing complexes (RISCs) to inhibit the expression of target genes. The introduction of RNAi agents is a genetic manipulation as defined herein, even though it does not necessarily alter the chromosomal structure of a cell. Similarly, the introduction of, for example, antisense RNA, is also a genetic manipulation as this term is used herein, as is the introduction of RNA-guided nucleases, such as Cas nucleases, that cleave target RNAs. Gene "inactivation" is a part of genetic manipulation that modifies chromosomal DNA so that a target gene is not expressed. Such inactivation can include, among other things, the deletion of all or part of a gene or its coding sequence, the insertion of a sequence that disrupts the expression of the target gene, and the replacement of the coding sequence with a sequence that encodes another polypeptide.As used herein, the term "genetic manipulation" or "genetic modification" can also refer to one or more changes to nucleic acids, for example, nucleic acids in the genome of an organism. For example, genetic modification can refer to the alteration, addition, and / or deletion of a gene or a portion of a gene or other nucleic acid sequence. A genetically modified cell can also refer to a cell in which a gene or a portion of a gene has been added, deleted, and / or modified. A genetically modified cell can also refer to a cell with an added nucleic acid sequence that is not a gene or a portion of a gene. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms and mechanisms that result in the permanent knock-in, knock-down, or knock-out of a targeted gene or portion of a gene or nucleic acid sequence. Genetic modifications include, for example, both transient knock-in and mechanisms that result in the permanent knock-in of a nucleic acid sequence.

[0289] In some embodiments, genetic manipulation does not necessarily involve permanent modification of the genome of cardiomyocytes. For example, it may be beneficial to knock down the expression of one or more of HCN4, CACNA1H, and SLC8A1 through RNAi or another transient genetic manipulation (e.g., RNA-specific Cas nuclease, antisense expression, etc.). RNAi or other inhibitory molecules can be administered to cardiomyocytes (e.g., in one of a number of different lipid complexes, among other delivery options) or expressed from a construct administered to or contacted with cardiomyocytes. In one embodiment, cardiomyocytes can be transiently transfected with one or more constructs encoding RNAi molecules or other targeted gene inhibitors (e.g., encoding the expression of shRNA). In such cases, over time, unless there is active selection for the construct, the transfected constructs are expected to be lost, resulting in transient expression of the inhibitor. Similar transient expression from a construct encoding KCNJ2 expression can also be used to stimulate KCNJ2 activity. In some cases, such transient knockdown or increase in ion channel activity may be sufficient to provide therapeutic benefit, provided that transplant arrhythmias generally resolve over a period of several weeks (unless, of course, the subject succumbs to the arrhythmia or its sequelae).

[0290] As used herein, "knockout," "knockdown," or "knockdown" refer to genetic modifications that result in the absence and reduced expression of the edited gene, respectively. As used herein, "knockdown" refers to a reduction in the expression of a target mRNA or the corresponding target protein. Knockdown is generally reported relative to the level present after administration or expression of a control molecule (e.g., a non-targeting shRNA, siRNA, guide RNA, or miRNA) that does not mediate a reduction in the expression level of the RNA. In some embodiments, the knockdown of the target gene is achieved by shRNA, siRNA, miRNA, or CRISPR interference (CRISPRi). In some embodiments, the knockdown of the target gene is achieved by a protein-based method, such as the degron method. In some embodiments, the knockdown of the target gene is achieved by genetic modifications including shRNA, siRNA, miRNA, or the use of a gene editing system (e.g., CRISPR / Cas). Knockdown is generally evaluated by measuring mRNA levels using quantitative polymerase chain reaction (qPCR) amplification, or by measuring protein levels by Western blot or enzyme-linked immunosorbent assay (ELISA).Analyzing protein levels provides an assessment of both mRNA cleavage and translation inhibition.Additional techniques for measuring knockdown include RNA solution hybridization, nuclease protection, Northern hybridization, gene expression monitoring by microarray, antibody binding, radioimmunoassay and fluorescence-activated cell analysis.Based on the details described herein, those skilled in the art will easily understand how to use exemplary gene editing systems (e.g., CRISPR / Cas) to knock out target polynucleotide sequences or parts thereof.

[0291] As used herein, "indel" refers to a mutation resulting from an insertion, deletion, or a combination thereof. As understood by those skilled in the art, an indel in the coding region of a genomic sequence will result in a frameshift mutation if the length of the indel is not a multiple of three. In some embodiments, the alteration is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one nucleotide. For example, the CRISPR / Cas system can be used to induce indels or point mutations of any length in a target polynucleotide sequence using gene editing, base editing, or prime editing. The term "base editing" refers to a method of programmably converting one base pair to another at a targeted locus, in some cases without double-stranded DNA breaks and in other cases without single-stranded DNA breaks. In some embodiments, base editing utilizes catalytically impaired Cas9, which has broad PAM sequence recognition to recognize the target DNA site and a window of based editing within and / or outside the protospacer sequence. The term "prime editing" refers to a method for gene editing that utilizes a programmable polymerase (such as, but not limited to, napDNAbps as described in International Publication No. 2020191242) and a specific guide RNA. In some embodiments, the guide RNA comprises a DNA synthesis template for encoding (or deleting) genetic information to be incorporated into the target DNA sequence. As will be recognized by those skilled in the art, base editing and prime editing are useful for regulating (e.g., reducing, eliminating, increasing, and enhancing) the expression of the described polynucleotides and polypeptides.

[0292] In some embodiments, the term "knock out," "knock-out," or "knockout" includes deleting all or part of a target polynucleotide sequence to prevent translation or function of the target polynucleotide sequence. For example, knockout can be achieved by altering the target polynucleotide sequence by inducing an insertion or deletion ("indel") in the target polynucleotide sequence, including in a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Based on the details described herein, one skilled in the art will readily understand how to use the gene editing system (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a portion thereof.

[0293] In some embodiments, genetic modification or alteration results in knockout or knockdown of a target polynucleotide sequence or a portion thereof. Using a gene editing system (e.g., CRISPR / Cas) to knock out a target polynucleotide sequence or a portion thereof can be useful for various applications. For example, knocking out a target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with the expression of the target polynucleotide sequence or for changing the genotype or phenotype of a cell (e.g., by knocking out a mutant allele in the cell ex vivo and introducing the cells containing the knocked-out mutant allele into a subject). In some cases, as used herein, "knockout" includes deleting all or part of a target polynucleotide sequence to disrupt the function of the target polynucleotide sequence. For example, knockout can be achieved by altering the target polynucleotide sequence by introducing an indel into the target polynucleotide sequence, in a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Based on the details described herein, those skilled in the art will easily understand how to use gene editing systems (e.g., CRISPR / Cas systems) to knock out target polynucleotide sequences or parts thereof. In some embodiments, the modification results in knocking out target polynucleotide sequences or parts thereof. Using the CRISPR / Cas system of the present disclosure to knock out target polynucleotide sequences or parts thereof can be useful for various applications. For example, knocking out target polynucleotide sequences in cells can be carried out in vitro for research purposes.For ex vivo purposes, knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in cells ex vivo and introducing those cells containing the knocked-out mutant allele into a subject).

[0294] As used herein, "knock-in," "knock-in," or "knockin" refers to a genetic modification resulting from the insertion of a DNA sequence into a chromosomal locus in a host cell. This results in increased levels of expression of the knocked-in gene, portion of a gene, or product into which the nucleic acid sequence has been inserted, e.g., increased RNA transcript levels and / or encoded protein levels. As will be understood by those skilled in the art, this can be achieved in several ways, including inserting or adding one or more additional copies of a gene or portion thereof into the host cell, or altering the regulatory components of an endogenous gene to increase protein expression or inserting a specific nucleic acid sequence whose expression is desired. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer, adding other regulatory elements, or modifying other gene expression sequences. The CRISPR / Cas system can be used to knock in sequences, whether by homologous DNA repair using a template with homology arms, or prime editing, or gene writing where specific sequences are edited. In some instances, the term "knock-in" refers to the process of adding genetic function to a host cell. This results in an increase in the level of the knocked-in gene product, such as RNA or the encoded protein.As will be understood by those skilled in the art, this can be achieved in several ways, including adding one or more additional copies of the gene to the host cell, or changing the regulatory components of the endogenous gene to increase protein expression.This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[0295] "Modulation" of gene expression refers to a change in the expression level of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Modulation can also be complete, i.e., gene expression is completely inactivated or activated to wild-type levels or above, or modulation can be partial, with gene expression being partially reduced or partially activated to a percentage of wild-type levels.

[0296] In further or alternative aspects, the methods and compositions described herein can include altering a target polynucleotide sequence using any method available to those skilled in the art, for example, using a nuclease system such as a TAL effector nuclease (TALEN) or zinc finger nuclease (ZFN) system. While examples of methods utilizing CRISPR / Cas (e.g., Cas9 and Cpf1) and TALEN are described in detail herein, it should be understood that the technology is not limited to the use of these methods / systems. Other targeting methods for reducing or eliminating expression in target cells known to those skilled in the art can be used herein. The methods provided herein can be used to alter a target polynucleotide sequence in a cell. The methods for generating modified cells contemplate altering a target polynucleotide sequence in a cell for any purpose. In some embodiments, the target polynucleotide sequence in a cell is altered to produce a mutant cell. As used herein, a "mutant cell" refers to a cell having a resulting genotype that differs from its original genotype. In some instances, a "mutated cell" exhibits a mutant phenotype, for example, when a normally functioning gene is altered using a gene editing system (e.g., CRISPR / Cas).

[0297] The terms "operatively linked" or "operably linked" are used interchangeably to refer to the juxtaposition of two or more components (such as sequence elements) where the components are positioned in a manner that allows for both components to function normally and allows for the potential for at least one of the components to mediate a function exerted on at least one of the other components. Illustratively, a transcription control sequence, such as a promoter, is operably linked to a coding sequence if it modulates the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcription control sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to the coding sequence. For example, an enhancer is a transcription control sequence operably linked to a coding sequence, even if the enhancer and coding sequence are not contiguous.

[0298] As used herein, a "genetically modified cell" refers to a cell that carries heterologous genetic material or constructs or contains a genome that has been engineered by mutation, including, but not limited to, site-specific mutation. The introduction of heterologous genetic material generally results in changes in gene or protein expression compared to unmodified cells. The introduction of RNA, as well as the introduction of vectors that do not integrate or replicate within the cell, can transiently promote the expression of foreign or heterologous products. The introduction of constructs that integrate into the cell's genome or replicate with the cell's nucleic acid will be more stable through successive cell divisions. In one embodiment, genetic modification is in addition to, or separate from, the introduction of a construct or constructs that reprogram somatic cells into a stem cell phenotype, such as an iPS cell phenotype. Genetic modifications are known to those skilled in the art and can include, but are not limited to, the introduction of genetic material via a viral vector, or modification using CRISPR / Cas or similar systems for site-specific recombination or random integration.

[0299] As used herein, the term "overexpressed" refers to expression of a target gene product or polypeptide at levels above those that occur in cells that are not genetically engineered for that target. Overexpression of a target gene or polypeptide results in increased expression of that target gene or polypeptide, as the term "increased" is used herein.

[0300] As used herein, the term "vector" or "construct" refers to a nucleic acid construct designed for delivery into 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 can replicate and introduce a gene sequence into a cell when associated with appropriate regulatory elements. Vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, and the like. Furthermore, a "vector" or "construct" can introduce a gene sequence into a target cell. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and introducing a gene sequence into a target cell. Thus, the term includes cloning and expression vehicles as well as integration vectors. Methods for introducing vectors or constructs into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and / or viral vector-mediated transfer.

[0301] The term "substantially pure" with respect to a particular cell population refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the total cell population. That is, the terms "substantially pure" or "essentially purified" with respect to a population of cardiomyocytes refer to a population of cells that contains less than about 20%, more preferably less than about 15%, less than about 10%, less than about 8%, less than about 7%, and most preferably less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than 1% of cells that are not cardiomyocytes, respectively.

[0302] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to, "peptide," "dipeptide," "tripeptide," "protein," "enzyme," "amino acid chain," and "contiguous amino acid sequence" are all included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term further includes polypeptides that have undergone one or more post-translational modifications, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translational processing, or modification by the inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structure. For example, single-letter and three-letter abbreviations are widely used to describe amino acids: alanine (A; Ala), arginine (R; Arg), asparagine (N; Asn), aspartic acid (D; Asp), cysteine ​​(C; Cys), glutamine (Q; Gln), glutamic acid (E; Glu), glycine (G; Gly), histidine (H; His), isoleucine (I; Ile), leucine (L; Leu), methionine (M; Met), phenylalanine (F; Phe), proline (P; Pro), serine (S; Ser), threonine (T; Thr), tryptophan (W; Trp), tyrosine (Y; Tyr), valine (V; Val) and lysine (K; Lys). The amino acid residues provided herein are preferably in the "L" isomeric form. However, any L-amino acid residue can be substituted by residues in their "D" isomeric form, provided the desired properties of the polypeptide are retained.

[0303] As used herein, the terms "expression" or "expressed" or "positive for" refer to a cell (e.g., a cardiomyocyte) that has a detectable level of a nucleic acid, vector, or polypeptide. The nucleic acid, vector, or polypeptide can be detected by any method available to those skilled in the art. For example, a polypeptide described herein can be expressed by a cardiomyocyte after contact with a vector or agent that induces expression of the polypeptide. Expression can be transient or stable expression by the cardiomyocyte.

[0304] As used herein, the term "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used, for example, to select cells containing a characteristic of interest and may vary depending on the specific cell. A marker is a characteristic, whether morphological, structural, functional, or biochemical (enzymatic) feature of a cell of a particular cell type or a molecule expressed by a cell type. In one aspect, such a marker is a protein. Such proteins can have epitopes for antibodies or other binding molecules available in the art. However, markers can consist of any molecule found in or on a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nucleus-to-cytoplasm ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to take up or exclude a particular dye, the ability to migrate under certain conditions, and the ability to differentiate along a particular lineage. Markers can be detected by any method available to those skilled in the art. A marker can also be the absence of a morphological feature or the absence of a protein, lipid, etc. A marker can be a panel of specific features of the presence and / or absence of a polypeptide in combination with other morphological or structural features. In one embodiment, the marker is a cell surface marker.

[0305] The terms "differentiate" or "differentiated" are relative terms indicating that a "differentiated cell" is a cell that has progressed further down the developmental pathway than its progenitor cell. Thus, in some embodiments, stem cells as defined herein can differentiate into lineage-restricted progenitor cells (e.g., human cardiac progenitor cells or mid-streak cardiogenic mesoderm progenitor cells) that can subsequently differentiate further down the pathway into other types of progenitor cells (such as tissue-specific progenitors, such as cardiomyocyte precursors), and then into terminally differentiated cells that play a characteristic role in a tissue type and may or may not retain the ability to further proliferate. Methods for in vitro differentiation of stem cells into cardiomyocytes are known in the art and / or described herein below.

[0306] As used herein, the term "mature" or "mature phenotype" or "mature cardiomyocyte" refers to a cellular phenotype that, when applied to cardiomyocytes, comprises a phenotype similar to that of adult cardiomyocytes and does not comprise at least one characteristic of fetal cardiomyocytes. In some embodiments, markers that indicate increased maturity of in vitro differentiated cells include, but are not limited to, electrical maturity, metabolic maturity, genetic maturity, and contractile maturity.

[0307] As used herein, the terms "treating," "treatment," and "administering" are used interchangeably in the context of placing a composition described herein into a subject by a method or route that results in at least partial localization of the composition described herein at a desired site, such as the heart or a region thereof, so that a desired effect occurs. The agent, cardiomyocyte, or composition described herein can be administered by any suitable route that results in delivery to the desired location in the subject. The half-life of the agent after administration to the subject can be short-term, such as minutes, hours, or days, or long-term, such as 24 hours to several days to several years. In some embodiments of any aspect, the term "treatment" refers to administration of a composition described herein comprising cardiomyocytes in which HCN4, CACNA1H, and SLC8A1 activity is inhibited and KCNJ2 activity is stimulated. Administration can be by direct injection into a subject in need thereof (e.g., administered directly to target cells or tissue) or by contacting cardiomyocytes via intracardiac injection. Administration can be transient, local, or systemic. These terms also include administering an effective amount of the cells described herein to a subject so that the subject experiences at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical result. In the present technology, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, attenuation of the extent of the disease, a stabilized (i.e., not worsening) state of the disease, a delay or slowing of the progression of the disease, and a reduction or amelioration and remission (whether partial or complete) of the disease state, whether detectable or undetectable. Treating can refer to prolonging survival compared to the expected survival in the absence of treatment. Thus, those skilled in the art will recognize that treatment can improve disease symptoms but may not completely cure the disease. In some embodiments, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.

[0308] In some embodiments, the terms "treating" and "treatment" of cardiac disorders, heart diseases, or cardiac damage (e.g., myocardial infarction) referred to herein refer to therapeutic interventions that enhance cardiac function, and / or enhance cardiomyocyte engraftment, and / or enhance cardiomyocyte transplantation or graft vascularization in the treated area, thus improving, for example, cardiac function. That is, cardiac "treatment" refers to cardiac function (e.g., enhanced function within the infarct) and / or other sites treated with the compositions described herein. For example, when assessed by measuring left ventricular end-systolic diameter (LVESD) or cardiac output, a therapeutic approach that improves cardiac function by at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100% or more, for example, 2-fold, 5-fold, 10-fold or more, up to and including complete function, compared to the function before treatment, is considered an effective treatment. An effective treatment need not cure or directly affect the underlying cause of the heart disease or disorder to be considered an effective treatment.

[0309] In some embodiments, "treatment" can be evaluated for effectiveness by assessing beneficial or desired clinical results, whether detectable or undetectable, including, but not limited to, alleviation of one or more symptoms, attenuation of the extent of the disease, a stabilized (i.e., not worsening) state of the disease, delay or slowing of the progression of the disease, alleviation or amelioration and remission (whether partial or complete) of the disease state. Treating can refer to prolonging survival compared to the expected survival in the absence of treatment. Thus, those skilled in the art will recognize that treatment may improve cardiac disorders, cardiac diseases, or cardiac damage, but may not completely cure the disease. In some embodiments, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of a cardiac disorder, cardiac disease, or cardiac damage.

[0310] The term "effective amount" as used herein refers to the amount of a population of cardiomyocytes required to alleviate at least one or more symptoms of a disease or disorder, including, but not limited to, injury, disease, or disorder. An "effective amount" refers to an amount of a composition sufficient to provide a desired effect, for example, treating a subject with an infarcted area after myocardial infarction, improving cardiomyocyte engraftment, preventing the development of heart failure after cardiac injury, enhancing vascularization of a graft, etc. Thus, the term "therapeutically effective amount" refers to an amount of human cardiomyocytes or a composition of such cells that is sufficient to promote a particular effect when administered to a typical subject, such as one with or at risk for a cardiac disease or disorder. An effective amount as used herein also includes an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. It is understood that an appropriate "effective amount" for any given case can be determined by one skilled in the art using routine experimentation.

[0311] For the purposes of the present technology, beneficial or desired clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, attenuation of the extent of the disease, a stabilized (i.e., not worsening) state of the disease, delay or slowing of disease progression, alleviation or amelioration and remission (whether partial or complete) of the disease state, whether detectable or undetectable.

[0312] As used herein, the term "transplant arrhythmia" or "EA" refers to a new and abnormal heart rhythm or heart rate that occurs after the administration of cardiac cell or cardiomyocyte transplants, and can be a serious complication of cardiac remuscularization therapy.Transplant arrhythmia is observed after cardiac graft transplantation and generally persists transiently for several days to several weeks.Transplant arrhythmia can cause sudden cardiac death and heart failure in subjects.

[0313] As used herein, the term "contacting," when used in reference to a cell, encompasses both the introduction of an agent, surface, hormone, etc. into the cell in a manner that allows for physical contact between the cell and the agent, surface, hormone, etc., and the introduction of an element, such as a genetic construct or vector, that allows for expression of an agent, such as an miRNA, polypeptide, or other expression product in the cell. It should be understood that a cell that has been genetically modified to express an agent has been "contacted" with that agent, as well as any progeny of that cell that express the agent.

[0314] As used herein, the term "disease" or "disorder" refers to a disease, syndrome, or disorder that is caused, in part or in whole, directly or indirectly, by one or more abnormalities in a subject's genome, physiology, behavior, or health. The disease or disorder may be a cardiac disease or disorder.

[0315] As used herein, the term " cardiac disease " refers to the disease that affects the cardiac tissue of the subject.Non-limiting examples of cardiac disease include cardiomyopathy, cardiac arrhythmia, myocardial infarction, heart failure, cardiac hypertrophy, long QT syndrome, arrhythmogenic right ventricular dysplasia (ARVD), catecholamine-induced polymorphic ventricular tachycardia (CPVT), Barth syndrome and Duchenne muscular dystrophy.

[0316] As used herein, the term "transplant arrhythmia" refers to a heart rate or rhythm disturbance caused by or associated with the introduction or generation of new myocardium in a subject. A key feature of transplant arrhythmia is that the stimulus originates from the engraftment site, rather than from the sinoatrial node or atrioventricular node. Rhythm disturbance is any repeated or long-term deviation from normal sinus rhythm. Heart rate disturbances include deviations above or below the subject's normal resting heart rate by at least 10% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more) when new cardiomyocytes are introduced or introduced into the subject's cardiac tissue. In one embodiment, transplant arrhythmia is caused by or associated with the introduction of exogenous cardiomyocytes, including, but not limited to, in vitro differentiated cardiomyocytes, into cardiac tissue, such as in the transplantation of cardiomyocytes administered to promote infarct repair or to enhance cardiac function in cardiomyopathy. In another embodiment, the transplant arrhythmia is a heart rate greater than 100 beats per minute. In another embodiment, the heart rate or rhythm disturbance is prolonged, for example, lasting for more than 5% of the day or observation period. In some embodiments, the reduction in transplant arrhythmia after transplantation of the cardiomyocytes (including in vitro differentiated human cardiomyocytes) described herein comprises a reduction of at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to before transplantation.The reduction of transplant arrhythmia can also include a reduction of at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold or more compared to before transplantation.In some embodiments, before transplantation refers to before any cell transplantation.In some embodiments, before transplantation includes cases where the previous cell transplantation does not include cells that have at least partially inhibited HCN4, CACNA1H, and SLC8A1 activity and at least partially stimulated KCNJ2 activity (as described herein).

[0317] The terms "patient," "subject," or "individual" are used interchangeably herein and refer to animals, particularly humans, e.g., humans from which cells can be obtained and / or humans to whom treatment, including prophylactic treatment, using the cells described herein is provided. As used herein, the term "subject" refers to humans and non-human animals. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include all vertebrates, for example, mammals such as non-human primates (e.g., higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows, and non-mammals such as chickens, amphibians, and reptiles. In one embodiment of any aspect, the subject is a human. In another embodiment of any aspect, the subject is an experimental animal or animal surrogate for a disease model. In another embodiment of any aspect, the subject is a domestic animal, including pets (e.g., dogs, cats, rats, guinea pigs, hamsters, etc.). The subject may have previously been treated for the disease or may not have been treated for the disease. The subject may have previously been diagnosed with the disease or may not have been diagnosed with the disease. Advantageously, however, the subject is a mammal, such as a human or a domestic mammal, such as a dog, cat, horse, or other mammal, such as a livestock mammal, such as a cow, sheep, pig, etc.

[0318] As used herein, the terms "transplant," "implant," "transplant," "engraft," "graft," "transplantation," "administer," "introduce," or "implant" are used in the context of placing cells described herein, e.g., stem cell-derived cardiomyocytes, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, so that a desired effect occurs. Cells, e.g., cardiomyocytes or their differentiated progeny (e.g., cardiac fibroblasts, etc.) and cardiomyocytes, can be implanted directly into the heart or administered by any suitable route that results in delivery to a desired location in a subject, with at least some of the implanted cells or components of the cells surviving. The survival period of the cells after administration to a subject can range from short-term, e.g., a few hours, e.g., 24 hours to a few days, to several years, i.e., long-term engraftment. As one skilled in the art will appreciate, long-term engraftment of cardiomyocytes is desirable because cardiomyocytes do not proliferate to an extent that allows the heart to heal from acute injury, including cell death. In other embodiments, the cells can be administered via an indirect systemic route, such as intraperitoneal or intravenous.

[0319] As used herein, the term "scaffold" refers to a structure comprising a biocompatible material that provides a suitable surface for cell adhesion and growth. A scaffold can further provide mechanical stability and support. A scaffold can be of a particular shape or form to influence or delimit the three-dimensional shape or form assumed by a population of growing cells. Such shapes or forms include, but are not limited to, thin films (e.g., forms having two dimensions substantially greater than the third), ribbons, cords, sheets, flat disks, cylinders, spheres, three-dimensional amorphous shapes, and the like.

[0320] As used herein, "substrate" refers to a structure comprising a biocompatible material that provides a suitable surface for cell adhesion and growth. Nanopatterned or micropatterned substrates can further provide mechanical stability and support. The substrate can be of a particular shape or form to influence or delimit the three-dimensional shape or form assumed by the growing cell population. Such shapes or forms include, but are not limited to, thin films (e.g., forms having two dimensions substantially greater than the third dimension), ribbons, cords, sheets, flat disks, cylinders, spheres, three-dimensional amorphous shapes, and the like. The substrate can be nanopatterned or micropatterned to enable the formation of engineered tissues on the substrate.

[0321] As used herein, the term "implantable in a subject" refers to any non-living (e.g., acellular) implantable structure that does not generate a discernible immune response in the host organism upon implantation. Thus, the implantable structure should not be, or contain, an irritant, or contain, for example, LPS, etc.

[0322] The term "agent" as used herein refers to any compound or substance, including, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. An "agent" can be any chemical entity, entity, or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments of any aspect, the agent is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, protein, oligonucleotide, ribozyme, DNAzyme, glycoprotein, siRNA, lipoprotein, oligomer of nucleic acid, amino acid, or carbohydrate, including, but not limited to, aptamer, as well as modifications and combinations thereof. In certain embodiments, the agent is a small molecule having a chemical moiety. For example, the chemical moiety can include unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties, including macrolides, leptomycin, and their related natural products or analogs. The compound can be known to have the desired activity and / or properties, or can be selected from a library of diverse compounds.

[0323] The agent can be a molecule from one or more chemical classes, e.g., organic molecules, which can include organometallic molecules, inorganic molecules, genetic sequences, etc. The agent can also be a fusion protein from one or more proteins, a chimeric protein (e.g., domain swapping or homologous recombination of functionally important regions of related or different molecules), a synthetic protein, or other protein variant containing substitutions, deletions, insertions, and other variants.

[0324] The term "pharmaceutically acceptable" is used herein to describe compounds, materials, compositions, carriers, and / or dosage forms that 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 challenge, commensurate with a reasonable benefit / risk ratio.

[0325] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant decrease or reduction in a characteristic, level, or other parameter. In some embodiments, "reduce," "reduction," "reduce," or "inhibit" typically refers to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a decrease of 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 about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease may preferably be to a level that is accepted as within the normal range for individuals without a given disorder.

[0326] The terms "increased," "increase," "increases," "enhance," or "activate" are all used herein to generally refer to an increase in a property, level, or other parameter by a statistically significant amount. For the avoidance of doubt, the terms "increased," "increase," "enhance," or "activate" mean an increase of at least 10% compared to a base 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% compared to a base level, or an increase up to and including 100%, or any increase between 10 and 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 1000-fold, or more, compared to a base level. As used herein, the term "modulate" refers to an effect that includes increasing or decreasing a given parameter, as those terms are defined herein.

[0327] As used herein, a "reference level" refers to a normal, otherwise unaffected cell population or tissue (e.g., a biological sample obtained from a healthy subject or a biological sample obtained from a subject at a prior time point, e.g., a biological sample obtained from a patient before they have been diagnosed with a disease, or a biological sample that has not been contacted with a composition, polypeptide, or nucleic acid encoding such a polypeptide disclosed herein).

[0328] As used herein, a "suitable control" or "suitable control cells" or "other controls" or "other control cells" refers to untreated, otherwise identical cells or populations (e.g., biological samples that have not been contacted with an agent or composition described herein, or have not been contacted in the same manner, e.g., for a different period of time, as compared to non-control cells). This may include cardiomyocytes or other cells that have not been manipulated by one or more inhibitor drugs and / or genetic manipulation.

[0329] As used herein, the term "phenotypic characteristic" as applied to in vitro differentiated cells (e.g., cardiomyocytes) or cultures of in vitro differentiated cells refers to any of the parameters described herein as a measure of cell function. A "change in phenotypic characteristic," as described herein, is indicated by a statistically significant increase or decrease in a functional property relative to a baseline level or appropriate control.

[0330] The terms "statistically significant" or "significantly" refer to statistical significance, and generally mean a difference of 2 standard deviations (2 SD) or greater.

[0331] As used herein, the term "comprising" means that other elements may be present in addition to the defined elements set forth. The use of "comprising" indicates inclusion rather than limitation.

[0332] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in that description of an embodiment.

[0333] As used herein, the term "consisting essentially of" refers to 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 characteristics of that embodiment of the invention.

[0334] The singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to represent a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0335] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0336] Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0337] It should be noted that the appended claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features that may be readily separated from or combined with the features of any other embodiment without departing from the scope or spirit of the technology. Any described method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the technology, representative exemplary methods and materials are now described.

[0338] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this technology belongs.When a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value within that stated range, are encompassed within the technology.The upper and lower limits of these smaller ranges can be independently included in the smaller ranges, and are also encompassed within the technology, subject to any specifically excluded limit in the stated range.When a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also encompassed within the technology.Certain ranges are herein indicated by numerical values ​​preceded by the term "about".The term "about" is used herein to provide literal support for the exact number preceded by the term "about", as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is near or approximately a specifically recited number, the unrecited near or approximately number may be a number that, in the context indicated, provides a substantial equivalent to the specifically recited number.

[0339] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter for which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the technology described herein is not entitled to antedate such publication by virtue of prior art. Furthermore, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0340] Before further describing the present technology, it should be understood that the present technology is not limited to the particular embodiments described, and therefore may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present technology is limited only by the appended claims. It should also be understood that the headings used herein are not limiting, but are merely intended to guide the reader, but that the subject matter generally applies to the technology disclosed herein.

[0341] cardiovascular disease In some aspects, provided herein is a method for treating and / or preventing cardiac damage or cardiac disease or disorder in a subject in need thereof.The methods described herein can be used to treat, improve, prevent, or slow the progression of many diseases or their symptoms, such as those that cause pathological damage to the structure and / or function of the heart.

[0342] Cardiovascular disease is the disease that affects the heart and / or circulatory system of an object.Such heart disease or heart-related disease includes but is not limited to myocardial infarction, cardiac arrhythmia, heart failure, atherosclerotic heart disease, cardiomyopathy, congenital heart defect (such as non-compaction cardiomyopathy, septal defect, hypoplastic left heart), hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac hypertrophy, myocarditis, arrhythmogenic right ventricular dysplasia (ARVD), long QT syndrome, catecholamine-induced polymorphic ventricular tachycardia (CPVT), Barth syndrome, valve stenosis, regurgitation, ischemia, fibrillation, polymorphic ventricular tachycardia and muscular dystrophy and cardiac hypertrophy such as Duchenne or related heart disease.In some embodiments, the methods described herein can be used to treat, improve, prevent, or slow down the progression of cardiovascular disease. In some embodiments, the methods described herein can be used to treat, ameliorate, prevent, or slow the progression of myocardial infarction, cardiac arrhythmias, heart failure, atherosclerotic heart disease, cardiomyopathies, congenital heart defects (e.g., noncompaction cardiomyopathy, septal defects, hypoplastic left heart), hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac hypertrophy, myocarditis, ARVD, long QT syndrome, CPVT, Barth syndrome, valvular stenosis, regurgitation, ischemia, fibrillation, polymorphic ventricular tachycardia, and muscular dystrophies such as Duchenne or related heart diseases and cardiac hypertrophy.

[0343] The term "cardiac event" refers to the occurrence of myocardial damage, myocardial infarction, ventricular fibrillation, stenosis, arrhythmia, etc. In some embodiments, the methods described herein can be used to treat, ameliorate, prevent, or slow the progression of a cardiac event.

[0344] Symptoms of cardiovascular disease may include, but are not limited to, fainting, fatigue, shortness of breath, chest pain and palpitations.Cardiovascular disease is generally diagnosed by physical examination, blood test, and / or electrocardiogram (EKG).Abnormal EKG is an indication that the subject has abnormal heart rhythm or cardiac arrhythmia.Methods for diagnosing arrhythmia are known in the art.In some embodiments, the methods described herein can be used to treat, improve, prevent, or slow the progression of cardiovascular disease symptoms.

[0345] Cardiac electrophysiological and contractile functions are tightly regulated processes. Disruption of ion channel regulation or contractile function in cardiac cells or tissues can lead to potentially fatal cardiac arrhythmias. Heart disease remains a leading cause of death worldwide.

[0346] Human stem cell-derived cardiomyocytes have emerged as a promising treatment for cardiovascular disease and cardiac damage resulting from myocardial infarction. However, the functional maturity of in vitro-differentiated cardiomyocytes in existing models is generally lacking, and these cardiomyocytes may cause arrhythmias after engraftment. Without wishing to be bound by theory, the introduction of exogenous cardiomyocytes with their own impulse-generating activity has the potential to disrupt the tightly regulated electrophysiological function of the heart and cause arrhythmias. While this effect is particularly well-known in in vitro-differentiated cardiomyocyte grafts, it may also occur, for example, when cardiomyocytes derived from other sources are transplanted into cardiac tissue.

[0347] In one aspect, the present invention describes compositions and methods for treating cardiovascular disease.In another aspect, the present invention describes a method for avoiding, preventing, treating, or improving transplant arrhythmia in a subject receiving a cardiac transplant of cardiomyocytes, the method comprising administering to a subject the in vitro differentiated human cardiomyocytes described herein, the pharmaceutical composition described herein, the transplant composition described herein, or contacting cardiac tissue with pharmacologically engineered or genetically engineered cardiomyocytes delivered via a cardiac delivery device described herein, or any combination thereof.In some embodiments of this aspect and all other aspects provided herein, the method comprises administering to a subject a combination of one or more pharmaceutical compositions and in vitro differentiated human cardiomyocytes comprising one or more genetic modifications.In one embodiment of this aspect and all other aspects provided herein, the subject is administered an HCN4 channel inhibitor such as ivabradine.In another embodiment of this aspect and all other aspects provided herein, the subject is administered a CACNA1H channel inhibitor selected from the group consisting of miblefadil or ML-218.

[0348] In some embodiments of any aspect, the subject has or is at risk of having cardiovascular disease or a cardiac event.

[0349] In some embodiments of any aspect, the subject with cardiovascular disease is in need of, has received, or has received a cardiac cell transplant. In some embodiments, the subject is at risk for, has, or has been diagnosed with transplant arrhythmia.

[0350] Cardiac ion channels Action potential (AP) propagation by a single cardiomyocyte occurs via the rhythmic opening and closing of ion channels according to their electrochemical gradient. APs are divided into distinct phases (0 to 4; see also Figure 1A in the Examples) depending on the abundance of ion channels present and the current present in that phase. Compared to adult ventricular cardiomyocytes, AP characteristics of hPSC-CMs include a more depolarized phase 4 (corresponding to the resting membrane potential) and shorter AP duration (phases 2 / 3) (see, e.g., Karbassi E, et al., "Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol. 2020 Jun;17(6):341-359, incorporated herein by reference in its entirety), which leads to increased generation of spontaneous action potentials (i.e., automaticity).

[0351] The depolarization and repolarization phases of the action potential are reflected in the electrocardiogram (ECG). The ECG measures the electrical activity of the heart over a period of time. The average atrial depolarization (P wave) and ventricular repolarization (QT interval) durations are evident on the ECG. Cardiac rhythm abnormalities can be detected by the ECG and can result in reduced pumping efficiency of the myocardium. The molecular mechanisms of cardiac action potential generation are well known in the art and are described, for example, by Roden, DM et al., "Cardiac ion channels," Annu. Rev. Physiol. 64, 431-475 (2002); Grant, AO, "Cardiac Ion Channels," Circ. Arrhythm. Electrophysiol. 2, 185-194 (2009). The molecular mechanisms of cardiac impulse propagation and associated arrhythmias are reviewed, for example, in Andre G. Kleber and Yoram Rudy et al., "Basic Mechanisms of Cardiac Impulse Propagation and Associated Arrhythmias," Physiological Reviews. 2004 84:2, 431-488, the entire contents of each of which are incorporated herein by reference.

[0352] Generally, in adult mammalian cardiomyocytes, phase 4 is the resting membrane potential of the cardiac cell, which is stabilized by inwardly rectifying potassium channels. Members of the inwardly rectifying potassium channel (Kir2.x) subfamily primarily mediate the cardiac IK1 current, although other inward rectifiers may also be involved in cardiac excitability. The resting membrane potential is typically -90 mV in healthy adult ventricular myocytes. Phase 0, the initial phase of the action potential, is stabilized by voltage-gated sodium channels, primarily Na. V The first phase is a rapid depolarization phase caused by the influx of sodium ions through the ATP-dependent ATPase 1.5 (encoded by SCN5A). The second phase is a rapid depolarization phase caused by the inactivation of sodium channels and the transient outward potassium current (I to ), e.g., K VThe second phase is a rapid repolarization phase in which there is activation of L-type calcium channels Ca 4.2 (encoded by KCND2). v The plateau phase is caused by calcium ion influx through the sodium-calcium exchanger (NCX1, encoded by SLC8A1) by exchanging intracellular calcium with extracellular sodium ions, and ITPR2 controls calcium release from the endoplasmic reticulum. Phase 3, the third phase of the action potential, is a rapid repolarization that restores the membrane voltage to the resting potential. This phase is primarily driven by the hERG (IK r ) also known as voltage-gated potassium channel K V 11.1 (encoded by KCNH2) and K V LQT1(IK s ), also known as KCNQ1, V Caused by 7.1.

[0353] Methods for measuring expression and function of ion channels and ionic currents are known in the art, for example, whole-cell patch clamp or microelectrode arrays. See, for example, Liang P, et al., "Patient-Specific and Genome-Edited Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Brugada Syndrome," J Am Coll Cardiol. Nov 8; 68(19):2086-2096 (2016); Germanguz, I. et al., "Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells," J. Cell. Mol. Med. 15, 38-51 (2011); and Schwartz, PJ, Crotti, L., Zipes, D. & Jalife,, J. Cardiac Electrophysiology: From Cell to Bedside. Elsevier / Saunders, (2009), the contents of each of which are incorporated herein by reference in their entirety. Such methods can be used, for example, to monitor the activity of cardiac cell ion channels and / or the modulation of such activity, e.g., via pharmacological or genetic means. Methods for characterizing the electrophysiological phenotype of in vitro differentiated cardiomyocytes are further discussed elsewhere below.

[0354] In contrast to adult cardiomyocytes, in vitro differentiated cardiomyocytes can express a functionally immature, fetal-like phenotype that is not physiologically equivalent to native adult cardiac tissue. Specifically, unlike adult cardiomyocytes, in vitro differentiated cardiomyocytes lack high levels of KCNJ2 expression or activity, resulting in an unstable RMP range of -50 to -60 mV. Furthermore, T-type calcium channels, which are prominent in cardiac conduction cells, are absent in adult ventricular cardiomyocytes but are detected in variable amounts in pluripotent stem cell-derived cardiomyocytes, most likely due to the heterogeneous differentiation of pluripotent stem cell-derived cardiomyocytes. For example, Bkaily et al., “Angiotensin II-induced increase of T-type Ca 2+ current and decrease of L-type Ca 2+ current in heart cells.”Peptides 26,1410-1417.(2005);Ono and Iijima,”Cardiac T-type Ca 2+ channels in the heart.” J. Mol. Cell. Cardiol. 48, 65-70. (2010), the contents of each of which are incorporated herein by reference in their entireties.

[0355] Immature cell-cell connections between pluripotent stem cell-derived cardiomyocytes also affect their electrophysiological function. See, e.g., Noorman, M. et al., "Cardiac cell-cell junctions in health and disease: electrical versus mechanical coupling. J. Mol. Cell. Cardiol. 47, 23-31 (2009), the contents of which are incorporated herein by reference in their entirety.

[0356] Further reviews of the functional and morphological differences between in vitro differentiated cardiomyocytes compared to adult cardiomyocytes are provided, for example, by Robertson, C., et al., "Concise review: Maturation phases of human pluripotent stem cell-derived cardiomyocytes," Stem Cells 31, 829-837 (2013); and Scuderi GJ and Butcher J, "Naturally engineered maturation of cardiomyocytes," Front Cell Dev Biol. (2017), the contents of each of which are incorporated herein by reference in their entirety. See, for example, Sartiani, L. et al., "Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: a molecular and electrophysiological approach," Stem Cells 25, 1136-1144 (2007); Protze, S., et al., "Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker," Nat Biotechnol 35, 56-68 (2017); and Ma, J., et al., "High purity human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents," Am. J. Physiol. Heart Circ. Physiol. 301, H2006-H2017 (2011), the contents of each of which are incorporated herein by reference in their entireties.

[0357] To overcome the challenges associated with the immature electrical phenotype exhibited by in vitro differentiated cardiomyocytes, the compositions and methods described herein can be used to generate safer in vitro differentiated cardiomyocytes that do not cause transplant arrhythmias when transplanted into a subject in need thereof.

[0358] In one aspect, the present invention describes the human cardiomyocyte differentiated in vitro, wherein HCN4, CACNA1H (Cav3.2) and SLC8A1 (NCX1) activity is inhibited, and KCNJ2 (Kir2.1) activity is stimulated.In one embodiment of this aspect and all other aspects provided herein, HCN4 channel is inhibited by using inhibitor drug ivabradine.In another embodiment of this aspect and all other aspects provided herein, CACNA1H channel is inhibited by using one or more inhibitor drugs selected from the group consisting of miblefadil or ML-218.

[0359] In another embodiment of this aspect and all other aspects described herein, the KCNJ2 polypeptide is overexpressed by genomic insertion of the KCNJ2 gene, the SLC8A1 polypeptide is inhibited by genomic modification of the SLC8A1 gene, the HCN4 polypeptide is inhibited by either genomic modification or treatment with a pharmacological agent (e.g., ivabradine), and the CACNA1H polypeptide is inhibited by either genomic modification or treatment with a pharmacological agent (e.g., miblefadil, ML-218, flunarizide).

[0360] In another aspect, described herein are in vitro differentiated human cardiomyocytes having reduced expression of one or more, two or more, or each of the genes encoding HCN4, CACNA1H, and SLC8A1.

[0361] In some embodiments, the KCNJ2 (Kir2.1) polypeptide is overexpressed.

[0362] The structure and function of HCN4, CACNA1H, SLC8A1, and KCNJ2 are discussed further below.

[0363] Funny Channel - HCN4: HCN or "Funny" or "(I f Hyperpolarization-activated cyclic nucleotide-gated potassium channels, also known as "hyperpolarization-activated" (HCN) channels, maintain pacemaker activity in the mammalian heart. There are four isoforms of HCN channels, HCN1-HCN4, encoded by the HCN gene. Specifically, HCN4 regulates rhythmic activity in both thalamocortical neurons and cardiac pacemaker cells. Pacemaker activity depends on a phase of spontaneous, slow membrane depolarization that occurs during cardiac action potentials (i.e., during diastole). As mentioned above, several ionic currents are mediated by diastolic membrane depolarization (e.g., T-type and L-type Ca channels). 2+ These currents contribute to the time course and gradient of the hyperpolarization-activated nonselective cation current I, some of which is directly controlled by the autonomic nervous system. f The activity of is regulated by cAMP. f This involves direct interaction with the cyclic nucleotide binding domain (cNBD) of the channel. Ultimately, this results in accelerated diastolic depolarization and electrical impulse generation. Blocking the Fanny channel with antiarrhythmic agents, such as ivabradine, results in an overall reduction in heart rate.

[0364] HCN4 is a mixed Na + and K. +It has permeability, activation upon hyperpolarization, and very slow kinetics. Mutations in HCN4 in humans and animals are associated with cardiac arrhythmias such as sick sinus syndrome and Brugada syndrome. See, for example, Milanesi, R. et al., "Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel," New Eng. J. Med. 354:151-157, (2006) and Crotti, L. et al., "Spectrum and prevalence of mutations involving BrS1-through BrS12-susceptibility genes in a cohort of unrelated patients referred for Brugada syndrome genetic testing," J. Am. Coll. Cardiol. 60:1410-1418, (2012). Further details regarding the structure and function of HCN4 are described, for example, in Ludwig, A. et al., "Two pacemaker channels from human heart with profoundly different activation kinetics," EMBO J. 18:2323-2329, (1999).

[0365] The structure of HCN4 contains six putative transmembrane segments, a pore region and a cyclic nucleotide binding domain (cNBD).The genomic DNA, transcript and polypeptide sequence of HCN4 are known in the art, for example, the human HCN4 genomic sequence is located at position c73368958-73319859 on human chromosome 15, and is GenBank accession number AJ238850, NCBI gene ID: 10021 and NCBI reference sequence NP_005468.1, human HCN4 transcript sequence NCBI reference sequence: NM_005477.3; and the human HCN4 polypeptide amino acid sequence described below: NCBI reference sequence NP_005468.1. >NP_005468.1 Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 [Homo sapiens] TIFF2024517810000002.tif121164

[0366] For purposes of the compositions and methods provided herein, the level or activity of the HCN4 channel, its orthologs, or variants can be manipulated, for example, by targeting the full-length gene or coding sequence for deletion or disruption, or by modifying a structural domain required for function (e.g., the pore-forming domain or cNBD domain of the channel), or by targeting ion transport through the channel via pharmacological or genetic modifications described herein. See, e.g., Nof, E., et al., "Point mutation in the HCN4 cardiac ion channel pore affecting synthesis, trafficking, and functional expression is associated with familial asymptomatic sinus bradycardia," Circulation 116:463-470, 2007, the entire contents of which are incorporated herein by reference.

[0367] While complete knockout is effective, in some embodiments, the level of the HCN4 gene or HCN4 polypeptide can be reduced by, for example, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to an appropriate control. In some embodiments, the activity of the HCN4 gene or HCN4 polypeptide is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to an appropriate control. In one embodiment of this aspect and all other aspects provided herein, HCN4 channel activity is reduced or inhibited using the inhibitor drug ivabradine.

[0368] Methods of modulating the level or activity of ion channels provided herein are further discussed elsewhere herein.

[0369] T-type calcium channel - CACNA1H: Voltage-gated Ca 2+ VGCCs are Ca second messengers important for many cellular responses. 2+VGCCs provide an important pathway for the influx of calcium ions. VGCCs have traditionally been classified based on their biophysical and pharmacological properties: L-, N-, P-, Q-, and R-type channels constitute the family of high-voltage-activated (HVA) channels, whereas T-type channels are classified as low-voltage-activated (LVA) channels. Calcium channels mediate the influx of calcium ions into cells upon membrane polarization and consist of a complex of α1, α2 / δ, β, and γ subunits in a 1:1:1:1 ratio. The α1 subunit has 24 transmembrane segments and forms the pore through which ions pass into the cell. Multiple isoforms of each of the proteins in the complex exist, encoded by different genes or resulting from alternative splicing of the transcript. See, for example, Williams ME, et al., "Structure and functional characterization of a novel human low-voltage activated calcium channel," J Neurochem. 1999 Feb;72(2):791-9, and Zhang JF et al., "Distinctive pharmacology and kinetics of cloned neuronal Ca channels," the contents of each of which are incorporated herein by reference in their entirety. 2+ channels and their possible counterparts in mammalian CNS neurons.” Neuropharmacology 32, 1075-1088.

[0370] The gene CACNA1H is referred to herein as CACNA1H or Ca vIt encodes the α pore-forming subunit of the transient or T-type calcium channel, also called 3.2. The speciality of this type of channel is its opening at very negative potentials and voltage-dependent inactivation. T-type channels perform pacemaker functions in both central neurons and cardiac nodal cells and support calcium signaling in secretory cells and vascular smooth muscle. The structure and function of T-type calcium channels are known in the art. See, e.g., Cribbs L Let et al., "Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family," Circ. Res. 83:103-109 (1998); and Hansen PB, "Functional importance of T-type voltage-gated calcium channels in the cardiovascular and renal system: news from the world of knockout mice," Am J Physiol Regul Integr Comp Physiol. 2015 Feb 15;308(4):R227-37, the contents of each of which are incorporated herein by reference in their entirety.

[0371] The genomic DNA, transcript and polypeptide sequences of CACNA1H and Cav3.2 are known in the art; see, for example, the human CACNA1H genomic sequence located at positions 1153106-1221769 on human chromosome 16, Genbank accession AAK61268.1, NCBI gene ID: 8912 and NCBI reference sequence NG_012647.1; the human CACNA1H transcript sequence NCBI reference sequence: NM_021098.3; and the human CACNA1H (Cav3.2) polypeptide amino acid sequence described below: NCBI reference sequence NP_066921.2. >NP_066921.2 Voltage-gated T-type calcium channel subunit α-1H isoform a [Homo sapiens] TIFF2024517810000003.tif244164

[0372] T-type calcium channel, CACNA1H, Ca v 3.2, the level or activity of its orthologs or variants can be manipulated, for example, by targeting the full-length gene or coding sequence for deletion or disruption, or by modifying structural domains required for function (e.g., pore-forming domains), or by targeting ion transport through the channel via pharmacological or genetic modifications described herein. For further details regarding the structure and function of CACNA1H (Cav3.2), see, for example, Rzhepetskyy Y, et al., "Cav3.2 / Stac1 molecular complex controls T-type channel expression at the plasma membrane." Channels (Austin). 2016 Sep 2; 10(5): 346-354, the entire contents of which are incorporated herein by reference.

[0373] Although complete knockout is effective, in some embodiments, the level of CACNA1H gene or Cav3.2 polypeptide can be reduced by, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to an appropriate control.In some embodiments, the activity of CACNA1H gene or Cav3.2 polypeptide is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to an appropriate control.In one embodiment of this aspect and all other aspects provided herein, CACNA1H channel activity is reduced or inhibited using one or more inhibitor drugs selected from the group consisting of miblefazil or ML-218.

[0374] Methods for modulating the level or activity of CACNA1H are further discussed elsewhere herein.

[0375] Sodium-calcium exchanger - SLC8A1: The sodium-calcium exchanger or NCX1 (also known as solute transporter family 8 member A1, herein referred to as SLC8A1) is encoded by the SLC8A1 gene. The cardiac sodium / calcium exchanger (NCX1) is a bidirectional calcium transporter that contributes to the electrical activity of the heart. Specifically, it transports Ca 2+ The Ca concentration alternates between high levels during contraction and low levels during relaxation. 2+ The increase in concentration is primarily due to Ca from intracellular stores. 2+ However, some Ca 2+ also enters the cell through the sarcolemma (cell membrane). 2+ Ca is sequestered in intracellular stores. To prevent overload of intracellular stores, Ca 2+ must be extruded from the cell. + -Ca 2+When relaxed, the exchanger 2+ The primary mechanism by which cardiac Na(+)-Ca(2+) exchanger is extruded from cells is described in detail in the following sections: Shieh, B.-H., Xia, Y., Sparkes, RS, Klisak, I., Lusis, AJ, Nicoll, DA, Philipson, KD, "Mapping of the gene for the cardiac sarcolemmal Na(+)-Ca(2+) exchanger to human chromosome 2p21-p23," Genomics 12:616-617, (1992); and Kang, T.M., Hilgemann, DW, "Multiple transport modes of the cardiac Na(+) / Ca(2+) exchanger," Nature 427:544-548, (2004), the contents of each of which are incorporated herein by reference in their entirety.

[0376] The structure and function of NCX1, including human genomic DNA, transcript, and polypeptide sequences, are known; see, e.g., the human SLC8A1 genomic sequence is located at position c40512452-40094523 on human chromosome 2, Genbank accession AF128524, NCBI Gene ID: 6546, and NCBI Reference Sequence NC_000002.12; the human SLC8A1 transcript sequence NCBI Reference Sequence: NM_021097.5; and the human SLC8A1 (NCX1) polypeptide amino acid sequence described below: NCBI Reference Sequence NP_066920.1. >NP_066920.1 Sodium / calcium exchanger 1 isoform A precursor [Homo sapiens] TIFF2024517810000004.tif100164

[0377] The levels or activity of SLC8A1, NCX1, their orthologs or variants can be manipulated, for example, by targeting the full-length gene or coding sequence for deletion or disruption, or by modifying, for example, structural domains required for function, or by targeting ion transport through the channel via pharmacological or genetic modifications described herein. For further details regarding the structure and function of NCX1, see, for example, Iwamoto, T., et al., "Salt-sensitive hypertension is triggered by Ca(2+) entry via Na+ / Ca+ exchanger type-1 in vascular smooth muscle," Nature Med. 10:1193-1199, (2004); Langenbacher, A. D. et al., "Mutation in sodium-calcium exchanger 1 (NCX1) causes cardiac fibrillation in zebrafish," Proc. Nat. Acad. Sci. 102:17699-17704, (2005); and Wakimoto, K., et al., "Targeted disruption of Na(+) / Ca(2+) exchanger gene leads to cardiomyocyte apoptosis and defects in See also, "heartbeat." J. Biol. Chem. 275:36991-36998, (2000).

[0378] Although complete knockout is effective, in some embodiments, the level of the SLC8A1 gene or NCX1 polypeptide can be reduced by, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to an appropriate control. In some embodiments, the activity of the SLC8A1 gene or NCX1 polypeptide is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to an appropriate control. Methods for modulating the level or activity of the NCX1 ion channel are further discussed elsewhere herein.

[0379] Inwardly rectifying potassium channel 2 - KIR2.1 - KCNJ2 Inwardly rectifying potassium (Kir) channels are important regulators of resting membrane potential and cell excitability. Kir channel activity is critically dependent on the integrity of the channel's interaction with phosphatidylinositol 4,5-bisphosphate (PIP(2)). See, for example, Lopes, CMB, et al., "Alterations in conserved Kir channel-PIP(2) interactions underlying channelopathies," Neuron 34:933-944, (2002); and Donaldson, MR, et al., "PIP2 binding residues of Kir2.1 are common targets of mutations causing Andersen syndrome," Neurology 60:1811-1816, (2003), the entire contents of each of which are incorporated herein by reference.

[0380] Specifically, the gene KCNJ2 encodes the inwardly rectifying potassium channel Kir2.1 (also referred to herein as KCNJ2), which is expressed in the heart, lung, brain, placenta, and skeletal muscle. Kir2.1 is responsible for developmental signaling and regulates cellular excitability in the heart. See, for example, Plaster, N et al., "Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome," Cell 105:511-519, (2001), the entire contents of which are incorporated herein by reference. Therefore, mutations in KCNJ2 can lead to cardiovascular diseases such as Andersen's syndrome, familial atrial fibrillation, and short QT syndrome.

[0381] The structure and function of KCNJ2 (Kir2.1), including human genomic DNA, transcript, and polypeptide sequences, are known in the art, for example, the human KCNJ2 genomic sequence is located at positions 5001-15510 on human chromosome 17, GenBank accession AF153819, NCBI Gene ID: 3759, and NCBI Reference Sequence NG_008798.1; the human cDNA sequence of KCNJ2: GenBank and NCBI accession numbers BC152811.1, AB528777.1, NM_000891.3, CP034495.1, AH009400.2, AF153820.1, and NG_008798.1; the human KCNJ2 transcript sequence NCBI Reference Sequence: NM_000891.3, and the human KCNJ2 (Kir2.1) polypeptide amino acid sequence set forth below: See also NCBI reference sequence NP_000882.1. >NP_000882.1 Inwardly rectifying potassium channel 2 [Homo sapiens] TIFF2024517810000005.tif45164

[0382] The level or activity of KCNJ2 (Kir2.1), its orthologs, or variants can be manipulated pharmacologically or by genetic modifications as described herein by modifying regulatory elements, increasing the copy number of the sequence encoding the polypeptide, or modifying ion transport through the channel. For further details, see, for example, Raab-Graham, K., Radeke, C.M., Vandenberg, C.A., "Molecular cloning and expression of a human heart inward rectifier potassium channel," Neuroreport 5:2501-2505, (1994); and Derst, C., et al., "Genetic and functional linkage of Kir5.1 and Kir2.1 channel subunits," FEBS Lett. 491:305-311, (2001), the contents of each of which are incorporated herein by reference in their entirety.

[0383] In some embodiments, the level of the KCNJ2 gene or Kir2.1 polypeptide is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to a suitable control, including, for example, at least 2X, 3X, 4X, 5X or more. In some embodiments, the activity of the KCNJ2 gene or Kir2.1 polypeptide is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including, for example, at least 2X, 3X, 4X, 5X or more, compared to a suitable control.

[0384] In addition to the channels and exchangers described above, it is contemplated herein that other ion channels or their regulators can be inhibited or activated to further enhance engraftment of in vitro differentiated cardiomyocytes. Methods for modulating the levels or activity of the ion channels and exchangers described herein are further discussed below.

[0385] Genetic modification of ion channel expression and function The expression of the ion channels and exchangers described herein (e.g., HCN4, CACNA1H, SLC8A1, and KCNJ2) can be altered, deleted, inhibited, overexpressed, or activated so that the in vitro differentiated cardiomyocytes described herein do not exhibit electrical disturbances that can cause transplant arrhythmias after transplantation into a subject. This can be achieved by standard gene editing of the target sequence, with or without treatment with one or more channel blockers, such as, for example, ivabradine, miblefazil, or ML-218. In some embodiments, methods for genetically modifying cells to knock out, knock down, or otherwise modify one or more genes, including the genes of ion channels and exchangers described herein (e.g., HCN4, CACNA1H, SLC8A1, and KCNJ2), include, for example, using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, and site-specific nucleases, including clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems, as well as nickase systems, base editing systems, prime editing systems, and gene writing systems known in the art. This can also be achieved by utilizing the gene editing system (e.g., CRISPR / Cas) of the present disclosure in any manner available to those skilled in the art.

[0386] In one embodiment of any aspect described herein, the expression of gene editing mechanism / system, for example, RNA-guided nuclease and guide sequence, TALEN, zinc finger nuclease, etc., is expressed under the control of cell type-specific regulatory sequence that promotes their expression in the cells of cardiomyocyte development lineage.As described elsewhere herein, this can be useful, for example, to provide knockout (or knockin) of sequence only after the cardiomyocyte differentiation program has begun.

[0387] The target sequence for gene editing can be determined by methods known in the art, and the method of inhibiting gene function in host cells is known in the art.Non-limiting examples of gene knockdown, inhibition and modification include, but are not limited to, the introduction of inhibitory nucleic acid, including the use of CRISPR / Cas system (for example, CRISPR / Cas9 system), transcription activator-like effector nuclease (TALENS), zinc finger nuclease, etc. for targeted gene cleavage, and the expression of inhibitory nucleic acid.Exemplary embodiments of the type of inhibitory nucleic acid can include, for example, siRNA, shRNA, miRNA and / or miRNA.Those skilled in the art can design and test inhibitory nucleic acid active substances or gene editing approaches that target the expression of HCN4, CACNA1H, and / or SLC8A1.

[0388] Methods for preparing and delivering gene editing systems are described, for example, in International Publication No. WO 2015 / 013583; U.S. Patent No. 10,640,789; U.S. Patent Application Publication No. 2019 / 0367948; U.S. Patent Application Publication No. 2017 / 0266320; U.S. Patent Application Publication No. 2018 / 0171361; U.S. Patent Application Publication No. 2016 / 0175462 and U.S. Patent Application Publication No. 2018 / 0195089, the contents of each of which are incorporated herein by reference in their entirety.

[0389] CRISPR / Cas system In some embodiments, the cells described herein are produced using CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system.CRISPR system was first discovered in prokaryotes (for example, bacteria and archaea) as a system involved in the defense against invading phage and plasmid, providing a form of adaptive immunity.Currently, CRISPR system has been improved and used as a general gene editing tool in research and clinical applications.Generally, CRISPR is a collective term for a gene modification system that uses enzymes and factors derived from the defense mechanism of prokaryotes against bacteriophage to precisely modify target gene sequence in a given cell type. A CRISPR gene editing system can include transcripts and other elements involved in expression of or directing activity of a Cas gene, including sequences encoding a Cas nuclease gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0390] The guide sequence of a CRISPR system is designed to have complementarity to a target sequence (e.g., HCN4, CACNA1H, and / or SLC8A1, as described herein). Depending on the exact system used, the target sequence can include any DNA or RNA polynucleotide sequence. Hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. An active CRISPR complex results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). Perfect complementarity between the target sequence and the guide sequence is not necessarily required, as long as there is sufficient complementarity to allow hybridization and promote the formation of an active CRISPR complex.

[0391] CRISPR / Cas systems generally contain at least two components: one or more guide RNAs (gRNAs) and a Cas protein. The Cas protein is a nuclease that introduces a double-strand break (DSB) into the target site. CRISPR / Cas systems are divided into two major classes: Class 1 systems use a complex of multiple Cas proteins to degrade nucleic acids; Class 2 systems use a single large Cas protein for the same purpose. Class 1 systems are divided into types I, III, and IV; Class 2 systems are divided into types II, V, and VI. Different Cas proteins adapted for gene editing applications include Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (C2c10), and Cas12g. Cas proteins include, but are not limited to, Cas12h, Cas12i, Cas12k (C2c5), Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, C2c4, C2c8, C2c9, Cmr5, Cse1, Cse2, Csf1, Csm2, Csn2, Csx10, Csx11, Csy1, Csy2, Csy3, and Mad7. The most widely used Cas protein is type II Cas, and is described herein as an example. These Cas proteins can be derived from or originate from different source species. For example, Cas9 can be derived from Streptococcus pyogenes or Staphylococcus aureus (S. aureus).

[0392] In the genome of the original microorganism, the type II CRISPR system integrates sequences from the invaded DNA between CRISPR repeats encoded as an array in the host genome. Transcripts from the CRISPR repeat array are processed into CRISPR RNAs (crRNAs), each of which contains a variable sequence transcribed from the invaded DNA known as a "protospacer" sequence and a portion of the CRISPR repeats. Each crRNA hybridizes with a second trans-activating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer-encoded portion of the crRNA guides the Cas9 complex to cleave complementary target DNA sequences if they are adjacent to a short sequence known as a "protospacer adjacent motif" (PAM).

[0393] Since its discovery, the CRISPR system has been modified to induce sequence-specific DSBs and targeted genome editing in a wide range of cells and organisms, from bacteria to eukaryotic cells, including human cells. In its use in gene editing applications, artificially designed synthetic gRNAs have replaced the original crRNA:tracrRNA complex. For example, the gRNA can be a single guide RNA (sgRNA) composed of a crRNA, a tetraloop, and a tracrRNA. The crRNA typically contains a user-designed complementary region (also known as a spacer, typically approximately 20 nucleotides long) designed to recognize the target DNA of interest. The tracrRNA sequence contains a scaffold region for Cas nuclease binding. The crRNA and tracrRNA sequences are connected by a tetraloop, each with a short repeat sequence for hybridization with the other, thereby generating a chimeric sgRNA. The genome target of the Cas nuclease can be changed simply by changing the spacer or complementary region sequence present in the gRNA. The complementary region guides the Cas nuclease to the target DNA site through standard RNA-DNA complementary base-pairing rules.

[0394] For Cas nucleases to function, a PAM must be present immediately downstream of the target sequence in genomic DNA. Recognition of the PAM by the Cas protein is thought to destabilize the adjacent genomic sequence, allowing the gRNA to interrogate the sequence and, if a matching sequence is present, result in gRNA-DNA pairing. The specific sequence of the PAM varies depending on the species of the Cas gene. For example, the most commonly used Cas9 nuclease from Streptococcus pyogenes recognizes a PAM sequence of 5'-NGG-3' or, at a lower rate, a PAM sequence of 5'-NAG-3', where "N" can be any nucleotide. Other Cas nuclease variants with alternative PAMs have also been characterized and successfully used for genome editing, as summarized in Table 1 below.

[0395] Table 1. Exemplary Cas nuclease variants and their PAM sequences TIFF2024517810000006.tif54163R=A or G; Y=C or T; W=A or T; V=A, C, or G; N=any base

[0396] Any CRISPR / Cas system that can change target polynucleotide sequence in cells can be used.Such CRISPR-Cas system can use various Cas proteins (Haft et al.PLoS Comput Biol.2005;1(6)e60).The molecular mechanism of such Cas protein that enables CRISPR / Cas system to change target polynucleotide sequence in cells includes RNA binding protein, endonuclease and exonuclease, helicase and polymerase.In some embodiments, CRISPR / Cas system is CRISPRI type system.In some embodiments, CRISPR / Cas system is CRISPRII type system.In some embodiments, CRISPR / Cas system is CRISPRRV type system.

[0397] The gene editing (e.g., CRISPR / Cas) system described herein can be used to change any target polynucleotide sequence in a cell. Those skilled in the art will readily understand that the desired target polynucleotide sequence to be changed in any particular cell can correspond to any genomic sequence whose expression is associated with a disorder or otherwise facilitates the entry of pathogens into the cell. For example, the desired target polynucleotide sequence to be changed in a cell can be a polynucleotide sequence corresponding to a genomic sequence containing a single polynucleotide polymorphism associated with a disease.

[0398] In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

[0399] In some embodiments, the Cas nuclease can include one or more mutations to alter the activity, specificity, recognition, and / or other characteristics of the Cas nuclease. For example, the Cas nuclease can have one or more mutations that alter its fidelity to reduce off-target effects (e.g., eSpCas9, SpCas9-HF1, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9). As another example, the Cas nuclease can have one or more mutations that alter its PAM specificity.

[0400] In some embodiments, the CRISPR / Cas systems provided herein include a Cas protein and at least one or two ribonucleic acids capable of hybridizing to a target motif in a target polynucleotide sequence, guiding the Cas protein to the target motif in the target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues (i.e., a polymer of amino acids) linked by peptide bonds, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0401] In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some examples, the substitutions and / or modifications can prevent or reduce proteolysis and / or extend the half-life of the polypeptide within a cell. In some embodiments, the Cas protein can comprise peptide bond replacements (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein can comprise naturally occurring amino acids. In some embodiments, the Cas protein can comprise alternative amino acids (e.g., D-amino acids, β-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein can comprise modifications to include moieties (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0402] In some embodiments, the Cas protein comprises a core Cas protein, an isoform thereof, or any Cas-like protein having a function or activity similar to any Cas protein or its isoform. Exemplary Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. In some embodiments, the Cas protein comprises an Escherichia coli (E. coli) subtype Cas protein (also known as CASS2). Exemplary E. coli subtype Cas proteins include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Ypest subtype Cas protein (also known as CASS3). Exemplary Ypest subtype Cas proteins include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises a Nmeni subtype Cas protein (also known as CASS4). Exemplary Cas proteins of the Nmeni subtype include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5).Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of the Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6. See, e.g., Klompe et al., Nature 571, 219-225 (2019); Strecker et al., Science 365, 48-53 (2019). In some embodiments, the Cas protein comprises a Cas protein of the type I subtype. Type I CRISPR / Cas effector proteins are a subtype of class 1 CRISPR / Cas effector proteins. Examples include, but are not limited to, Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Csel, Csel2, Csyl, Csy2, Csy3, and / or GSU0054. In some embodiments, the Cas protein comprises Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Csel, Csel2, Csyl, Csy2, Csy3, and / or GSU0054. In some embodiments, the Cas protein comprises a type II subtype Cas protein. Type II CRISPR / Cas effector proteins are a subtype of class 2 CRISPR / Cas effector proteins. Examples include, but are not limited to, Cas9, Csn2, and / or Cas4. In some embodiments, the Cas protein comprises Cas9, Csn2 and / or Cas4. In some embodiments, the Cas protein comprises a type III subtype Cas protein.Type III CRISPR / Cas effector proteins are a subtype of Class 1 CRISPR / Cas effector proteins. Examples include, but are not limited to, Cas10, Csm2, Cmr5, Cas10, Csx11, and / or Csx10. In some embodiments, the Cas protein comprises Cas10, Csm2, Cmr5, Cas10, Csx11, and / or Csx10. In some embodiments, the Cas protein comprises a Type IV subtype Cas protein. Type IV CRISPR / Cas effector proteins are a subtype of Class 1 CRISPR / Cas effector proteins. Examples include, but are not limited to, Csf1. In some embodiments, the Cas protein comprises Csf1. In some embodiments, the Cas protein comprises a Type V subtype Cas protein. Type V CRISPR / Cas effector proteins are a subtype of Class 2 CRISPR / Cas effector proteins. For examples of type V CRISPR / Cas systems and their effector proteins (e.g., Cas12 family proteins such as Cas12a), see, e.g., Shmakov et al., Nat Rev Microbiol. 2017;15(3):169-182: "Diversity and evolution of class 2 CRISPR-Cas systems." Examples include, but are not limited to, the Cas12 family (Cas12a, Cas12b, Cas12c), C2c4, C2c8, C2c5, C2c10, and C2c9, as well as CasX (Cas12e) and CasY (Cas12d). See also, e.g., Koonin et al., Curr Opin Microbiol. 2017;37:67-78: "Diversity, classification, and evolution of CRISPR-Cas systems." In some embodiments, the Cas protein comprises a Cas12 protein, such as Cas12a, Cas12b, Cas12c, Cas12d and / or Cas12e.

[0403] In some embodiments, the Cas protein comprises any one of the Cas proteins described herein, or a functional portion thereof. As used herein, "functional portion" refers to a portion of a peptide that retains its ability to form a complex with at least one ribonucleic acid (e.g., a guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA-binding domain, at least one RNA-binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked Cas12a (also known as Cpf1) protein functional domains selected from the group consisting of a DNA-binding domain, at least one RNA-binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of an HNH nuclease domain. In some embodiments, the functional portion of the Cas12a protein comprises a functional portion of a RuvC-like domain.

[0404] In some embodiments, exogenous Cas proteins can be introduced into cells in the form of polypeptides. In some embodiments, Cas proteins can be conjugated or fused to cell-penetrating polypeptides or cell-penetrating peptides. As used herein, "cell-penetrating polypeptides" and "cell-penetrating peptides" refer to polypeptides or peptides that facilitate the uptake of molecules into cells, respectively. Cell-penetrating polypeptides can contain detectable labels.

[0405] In some embodiments, the Cas protein can be conjugated or fused to a charged protein (e.g., having a positive, negative, or overall neutral charge). Such attachment can be covalent. In some embodiments, the Cas protein can be fused to the highly positively charged GFP to significantly increase the ability of the Cas protein to penetrate cells (Cronican et al. ACS Chem Biol. 2010;5(8):747-52). In some embodiments, the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into cells. Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a highly positively charged GFP. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a PTD. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a tat domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to an oligoarginine domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a penetratin domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a highly positively charged GFP.

[0406] In some embodiments, the Cas protein can be introduced into a cell containing a target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein. The method of introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection with a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises a modified DNA described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA described herein (e.g., a synthetic modified mRNA).

[0407] In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic, modified mRNA) described herein.

[0408] The methods disclosed herein contemplate the use of any ribonucleic acid that can guide a Cas protein to a target motif in a target polynucleotide sequence and hybridize to the target motif in the target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises a tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises a CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that guides a Cas protein to a target motif in a target polynucleotide sequence in a cell and hybridizes to the target motif in the target polynucleotide sequence in the cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that guides a Cas protein to a target motif in a target polynucleotide sequence in a cell and hybridizes to the target motif in the target polynucleotide sequence in the cell. In some embodiments, both of the one to two ribonucleic acids comprise a guide RNA that guides a Cas protein to a target motif in a target polynucleotide sequence in a cell and hybridizes to the target motif in the target polynucleotide sequence in the cell. The ribonucleic acids provided herein can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR / Cas system used and the sequence of the target polynucleotide, as will be understood by those skilled in the art. The one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to the deoxyribonucleic acid motif recognized by the Cas protein.

[0409] In some embodiments, each of the one to two ribonucleic acids comprises a guide RNA that guides the Cas protein to a target motif in a target polynucleotide sequence in the cell and hybridizes to the target motif in the target polynucleotide sequence in the cell.

[0410] In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to sequences on the same strand of a target polynucleotide sequence and / or hybridize to sequences on the same strand of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to sequences on opposite strands of a target polynucleotide sequence and / or hybridize to sequences on the same strand of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are not complementary to sequences on opposite strands of a target polynucleotide sequence and / or do not hybridize to sequences on the same strand of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to overlapping target motifs of a target polynucleotide sequence and / or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to offset target motifs of the target polynucleotide sequence.

[0411] In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one to two ribonucleic acids are introduced into the cell via viral transduction (e.g., lentiviral transduction). In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic, modified mRNA) described herein.

[0412] If gene editing is desired, an editing sequence or editing template polynucleotide can be used for recombination into a targeted locus containing the target sequence. In some embodiments, the recombination is homologous recombination.

[0413] Base editing is another approach for changing endogenous genes as described herein.Base editing can be used to introduce point mutations into cellular DNA without double-strand breaks.In some embodiments, the method for changing endogenous nucleic acid described herein is by cytosine base editing, adenine base editing, antisense oligonucleotide-dependent A to I RNA editing or Cas13 base editing.Base editing methods are known in the art and are described, for example, in Rees et al.Nature Rev Genet.19(12);770-788(2018) and Kopmor et al., Nature 533,420-424(2016), which are incorporated herein by reference in their entirety.

[0414] The CRISPR system or base editing elements can be combined in a single vector and can be arranged in any suitable orientation, such as one element being 5' ("upstream" of the second element) or 3' ("downstream" of the second element) relative to the second element. The coding sequence of one element can be located on the same or opposite strand of the coding sequence of the second element and can be oriented in the same or opposite direction. In some embodiments, a single promoter drives the expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and tracr sequence embedded in one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

[0415] ZFN Zinc finger nucleases (ZFNs) are fusion proteins containing an array of site-specific DNA-binding domains adapted from zinc finger-containing transcription factors attached to the endonuclease domain of the bacterial FokI restriction enzyme. ZFNs can have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) DNA-binding or zinc finger domains. See, for example, Carroll et al., Genetics Society of America (2011) 188:773-782; Kim et al., Proc. Natl. Acad. Sci. USA (1996) 93:1156-1160. Each zinc finger domain is a small protein structural motif stabilized by one or more zinc ions and typically recognizes a 3-4 bp DNA sequence. Thus, tandem domains can potentially bind to unique, extended nucleotide sequences within a cell's genome.

[0416] Various zinc fingers of known specificity can be combined to create multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp. A variety of selection and modular assembly techniques are available for generating zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid system, bacterial one-hybrid and two-hybrid systems, and mammalian cells. Zinc fingers can be engineered to bind to specific nucleic acid sequences. Criteria for engineering zinc fingers to bind to specific nucleic acid sequences are known in the art. For example, see Sera et al., Biochemistry (2002) 41: 7074-7081; Liu et al., Bioinformatics (2008) 24: 1850-1857.

[0417] ZFNs containing a FokI nuclease domain or other dimeric nuclease domains function as dimers. Therefore, a pair of ZFNs is required to target non-palindromic DNA sites. Two individual ZFNs must bind opposite strands of DNA with their nucleases appropriately spaced apart. See Bitinaite et al., Proc. Natl. Acad. Sci. USA (1998) 95:10570-10575. To cleave a specific site in the genome, a pair of ZFNs is designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand. Upon binding of the ZFNs on both sides of the site, the nuclease domains dimerize and cleave the DNA at that site, generating a DSB with a 5' overhang. Homologous recombination (HDR) can then be used to introduce the specific mutation with the aid of a repair template containing the desired mutation flanked by homology arms. The repair template is usually an exogenous double-stranded DNA vector introduced into the cell. See Miller et al., Nat. Biotechnol. (2011) 29:143-148; Hockemeyer et al., Nat. Biotechnol. (2011) 29:731-734.

[0418] TALEN In some embodiments, the cells described herein are generated using the Transcription Activator-Like Effector Nuclease (TALEN) method. By "TALE nuclease" (TALEN) is meant a fusion protein consisting of a nucleic acid binding domain, typically derived from a transcription activator-like effector (TALE), and a single nuclease catalytic domain for cleaving a nucleic acid target sequence. TALENs are another example of artificial nucleases that can be used to edit target genes. TALENs are derived from a DNA-binding domain called a TALE repeat, which typically contains a tandem array of 10-30 repeats that bind and recognize extended DNA sequences. Each repeat is 33-35 amino acids long, with two adjacent amino acids (called repeat variable dimers, or RVDs) conferring specificity for one of four DNA base pairs. Thus, there is a one-to-one correspondence between the repeat and the base pairs in the target DNA sequence. The catalytic domain is preferably a nuclease domain, more preferably a domain with endonuclease activity, such as I-TevI, ColE7, NucA, and Fok-I. In certain embodiments, the TALE domain can be fused to a meganuclease, such as I-CreI and I-OnuI, or functional variants thereof. In a more preferred embodiment, the nuclease is a monomeric TALE nuclease. A monomeric TALE nuclease is a TALE nuclease that does not require dimerization for specific recognition and cleavage, such as the fusion of engineered TAL repeats with the catalytic domain of I-TevI, as described in WO2012138927. Transcription activator-like effector (TALE) is a protein from the bacterial species Xanthomonas, which contains multiple repeats, each containing a dinucleotide repeat (RVD) at positions 12 and 13 specific for each nucleotide base of a nucleic acid target sequence. TALEN kits are commercially available.

[0419] TALENs are artificially created by fusing one or more TALE DNA binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) to a nuclease domain, such as a FokI endonuclease domain. See Zhang, Nature Biotech. (2011) 29:149-153. Several mutations to FokI have been created for use in TALENs, which improve, for example, cleavage specificity or activity. See Cermak et al., Nucl. Acids Res. (2011) 39:e82; Miller et al., Nature Biotech. (2011) 29:143-148; Hockemeyer et al., Nature Biotech. (2011) 29:731-734; Wood et al., Science (2011) 333:307; Doyon et al., Nature Methods (2010) 8:74-79; Szczepek et al., Nature Biotech (2007) 25:786-793; Guo et al., J. Mol. Biol. (2010) 200:96. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains to be properly oriented and spaced apart for sites in the target genome. Both the number of amino acid residues between the TALE DNA binding domain and the FokI nuclease domain and the number of bases between two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al., Nature Biotech. (2011) 29:143-148.

[0420] By combining engineered TALE repeat with nuclease domain, it can create the site-specific nuclease that is specific to any desired DNA sequence.Similar to ZFN, TALEN can be introduced into cell to generate DSB at desired target site in genome, and therefore can be used to knock out gene or knock in mutation in the similar HDR-mediated pathway.See Boch, Nature Biotech.(2011)29:135-136;Boch et al., Science(2009)326:1509-1512;And Moscou et al., Science(2009)326:3501.

[0421] In some embodiments, cells are engineered using zinc finger nucleases (ZFNs). A "zinc finger binding protein" is a protein or polypeptide that binds DNA, RNA, and / or proteins, preferably in a sequence-specific manner, as a result of stabilization of the protein structure through the coordination of zinc ions. The term zinc finger binding protein is often abbreviated as zinc finger protein or ZFP. Individual DNA-binding domains are typically referred to as "finger(s)." ZFPs have at least one finger, typically two, three, or six fingers. Each finger binds 2-4 base pairs of DNA, typically 3 or 4 base pairs. ZFPs bind to nucleic acid sequences called target sites or target segments. Each finger typically contains a zinc-chelating DNA-binding subdomain of about 30 amino acids. Studies have demonstrated that this class of single zinc finger consists of an α-helix containing two invariant histidine residues that coordinate zinc with two cysteine ​​residues in a single β-turn (see, e.g., Berg & Shi, Science 271:1081-1085 (1996)).

[0422] Homing endonucleases In some embodiments, the cells described herein are generated using a homing endonuclease. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and generate single- or double-strand breaks. Homing endonucleases are highly specific and recognize DNA target sites ranging in length from 12 to 45 base pairs (bp), typically ranging in length from 14 to 40 bp. The homing endonuclease can correspond to, for example, LAGLIDADG endonuclease, HNH endonuclease, or GIY-YIG endonuclease. In some embodiments, the homing endonuclease can be an I-CreI variant.

[0423] Meganuclease In some embodiments, the cells described herein are produced using meganucleases. Meganucleases are enzymes of the endonuclease family that are characterized by their ability to recognize and cleave large DNA sequences (14 to 40 base pairs). Meganucleases, by definition, are sequence-specific endonucleases that recognize large sequences (Chevalier, BS and BL Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). Meganucleases can cleave unique sites in living cells, thereby enhancing gene targeting by more than 1000-fold in the vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21, 5034-5040; Rouet et al., Mol. Cell. Biol., 1994, 14, 8096-8106; Choulika et al., Mol. Cell. Biol., 1995, 15, 1968-1973; Puchta et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 5055-5060; Sargent et al., Mol. Cell. Biol., 1997, 17, 267-77; Donoho et al., (e.g., Elliott et al., Mol. Cell. Biol., 1998, 18, 4070-4078; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101; Cohen-Tannoudji et al., Mol. Cell. Biol., 1998, 18, 1444-1448). Meganucleases are classified into families based on their structural motifs that affect nuclease activity and / or DNA recognition. The most widespread and best-known meganucleases are the LAGLIDADG family of proteins, whose name is derived from a conserved amino acid sequence. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.On the other hand, GIY-YIG family members possess a GIY-YIG module, which is 70-100 residues long and contains four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey et al., Nature Struct. Biol. (2002) 9:806-811. His-Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines spanning a region encompassing several hundred amino acid residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774. Members of the NHN family are defined by a motif containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.

[0424] Because the high specificity required makes it unlikely to identify a natural meganuclease for a particular target DNA sequence, various methods, including mutagenesis and high-throughput screening methods, have been used to create meganuclease variants that recognize unique sequences. Strategies for engineering meganucleases with altered DNA binding specificity, for example, that bind to a given nucleic acid sequence, are known in the art. For example, Chevalier et al.,Mol.Cell.(2002)10:895-905;Epinat et al.,Nucleic Acids Res(2003)31:2952-2962;Silvaet al.,J Mol.Biol.(2006)361:744-754;Seligman et al.,Nucleic Acids Res(2002)30:3870-3879;Sussman et al.,J Mol Biol(2004)342:31-41;Doyon et al.,J Am Chem Soc(2006)128:2477-2484;Chen et al.,Protein Eng Des Sel(2009)22:249-256;Arnould et al.,J Mol See Biol. (2006) 355:443-458; and Smith et al., Nucleic Acids Res. (2006) 363(2):283-294.

[0425] Similar to ZFN and TALEN, meganuclease can create DSB in genomic DNA, which, if improperly repaired, for example, through NHEJ, can create frameshift mutations, resulting in reduced expression of target genes in cells. Alternatively, meganuclease can be used to introduce foreign DNA into cells. Depending on the sequence of foreign DNA and chromosomal sequence, this process can be used to modify target genes. See Silva et al., Current Gene Therapy (2011) 11:11-27.

[0426] transposase In some embodiments, the cells described herein are produced using transposase.Transposase is an enzyme that binds to the end of transposon and catalyzes the transposon's movement to another part of the genome by cut-and-paste mechanism or replication transposition mechanism.By linking transposase to other systems such as CRISPER / Cas system, new gene editing tools can be developed to enable site-specific insertion or manipulation of genomic DNA.There are two known DNA integration methods using transposons: one uses catalytically inactive Cas effector protein and Tn7-like transposon.Transposase-dependent DNA integration does not induce DSB in the genome, and can ensure safer and more specific DNA integration.

[0427] Nickase In some embodiments, the cells described herein are produced using nickase. The nuclease domain of Cas, particularly Cas9 nuclease, can be independently mutated to generate an enzyme called DNA "nickase." Nickase can introduce single-strand breaks with the same specificity as conventional CRISPR / Cas nuclease systems, including CRISPR / Cas9. Nickase can be used to generate double-strand breaks, which can be used in gene editing systems (Mali et al., Nat Biotech, 31(9):833-838(2013); Mali et al. Nature Methods, 10:957-963(2013); Mali et al., Science, 339(6121):823-826(2013)). In some instances, when two Cas nickases are used, long overhangs are generated at each of the cut ends instead of blunt ends, allowing for precise gene integration and further control over insertion (Mali et al., Nat Biotech, 31(9):833-838 (2013); Mali et al. Nature Methods, 10:957-963 (2013); Mali et al., Science, 339(6121):823-826 (2013)). Because both nicking Cas enzymes must effectively nick their target DNA, paired nickases can have lower off-target effects compared to Cas-based systems that make double-strand breaks (Ran et al., Cell, 155(2):479-480 (2013); Mali et al., Nat Biotech, 31(9):833-838 (2013); Mali et al. Nature Methods, 10:957-963 (2013); Mali et al., Science, 339(6121):823-826 (2013)).

[0428] RNA silencing or RNA interference In some embodiments, the cells provided herein are produced by using RNA silencing or RNA interference (also called RNAi, siRNA) to knock down (for example, reduce, eliminate or inhibit) the expression of polypeptide.Useful RNAi methods include those that utilize synthetic RNAi molecules, small interfering RNA (siRNA), PIWI-interacting RNA (piRNA), short hairpin RNA (shRNA), microRNA (miRNA) and other transient knockdown methods recognized by those skilled in the art.Reagents for RNAi, including sequence-specific shRNA, siRNA, miRNA, etc., are commercially available.

[0429] In some embodiments, stem cells or in vitro differentiated cardiomyocytes described herein are transiently transfected with components of a gene editing system (such as by transient transfection of one or more vectors or transfection with RNA) and modified via the activity of a CRISPR or base editing complex to establish new cells or cell lines comprising cells containing modifications to host cell genes.

[0430] In some embodiments, it may be beneficial to express gene editing mechanism in a cell-, tissue- and / or development-specific manner.This may be particularly useful, for example, when transgene expression or gene knockout is harmful to cells at some stages of development or differentiation, but not at other stages.Therefore, when transgene or knockout is harmful to stem cells, such as pluripotent stem cells, it may be beneficial to use the regulatory element that is only active at the later stage of differentiation, for example, cardiomyocyte differentiation, to drive the expression of gene editing mechanism.

[0431] In addition to the methods provided above, overexpression of KCNJ2, also known as Kir2.1, by the transplanted cardiomyocytes described herein can be achieved by contacting the cells with a nucleic acid encoding KCNJ2 (i.e., Kir2.1) or a vector containing such a nucleic acid.

[0432] Stimulated activity of KCNJ2 / Kir2.1 can be achieved by any of a number of methods known in the art. Factors to consider include the fact that expression of KCNJ2 is toxic to pluripotent cells. Therefore, it is beneficial to drive stimulated expression or overexpression of KCNJ2 through a developmentally regulated sequence that allows expression in immature cardiomyocytes but not in pre-differentiated pluripotent stem cells, and preferably not in mature cardiomyocytes where the endogenous gene is active. HCN4 and CACNA1H are non-limiting examples of genes that are regulated in a manner that is beneficial for controlling or driving a KCNJ2 transgene. SLC8A1 is an example of a gene that is regulated in a manner that is not expected to be beneficial for KCNJ2 expression, as it is highly expressed early in the differentiation program before further increasing. Thus, in one embodiment, a construct encoding a KCNJ2 transgene can be operably linked to regulatory sequences (e.g., promoters, enhancers, 5'-, 3'- and / or internal genomic regulatory sequences, etc.) derived from HCN4 or CACNA1H to provide beneficial expression of a KCNJ2 (Kir2.1) polypeptide via the compositions and methods described herein.

[0433] In another embodiment, the sequence encoding KCNJ2 (Kir2.1) can be knocked into or replaced by one of the genes to be inactivated in the target cell, such as HCN4 or CACNA1H. This approach has the advantage of both inactivating HNC4 or CACNA1H and stimulating the ectopic expression of KCNJ2 in the same gene editing process.

[0434] In some embodiments, gene editing method is combined with the pharmacological inhibition of HCN4 or CACNA1H channel.In one embodiment of this aspect and all other aspects provided herein, HCN4 channel is inhibited by using inhibitor drug ivabradine.In another embodiment of this aspect and all other aspects provided herein, CACNA1H channel is inhibited by using one or more inhibitor drugs selected from the group consisting of miblefadil or ML-218.

[0435] Pharmacological modulation of ion channel function In addition to genetic manipulation, pharmacological agents that inhibit or activate the ion channels and exchangers described herein (e.g., HCN4, CACNA1H, SLC8A1, and KCNJ2) can be used, as further discussed below. In some embodiments, pharmacological alterations of ion channel function are used in combination with the gene editing methods described herein.

[0436] In some embodiments, the agent is a small molecule, polypeptide, antibody or aptamer.The term "small molecule" used herein refers to natural (i.e., found in nature) or non-natural (i.e., not found in nature) organic or inorganic molecules, which can include but are not limited to peptide, peptidomimetic, amino acid, amino acid analog, polynucleotide, polynucleotide analog, aptamer, nucleotide, nucleotide analog, organic or inorganic compound (including, for example, heteroorganic compounds and organometallic compounds) with molecular weight of less than about 10,000 g / mol, organic or inorganic compound with molecular weight of less than about 5,000 g / mol, organic or inorganic compound with molecular weight of less than about 1,000 g / mol, organic or inorganic compound with molecular weight of less than about 500 g / mol, and the salt, ester and other pharmaceutically acceptable forms of such compounds. Examples of "small molecules" synthesized in the laboratory include, but are not limited to, compounds described in Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill Education 12th Edition (ISBN-13:978-0071624428), the contents of which are incorporated herein by reference in their entirety.

[0437] In some embodiments, the agent is administered to the in vitro differentiated cardiomyocytes prior to engraftment. In some embodiments, the agent is administered to the subject undergoing treatment after engraftment. In some embodiments, the agent is contacted with the cells before they are transplanted and administered to the subject receiving the cells after transplantation or engraftment.

[0438] Non-limiting examples of small molecules that can be used to modulate the levels or activity of the ion channels described herein are shown in Table 2 below.

[0439] (Table 2) Agents that inhibit HCN4, CACNA1H, SLC8A1 or activate KCNJ2. TIFF2024517810000007.tif81158 * FDA-approved drugs

[0440] In some embodiments of any aspect, HCN4 is inactivated by an agent selected from the group consisting of ivabradine, zetabladine, and ZD 7288.

[0441] In some embodiments of any aspect, CACNA1H (Cav3.2) is inactivated by an agent selected from the group consisting of mibefradil, ML218, and flunarizide.

[0442] In some embodiments of any aspect, SLC8A1 (NCX1) is inhibited by an agent selected from the group consisting of SEA-0400, KB-R7943, CGP 37157, and 3',4'-dichlorobenzamyl.

[0443] In some embodiments of any aspect, KCNJ2 (Kir2.1) is activated by zacopride.

[0444] It is envisioned that one or more agents that target the activity of one or more of these channels can be used in place of or in combination with genetic engineering of such channels in cells for engraftment.

[0445] In some embodiments, the ion channel protein HCN4, Ca VThe activity of 3.2 (also referred to herein as CACNA1H), NCX1 (also referred to herein as SLC8A1), and Kir2.1 (also referred to herein as KCNJ2) can be modulated by a combination of pharmacological and genetic approaches. Essentially any combination can be used, but as one non-limiting example, HCN4 activity can be inhibited using, for example, ivabradine, while CACNA1H, SLC8A1, and KCNJ2 activity are modulated or manipulated through one or more genetic approaches (e.g., knockout, knockdown, overexpression, as appropriate). Other drugs that can inhibit HCN4 activity include, but are not limited to, zetabladine and ZD 7288. Non-limiting examples of drugs that can inhibit CACNA1H activity include mibefradil, ML218, and flunarizide. A non-limiting example of a drug that can activate KCNJ2 is zacopride. Non-limiting examples of drugs that can inhibit SLC8A1 (NCX1) include SEA-0400, KB-R7943, CGP 37157, and 3',4'-dichlorobenzamyl. These and other drugs that alter the activity of these ion channels can be used at concentrations known to inhibit or activate the target ion channel, depending on the case.

[0446] In another embodiment of this aspect and all other aspects described herein, the KCNJ2 polypeptide is overexpressed by genomic insertion of the KCNJ2 gene, the SLC8A1 polypeptide is inhibited by genomic modification of the SLC8A1 gene, the HCN4 polypeptide is inhibited by either genomic modification or treatment with a pharmacological agent (e.g., ivabradine), and the CACNA1H polypeptide is inhibited by either genomic modification or treatment with a pharmacological agent (e.g., miblefadil, ML-218, flunarizide) or any combination thereof.

[0447] Cardiomyocytes for cardiac engraftment The compositions and methods described herein use cardiomyocytes contacted with a genetic engineering or gene editing system (e.g., CRISPR / Cas9) or agent (e.g., an ion channel blocker or an antiarrhythmic agent) that prevents electrical disturbances when the cardiomyocytes are engrafted into a subject for the treatment of a cardiac disease or disorder (e.g., myocardial infarction or heart failure).

[0448] Cardiac engraftment is administered to the site of cardiac injury in the heart.Those skilled in the art can determine the site of injury by methods known in the art.The main goal of cardiac engraftment is to provide the injured myocardium with electrical and mechanical stability, which cannot be achieved by pharmaceutical treatment alone.

[0449] The cardiomyocytes described herein can be isolated from a human subject or differentiated from stem cells or cardiac progenitors. The following describes various sources and stem cells that can be used to prepare cardiomyocytes for engraftment into a subject.

[0450] Stem cells are cells that retain the ability to regenerate themselves through mitotic cell division and can differentiate into more specialized cell types. Three broad types of mammalian stem cells include embryonic stem (ES) cells found in blastocysts, induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells, and adult stem cells found in adult tissues. Other sources of pluripotent stem cells may include amniotic or placenta-derived stem cells. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0451] The cardiomyocytes useful in the compositions and methods described herein can be differentiated from, among others, embryonic stem cells and induced pluripotent stem cells.In one embodiment, the compositions and methods provided herein use human cardiomyocytes differentiated from embryonic stem cells.Alternatively, in some embodiments, the compositions and methods provided herein do not include the generation or use of human cardiogenic cells made from cells taken from living human embryos.

[0452] Embryonic stem cells: Embryonic stem cells and their recovery methods are well known in the art, and are described, for example, in Trounson AO Reprod Fertil Dev (2001) 13:523, Roach ML Methods Mol Biol (2002) 185:1 and Smith AG Annu Rev Cell Dev Biol (2001) 17:435.The term "embryonic stem cells" refers to the pluripotent stem cells of the inner cell mass of embryonic blastocyst (see, for example, U.S. Patent No. 5,843,780, U.S. Patent No. 6,200,806).Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Patent No. 5,945,577, U.S. Patent No. 5,994,619, U.S. Patent No. 6,235,970).

[0453] Cells derived from embryonic sources can include embryonic stem cells or stem cell lines obtained from stem cell banks or other recognized depositories. Other means of generating stem cell lines include using blastomere cells from early embryos (approximately the 8-cell stage) before blastocyst formation. Such techniques correspond to the preimplantation genetic diagnosis techniques routinely performed in assisted reproduction medical institutions. In this approach, single blastomere cells are co-cultured with established ES cell lines and then separated from them to form fully competent ES cell lines.

[0454] Undifferentiated embryonic stem (ES) cells are easily recognized by those skilled in the art and typically appear in two dimensions of a microscopic field in colonies of cells with a high nucleus / cytoplasm ratio and prominent nuclei. In some embodiments, the human cardiomyocytes described herein are not derived from embryonic stem cells or any other cells derived from an embryo.

[0455] Induced pluripotent stem cells (iPSC): In some embodiments, the compositions and methods described herein utilize cardiomyocytes differentiated in vitro from induced pluripotent stem cells. An advantage of using iPSCs to generate cardiomyocytes for the compositions described herein is that, if so desired, the cells can be derived from the same subject to whom the desired human cardiomyocytes will be administered. That is, somatic cells can be obtained from the subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into human cardiomyocytes (e.g., autologous cells) for administration to the subject. Because the cardiomyocytes (or their differentiated progeny) are essentially derived from an autologous source, the risk of engraftment rejection or allergic reaction is reduced compared to the use of cells from another subject or group of subjects. While this is an advantage of iPSCs, in alternative embodiments, cardiomyocytes useful in the methods and compositions described herein are derived from a non-autologous source (e.g., allogeneic). Furthermore, the use of iPSCs obviates the need for cells obtained from an embryonic source. Thus, in one embodiment, the stem cells used to generate cardiomyocytes for use in the methods and compositions described herein are not embryonic stem cells.

[0456] Although differentiation is generally irreversible under physiological conditions, several methods have been developed in recent years for reprogramming somatic cells into induced pluripotent stem cells. Exemplary methods are known to those skilled in the art and are briefly described herein below.

[0457] Reprogramming is the process of altering or reversing the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming is the process of reverting cell differentiation to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture can result in some loss of fully differentiated characteristics. However, simply culturing such cells encompassed by the term differentiated cells does not render them non-differentiated (e.g., undifferentiated) or pluripotent. The transition of differentiated cells to pluripotency requires a reprogramming stimulus that exceeds the stimulus that results in partial loss of differentiated characteristics when differentiated cells are placed in culture. Compared to primary cell parents, which generally only have the capacity for a limited number of divisions in culture, reprogrammed cells are also characterized by the ability to be passaged for long periods without losing their proliferative potential.

[0458] The cells to be reprogrammed can be partially or highly differentiated prior to reprogramming. Thus, the cells can be highly differentiated somatic cells and can be derived from adult or somatic stem cells.

[0459] In some embodiments, reprogramming involves the complete reversion of the differentiated state of a differentiated cell (e.g., a somatic cell) to a pluripotent or multipotent state. In some embodiments, reprogramming involves the complete or partial reversion of the differentiated state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in the expression of specific genes by the cell, which expression further contributes to reprogramming. In certain embodiments described herein, reprogramming a differentiated cell (e.g., a somatic cell) causes the differentiated cell to adopt an undifferentiated state capable of self-renewal and differentiation into cells of all three germ layer lineages. These are induced pluripotent stem cells (iPSCs or iPSCs).

[0460] Methods for reprogramming somatic cells into iPSCs are known in the art.See, for example, U.S. Patent No. 8,129,187; U.S. Patent No. 8,058,065; U.S. Patent Application Publication No. 2012 / 0021519; Singh et al. Front. Cell Dev Biol. (2015); and Park et al. Nature (2008), which are incorporated by reference in their entirety.Specifically, iPSCs are generated from somatic cells by introducing a combination of reprogramming transcription factors.Reprogramming factors can be, for example, nucleic acids, vectors, small molecules, viruses, polypeptides, or any combination thereof.Non-limiting examples of reprogramming factors include Oct4 (octamer-binding transcription factor-4), Sox2 (sex determining region Y)-box 2, Klf4 (Kruppel-like factor-4), and c-Myc. Additional factors (e.g., LIN 28+Nanog, Esrrb, Pax5 shRNA, C / EBPa, p53 siRNA, UTF1, DNMT shRNA, Wnt3a, SV40 LT(T), hTERT) or chemicals (e.g., BIX-01294, BayK8644, RG108, AZA, dexamethasone, VPA, TSA, SAHA, PD025901+CHIR99021(2i), A-83-01) have been found to replace one or other of the basic reprogramming factors or increase the efficiency of reprogramming.

[0461] The particular approach or method used to generate pluripotent stem cells from somatic cells (e.g., any cell of the body excluding germline cells; fibroblasts, etc.) is not critical to the claimed invention. Thus, any method of reprogramming somatic cells to a pluripotent phenotype would be suitable for use in the methods described herein.

[0462] The reprogrammed somatic cells disclosed herein express genes encoding alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; β-III-tubulin; α-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (embryonic stem cell-associated transcript 1 (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fth117; Sal14; The cells can express any of a number of pluripotent cell markers, including blastocyte transcription factor (Utf1); Rex1; p53; G3PDH; telomerase including TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box-containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3 / Stella; DPPA4; and other common markers for pluripotency. Other markers include Dnmt3L; Sox15; Stat3; Grb2; These cells may also be characterized by the downregulation of markers characteristic of the somatic cells from which the induced pluripotent stem cells are derived.

[0463] The efficiency of reprogramming (i.e., the number of reprogrammed cells) from a population of starting cells can be increased by the addition of various small molecules, as shown by Shi, Y., et al. (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al. (2008) Nature Biotechnology 26(7):795-797 and Marson, A., et al. (2008) Cell-Stem Cell 3:132-135. Some non-limiting examples of agents that increase reprogramming efficiency include soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

[0464] To confirm the induction of pluripotent stem cells for use with the methods described herein, isolated clones can be tested for the expression of one or more stem cell markers. Such expression in cells derived from somatic cells identifies the cells as induced pluripotent stem cells. Stem cell markers can include, but are not limited to, SSEA3, SSEA4, CD9, Nanog, Oct4, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1, among others. In one embodiment, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include, for example, immunological methods that detect the presence of encoded polypeptides, such as RT-PCR and Western blot or flow cytometry analysis. In some embodiments, detection does not only involve RT-PCR, but also includes the detection of protein markers. Intracellular markers can best be identified by RT-PCR, whereas cell surface markers are easily identified by, for example, immunocytochemistry.

[0465] The pluripotent stem cell properties of isolated cells can be confirmed by testing the ability of iPSCs to differentiate into cells of each of the three germ layers. As an example, teratoma formation in nude mice can be used to evaluate the pluripotent properties of isolated clones. The cells are introduced into nude mice, and tumors arising from the cells are subjected to histology and / or immunohistochemistry. For example, the growth of tumors containing cells derived from all three germ layers further indicates that the cells are pluripotent stem cells.

[0466] Adult stem cell: Adult stem cell is stem cell derived from tissue of organism after birth or neonatal / post-neonatal period or adult organism.Adult stem cell is structurally different from embryonic stem cell not only in the marker that adult stem cell expresses or does not express compared with embryonic stem cell, but also by the existence of epigenetic difference, for example, difference in DNA methylation pattern.It is assumed that cardiomyocyte differentiated from adult stem cell can also be used for cardiac graft as described herein.Method for isolating adult stem cell is known in the art.For example, see U.S. Patent No. 9,206,393 and U.S. Patent Application Publication No. 2010 / 0166714, which are incorporated herein by reference in their entirety.

[0467] In vitro differentiation Various methods and compositions described herein use in vitro differentiated cardiomyocytes.The method for differentiating any cell type from ESC or iPSC is known in the art.For example, see LaFlamme et al., Nature Biotech 25:1015-1024 (2007), which describes the differentiation of cardiomyocytes, and is incorporated herein by reference in its entirety.

[0468] In some embodiments, the stepwise differentiation of ESCs or iPSCs into cardiomyocytes proceeds in the following order: ESCs or iPSCs>cardiogenic mesoderm>cardiomyocyte precursor cells>cardiomyocytes (see, for example, Lian et al. Nat Prot (2013); US Patent Application Publication Nos. 2017 / 0058263; 2008 / 0089874; 2006 / 0040389; US Patent Nos. 10,155,927; 9,994,812; and 9,663,764, the contents of each of which are incorporated herein by reference in their entirety). Numerous protocols for differentiating ESCs and iPSCs into cardiomyocytes are known in the art. For example, to induce stepwise differentiation into cardiomyocytes, agents can be added to or removed from the cell culture medium. Non-limiting examples of factors and agents that can promote cardiomyocyte differentiation include small molecules (e.g., Wnt inhibitors, GSK3 inhibitors), polypeptides (e.g., growth factors), nucleic acids, vectors, and patterned substrates (e.g., nanopatterns). The addition of growth factors required for the development of the cardiovascular system, including but not limited to, fibroblast growth factor 2 (FGF2), transforming growth factor β (TGFβ) superfamily growth factors-activin A and BMP4, vascular endothelial growth factor (VEGF), and the Wnt inhibitor DKK-1, can also be beneficial in inducing differentiation along the cardiac lineage. Further examples of factors and conditions that help promote cardiomyocyte differentiation include, but are not limited to, B27 supplements lacking insulin, cell-conditioned medium, external electrical pacing, and nanopatterned substrates, among others.

[0469] As will be understood by those skilled in the art, in vitro differentiation of cardiomyocytes ultimately results in cells with the phenotypic and morphological characteristics of the desired cell type, but the differentiation steps of in vitro differentiation do not need to be the same as those that occur naturally in embryos. That is, it is specifically contemplated herein that during differentiation into cardiomyocytes, the stepwise differentiation approach utilized to produce such cells does not need to proceed through all of the precursor cell types identified during embryogenesis, and can essentially "skip" certain stages of development that occur during embryogenesis. For example, see International Publication No. 2018096343 regarding transcription factor-mediated reprogramming of hPSCs. It is also contemplated that cardiomyocytes derived from other cells, for example, through transdifferentiation, can also benefit from the regulation of the ion channel set described herein when used for transplantation.

[0470] Monitoring cardiomyocyte differentiation and functional characterization As will be appreciated by those skilled in the art, the in vitro differentiated cardiomyocytes described herein lack markers of hematopoietic or hemogenic cells, vascular endothelial cells, embryonic stem cells, or induced pluripotent stem cells. In one embodiment of the methods described herein, one or more cell surface markers are used to determine the degree of differentiation along the spectrum of embryonic stem cells or iPSCs, e.g., to fully differentiated cardiomyocytes.

[0471] In some embodiments, antibodies or similar agents specific for a given marker or set of markers can be used to separate and isolate desired cells using other methods known to those skilled in the art, including fluorescence-activated cell sorting (FACS), panning, magnetic particle selection, particle sorter selection, and density separation (Xu et al. (2002) Circ. Res. 91:501; U.S. Patent Application No. 20030022367) and separation based on other physical properties (Doevendans et al. (2000) J. Mol. Cell. Cardiol. 32:839-851). Negative selection can be performed, including selecting and removing cells with undesirable markers or characteristics, such as fibroblast markers, epithelial cell markers, etc.

[0472] Undifferentiated ES cells express genes that can be used as markers for detecting the presence of undifferentiated cells.Exemplary ES cell markers include stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-I-60, TRA-1-81, alkaline phosphatase, or those described in, for example, US Patent Application No. 2003 / 0224411 or Bhattacharya (2004) Blood 103(8):2956-64, each of which is incorporated herein by reference in its entirety.Exemplary markers expressed on cardiac progenitor cells include, but are not limited to, TMEM88, GATA4, ISL1, MYL4 and NKX2-5.

[0473] Exemplary markers expressed on cardiomyocytes include, but are not limited to, NKX2-5, MYH6, MYL7, TBX5, ATP2a2, RYR2, and cTnT.

[0474] In some embodiments, the desired cells (e.g., in vitro differentiated cardiomyocytes) are an enriched cell population. That is, the percentage (e.g., percent of cells) of in vitro differentiated cardiomyocytes in a population of cells is at least 10% of the total number of cells in the population. For example, an enriched population contains at least 15% definitive cardiomyocytes, and at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% of the population comprises in vitro differentiated human cardiomyocytes. In some embodiments, the population of cells is at least 100 cells, at least 500 cells, at least 1000 cells, at least 1 x 10 4 cells, at least 1 x 10 5 cells, at least 1 x 10 6 cells, at least 1 x 10 7 cells, at least 1 x 10 8 cells, at least 1 x 10 9 cells, at least 1 x 10 10 cells, at least 1 x 10 11 cells, at least 1 x 10 12 cells, at least 1 x 10 13 cells, at least 1 x 10 14 cells, at least 1 x 10 15 It may contain cells or more.

[0475] Confirmation of cardiomyocyte differentiation and maturation can be assessed by assaying sarcomere morphology and the structural characteristics of actin and myosin. Because the structure of the cardiac sarcomere is highly ordered, those skilled in the art can recognize these proteins (actin, myosin, α-actinin, titin) and use their arrangement in tissues or cultured cell populations as markers to identify mature myocytes and tissues. Developing cardiac cells undergo "sarcomere formation," creating new sarcomere units within the cell. The degree of sarcomere organization provides a measure of cardiomyocyte maturity.

[0476] Immunofluorescence assays and electron microscopy for α-actinin, β-myosin, actin, cTnT, tropomyosin, and collagen, among others, can be used to identify and measure sarcomere structure. Immunofluorescence images can be quantified for sarcomere alignment, pattern intensity, and sarcomere length. This can be achieved by staining for proteins within the sarcomere (e.g., α-actinin) and qualitatively or quantitatively determining whether the sarcomeres are aligned. For quantitative measurements of sarcomere alignment, several methods can be used, such as using a scanning gradient Fourier transform script to determine the location of proteins within the sarcomere. This is done by using each image captured by the microscope and camera for individual analysis. Directional derivatives can be used to calculate the image gradient for each segment to determine the local alignment of the sarcomere. Pattern intensity can be determined by calculating the maximum peak of the one-dimensional Fourier transform of the gradient direction. Sarcomere length can be calculated by measuring the intensity profile of the sarcomere along this same gradient direction.

[0477] Cell morphology can be used to identify structurally mature stem cell-derived cardiomyocytes. Non-limiting examples of morphological and structural parameters include, but are not limited to, sarcomere length, Z-band width, binucleation rate, nuclear eccentricity, cell area, and cell aspect ratio.

[0478] Cellular activity and maturity can be determined by many parameters such as electrical, metabolic or contractile maturity of cardiomyocytes.

[0479] Mature cardiomyocytes possess functional ion channels that allow for synchronization of myocardial contractions. The electrical function of cardiomyocytes can be measured by various methods. Non-limiting examples of such methods include whole-cell patch clamp (manual or automated), multi-electrode array, field potential stimulation, calcium imaging, and optical mapping, among others. Cardiomyocytes can be electrically stimulated during whole-cell current clamp or field potential recording to produce electrical and / or contractile responses. Furthermore, cardiomyocytes can be genetically modified to express, for example, channelrhodopsin, which allows for optical stimulation of the cells.

[0480] Measurement of the field potential and biopotential of cardiomyocytes can be used to determine the differentiation stage and cell maturity of cardiomyocytes.For example, but not limited to, the following parameters can be used to determine the electrophysiological function of cardiomyocytes: change in field potential duration (FPD), quantification of FPD, beat rate, beats per minute, rise rate, resting membrane potential, action potential amplitude, maximum diastolic potential, relaxation time constant, action potential duration (APD) at 90% repolarization, interspike interval, change in beat interval, current density, activation and inactivation kinetics.

[0481] Electrical maturity is determined by one or more of the following markers compared to baseline levels: increased gene expression of ion channel genes, increased sodium current density, increased inward rectifier potassium channel current density, decreased action potential frequency, decreased calcium wave frequency, and decreased field potential frequency. During disease states, the electrophysiological function of cardiomyocytes can be impaired. For example, compared to normal stem cell-derived cardiomyocytes, prolonged field potential duration (FPD) and prolonged action potential duration (APD) are indicators of cardiomyocytes with arrhythmic potential. As used herein, the term "field potential duration (FPD)" refers to the time from a given depolarization event to the last depolarization event, while the term "action potential duration (APD)" refers to the time between the opening of sodium channels (or phase 0) and the opening of potassium channels (phase 3) that bring the membrane to its resting potential. While "normal" or "prolonged" values ​​for FPD and APD depend on the cell line being tested, an FPD of approximately 200 ms and an APD of approximately 100 ms can be considered "normal" for hESC-CMs, and an FPD > 400 ms and an APD > 300 ms can be considered "prolonged" for such cells (see, e.g., Stem Cell Res Ther 12, 278 (2021)).

[0482] Compared with fetal cardiomyocytes, adult cardiomyocytes have been shown to have enhanced oxidative cell metabolism, characterized by increased mitochondrial function and spare respiratory capacity.Metabolic assays can be used to determine the differentiation stage and cell maturity of stem cell-derived cardiomyocytes described herein.Non-limiting examples of metabolic assays include cell bioenergetics assays (for example, Seahorse Bioscience XF Extracellular Flux Analyzer) and oxygen consumption tests.

[0483] Specifically, cellular metabolism can be quantified by oxygen consumption rate (OCR), OCR trace during fatty acid stress test, maximum change in OCR, maximum change in OCR after FCCP addition, and maximum respiratory capacity, among other parameters.

[0484] Furthermore, metabolic challenge or lactate enrichment assay can provide a measure of the maturation of stem cell-derived cardiomyocytes or the measure of the effect of various treatments on these cells.Most mammalian cells generally use glucose as their main energy source.However, cardiomyocytes can produce energy from different sources, such as lactate or fatty acid.In some embodiments, lactate is supplemented and glucose is depleted in the culture medium, or the ability of cells to use lactate or fatty acid as energy source is useful for identifying the variation of mature cardiomyocytes and their function.

[0485] The metabolic maturity of in vitro differentiated cardiomyocytes is determined by one or more of the following markers compared to baseline levels: Increased activity of mitochondrial function, increased fatty acid metabolism, increased oxygen consumption rate (OCR), increased phosphorylated ACC levels or activity, increased levels or activity of fatty acid binding protein (FABP), increased levels or activity of pyruvate dehydrogenase kinase-4 (PDK4), increased mitochondrial respiratory capacity, increased mitochondrial volume, and increased levels of mitochondrial DNA.

[0486] Contractility of cardiomyocytes can be measured by optical tracking methods, such as video analysis. In addition to optical tracking, impedance measurements can also be performed. For example, the cardiomyocytes described herein can have contractility or beating rate measurements determined by xCelligence™ real-time cell analysis (Acea Biosciences, Inc., San Diego, CA).

[0487] A useful parameter for determining cardiomyocyte function is the beating rate. Contraction frequency, beating rate, change in beating interval (ΔBI) or beating interval can be used to determine the stem cell differentiation stage, maturity of stem cell-derived cardiomyocytes, and the effect of a given treatment on beating rate. Beating rate can be measured by optical tracking. Beating rate typically increases in fetal cardiomyocytes and decreases as cardiomyocytes develop. Contraction parameters can also include, but are not limited to, contraction force, contraction velocity, relaxation velocity, contraction angle distribution, or contraction anisotropy ratio.

[0488] Contractile maturity is determined by one or more of the following markers compared to baseline levels: increased beat rate, increased contractile force, increased levels or activity of α-myosin heavy chain (α-MHC), increased levels or activity of sarcomeres, decreased circularity index, increased levels or activity of troponin, increased levels or activity of titin N2b, increased cell area, and increased aspect ratio.

[0489] Heart / cardiomyocyte grafts In one aspect, described herein is a method of transplanting cardiomyocytes, e.g., in vitro differentiated cardiomyocytes, comprising contacting cardiac tissue with human cardiomyocytes described herein, a pharmaceutical composition described herein, a transplant composition described herein, or delivering cardiomyocytes to a subject in need thereof using a cardiac delivery device described herein.

[0490] As used herein, the term "implant" or "transplantation" is used in the context of placing cells described herein, e.g., cardiomyocytes, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, so that a desired effect occurs. Cells, e.g., cardiomyocytes, or their differentiated progeny (e.g., cardiac fibroblasts, etc.) and cardiomyocytes, can be implanted directly or into the recipient's cardiac tissue, e.g., at or near the site, or into the cardiac tissue of a subject with cardiac disease. As those skilled in the art will understand, cardiomyocytes generally do not proliferate to the point where the heart can heal from acute injury, including cell death, so long-term engraftment of cardiomyocytes is desirable. In some embodiments, the cells are optionally implanted on or within a scaffold or biocompatible material that supports the survival of the implanted cardiomyocytes and / or, for example, helps hold the administered cells in a desired location for engraftment or promotes integration with the subject's native cardiac cells. Preferably, the cardiomyocytes are human stem cell-derived cardiomyocytes or in vitro differentiated cardiomyocytes described herein.

[0491] Scaffold composition: In one aspect, the cardiomyocytes described herein can be mixed with or cultured on a preparation that provides a scaffold or patterned substrate to support the cells. Such scaffolds or patterned substrates can provide a physical advantage in fixing the cells in place, e.g., after implantation, as well as a biochemical advantage in providing extracellular cues for further maturation or maintenance of phenotype, e.g., until the cells are established.

[0492] A scaffold is a structure comprising biocompatible materials, including but not limited to gels, sheets, or lattices, that can confine cells in a desired location while allowing the ingress or diffusion of factors necessary for survival and function into the environment. Many biocompatible polymers suitable for scaffolds are known in the art.

[0493] Biocompatible synthetic polymers, natural polymers and semi-synthetic polymers can be used to synthesize polymer particles that can be used as scaffold material.Generally, to carry out the method described herein, it is preferable that the scaffold is biodegradable, so that cardiomyocytes can be isolated from the polymer before implantation, or the scaffold degrades in the subject over time and does not need to be removed.Therefore, in one embodiment, the scaffold provides a temporary structure or matrix for the growth and / or delivery of cardiomyocytes to a subject in need thereof.In some embodiments, the scaffold allows human cells to grow in a shape suitable for implantation or administration into a subject in need thereof, thereby allowing the scaffold to be removed before implantation, reducing the risk of rejection or allergic reaction initiated by the scaffold itself.

[0494] Examples of polymers that can be used include natural and synthetic polymers, but synthetic polymers are preferred for their reproducibility and sustained release kinetics.Synthetic polymers that can be used include biodegradable polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA) and other polyhydroxy acids, poly(caprolactone), polycarbonate, polyamide, polyanhydride, polyphosphazene, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate and biodegradable polyurethane; non-biodegradable polymers such as polyacrylate, ethylene-vinyl acetate polymer and other acyl-substituted cellulose acetate and its derivatives; polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulfonated polyolefin and polyethylene oxide. Examples of biodegradable natural polymers include proteins such as albumin, collagen, fibrin, silk, synthetic polyamino acids and prolamines; polysaccharides such as alginate and heparin; and other naturally occurring biodegradable polymers of sugar units. Alternatively, a combination of the aforementioned polymers can be used. In one aspect, natural polymers that are not commonly found in the extracellular matrix can be used.

[0495] PLA, PGA, and PLA / PGA copolymers are particularly useful for forming biodegradable scaffolds. PLA polymers are typically prepared from cyclic esters of lactic acid. Both L(+) and D(-) forms of lactic acid can be used to prepare PLA polymers, as well as optically inactive DL-lactic acid mixtures of D(-) and L(+) lactic acid. Methods for preparing polylactides are well described in the patent literature. The following U.S. patents, the teachings of which are incorporated herein by reference, describe in detail suitable polylactides, their properties, and their preparation: U.S. Patent No. 1,995,970 to Dorough; U.S. Patent No. 2,703,316 to Schneider; U.S. Patent No. 2,758,987 to Salzberg; U.S. Patent No. 2,951,828 to Zeile; U.S. Patent No. 2,676,945 to Higgins; and U.S. Patent No. 2,683,136 to Trehu.

[0496] PGA is a homopolymer of glycolic acid (hydroxyacetic acid). In the conversion of glycolic acid to poly(glycolic acid), glycolic acid first reacts with itself to form the cyclic ester glycolide, which is then converted to a high molecular weight linear polymer in the presence of heat and a catalyst. PGA polymers and their properties are described in more detail in "Cyanamid Research Develops World's First Synthetic Absorbable Suture," Chemistry and Industry, 905 (1970).

[0497] Fibers may be formed by melt spinning, extrusion, casting, or other techniques well known in the polymer processing art. When used to remove the scaffold prior to implantation, preferred solvents are those that are either completely removed by processing or are biocompatible in the amount remaining after processing.

[0498] Polymers for use in matrices should meet the mechanical and biochemical parameters necessary to provide adequate support for cells with subsequent growth and proliferation. Polymers can be characterized for mechanical properties such as tensile strength using an Instron testing machine, polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC), and bond structure by infrared (IR) spectroscopy.

[0499] The substrate or scaffold may optionally be nanopatterned or micropatterned with grooves and ridges, for example, that allow or promote the growth, placement, or maturation of cardiac tissue on the scaffold. The scaffold may have any desired shape and may include a wide range of geometric shapes useful for the methods described herein. A non-limiting list of shapes includes, for example, patches, hollow particles, tubes, sheets, cylinders, spheres, and fibers, among others. The shape or size of the scaffold should not substantially interfere with cell growth, cell differentiation, cell proliferation, or any other cellular process, and the scaffold should not induce cell death, for example, via apoptosis or necrosis. Furthermore, care should be taken to ensure that the scaffold shape allows adequate surface area for the delivery of nutrients from the surrounding medium to the cells in the population so as not to compromise cell viability. The porosity of the scaffold may also be modified as desired by those skilled in the art.

[0500] In some embodiments, cell attachment to polymer is enhanced by coating the polymer with compounds such as basement membrane components, fibronectin, agar, agarose, gelatin, gum arabic, type I, type II, type III, type IV and type V collagen, laminin, glycosaminoglycans, polyvinyl alcohol, mixtures thereof, and other hydrophilic and peptide adhesive materials known to those skilled in the art of cell culture or tissue engineering.Examples of materials for coating polymer scaffolds include polyvinyl alcohol and collagen.As those skilled in the art will understand, Matrigel™ is not suitable for administration to human subjects, and therefore the compositions described herein do not include Matrigel™.

[0501] In some embodiments, it may be desirable to add bioactive molecules / factors to the scaffold. The matrices described herein can be used to deliver a variety of bioactive molecules.

[0502] In one embodiment, the bioactive factor comprises a growth factor, such as platelet-derived growth factor (PDGF), transforming growth factor α or β (TGFβ), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 7 (FGF7), fibroblast growth factor 10 (FGF10), epidermal growth factor (EGF / TGFα), and vascular endothelial growth factor (VEGF), some of which are also angiogenic factors.

[0503] These agents are known to those skilled in the art and are commercially available or described in the literature. Bioactive molecules can be incorporated into the matrix and released over time by diffusion and / or degradation of the matrix, or can be suspended in the cell suspension.

[0504] Pharmaceutically acceptable carrier The method of administering human cardiomyocytes to a subject as described herein involves the use of a therapeutic composition comprising such cells, which contains a physiologically tolerable carrier with the cell composition and, optionally, at least one additional bioactive agent, polypeptide, nucleic acid encoding said polypeptide, or factor described herein dissolved or dispersed therein as an active ingredient.

[0505] In preferred embodiments, the therapeutic composition is not substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless substantially immunogenic is desired. As used herein, the terms "pharmaceutically acceptable" and "physiologically tolerable" and their grammatical variations are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that these materials can be administered to or on a mammal without producing undesirable physiological effects, such as nausea, dizziness, stomach upset, transplant rejection, allergic reactions, and the like. A pharmaceutically acceptable carrier does not promote the development of an immune response against an agent mixed with the pharmaceutically acceptable carrier, unless so desired. The preparation of a composition containing 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 injectables, either as liquid solutions or suspensions, although solid forms suitable for solution or suspension in liquid prior to use may also be prepared.

[0506] Human transplant compositions can contain one or more pharmaceutically acceptable carriers or materials as excipients. In contrast, cell culture compositions (not intended for human transplantation) typically use research reagents such as cell culture medium as excipients. Cardiomyocytes can also be administered in FDA-approved matrices / scaffolds or in combination with FDA-approved drugs, as described above.

[0507] Generally, the compositions comprising cardiomyocytes described herein are administered as a suspension formulation in which the cells are mixed with a pharmaceutically acceptable carrier.Those skilled in the art will recognize that the pharmaceutically acceptable carrier used in the cell composition does not contain buffers, compounds, cryopreservatives, preservatives, or other agents in an amount that substantially interferes with the viability of the cells delivered to the subject.Cell-containing formulations can include, for example, an osmotic buffer that can maintain the integrity of the cell membrane, and optionally, nutrients that maintain cell viability or enhance engraftment upon administration.Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the human cardiac progenitor cells described herein using routine experimentation.

[0508] The cell compositions may also be emulsified or presented as liposomal compositions, so long as the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients may be mixed with excipients that are pharmaceutically acceptable, compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods described herein.

[0509] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing only the active ingredient and water, or containing both buffers, such as sodium phosphate, saline, or phosphate-buffered saline, at physiological pH values. In addition, aqueous carriers can contain one or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and in addition to water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in the cell compositions described herein that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0510] Cryopreservation: In some embodiments, the cardiomyocytes described herein, including cardiomyocytes in which HCN4, CACHA1H, and SLC8A1 are inhibited and KCNJ2 is activated, are cryopreserved, i.e., frozen for later thawing and administration. Cryopreservation and cryopreservation agents are well known in the art, and include, for example, suspensions of cells in media containing DMSO (e.g., 7.5% to 15% or about 7.5% to 15%) or glycerol (e.g., 10% or about 10%), among other cryopreservation agents. Mammalian cells, including cardiomyocytes, are generally slowly frozen, for example, by lowering the temperature to -70°C to -90°C at about -1°C / min. Storage can be performed at -80°C, for example, in an ultra-low temperature freezer, or, for example, on dry ice or under liquid nitrogen.

[0511] Dosage and Efficacy Provided herein is a method for treating heart disease, heart disorder, heart injury, heart failure or myocardial infarction, comprising administering cardiomyocytes to a subject in need thereof.In some embodiments, provided herein is a method and composition for preventing expected disorders, such as heart failure after myocardial injury.

[0512] Measured or measurable parameters include clinically detectable markers of disease, such as elevated or decreased levels of clinical or biological markers, as well as parameters related to clinically accepted scales of symptoms or markers for diseases or disorders. However, it will be understood that the total amount of the compositions and formulations disclosed herein to be used will be determined by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated.

[0513] In some embodiments, the subject is first diagnosed with a disease or disorder affecting the myocardium before administering the cells according to the methods described herein. In some embodiments, the subject is first diagnosed as being at risk for developing the disease (e.g., heart failure following myocardial injury) or disorder before administering the cells.

[0514] Those skilled in the art can determine the number of cardiomyocytes required for the graft. In some embodiments, about 10 million to about 10 billion cardiomyocytes are administered to the subject. For use in various aspects described herein, an effective amount of human cardiomyocytes is at least 1 x 10 3 , at least 1 x 10 4 , at least 1 x 10 5 , at least 5 × 10 5 , at least 1 x 10 6 , at least 2 × 10 6 , at least 3 × 10 6 , at least 4 × 10 6 , at least 5 × 10 6 , at least 6 × 10 6 , at least 7 × 10 6 , at least 8 x 10 6 , at least 9 x 10 6 , at least 1 x 10 7 , at least 1.1 x 10 7 , at least 1.2 × 10 7 , at least 1.3 × 10 7 , at least 1.4 × 10 7 , at least 1.5 x 10 7 , at least 1.6 × 10 7 , at least 1.7 x 10 7 , at least 1.8 x 10 7 , at least 1.9 × 10 7 , at least 2 × 10 7 , at least 3 × 10 7 , at least 4 × 10 7 , at least 5 × 10 7 , at least 6 × 10 7 , at least 7 × 10 7 , at least 8 x 107 , at least 9 x 10 7 , at least 1 x 10 8 , at least 2 × 10 8 , at least 5 × 10 8 , at least 7 × 10 8 , at least 1 x 10 9 , at least 2 × 10 9 , at least 3 × 10 9 , at least 4 × 10 9 , at least 5 × 10 9 or more cardiomyocytes.

[0515] In some embodiments, compositions comprising cardiomyocytes treated with any one or more of the polypeptides or nucleic acids encoding such polypeptides described herein allow for engraftment of cells in the heart at an efficiency at least 20% greater than engraftment when such cardiomyocytes are administered alone. In other embodiments, such efficiency is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, or at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold or more than the efficiency of engraftment when cardiomyocytes are administered alone without the polypeptides or nucleic acids encoding such polypeptides described herein.

[0516] In some embodiments, an effective amount of cardiomyocytes is administered to a subject by intracardiac administration or delivery. As defined herein, "intracardiac" administration or delivery refers to any administration route that a population of cardiomyocytes is administered to bring these cells into direct contact with the subject's myocardium, including but not limited to direct cardiac injection, intramyocardial injection, intrainfarct injection, and injection during surgery (e.g., cardiac bypass surgery, implantation of a cardiac minipump or pacemaker, etc.). In some such embodiments, cells are injected into the myocardium (e.g., cardiomyocytes) or into the chambers of the atrium and / or ventricle. In some embodiments, intracardiac delivery of cells includes the administration method in which cells are administered to a subject undergoing surgery, for example, as a cell suspension, via a single injection or multiple "mini" injections into the desired region of the heart.

[0517] The choice of formulation depends on the specific composition used and the number of cardiomyocytes to be administered, and such formulation can be adjusted by those skilled in the art.However, as an example, when the composition is cardiomyocytes in a pharmaceutically acceptable carrier, the composition can be a suspension of cells in a suitable buffer (for example, saline buffer) at an effective concentration of cells per mL of solution.The formulation can also include cell nutrients, simple sugars (for example, for osmotic pressure adjustment), or other components to maintain cell viability.Alternatively, the formulation can include a scaffold, such as a biodegradable scaffold.

[0518] In some embodiments, additional agents to aid in the treatment of the subject can be administered before or after treatment with cardiomyocytes as described. Such additional agents can be used to prepare the target tissue for administration of progenitor cells. Alternatively, additional agents can be administered after the cardiomyocytes to support the engraftment and growth of the administered cells into the heart or other desired administration site. In some embodiments, the additional agent comprises a growth factor such as VEGF or PDGF. Other exemplary agents can be used to reduce the load on the heart during cardiomyocyte engraftment (e.g., beta-blockers, drugs to lower blood pressure, etc.).

[0519] The effectiveness of treatment can be determined by those skilled in the art. However, as this term is used herein, treatment is considered to be "effective treatment" if, after treatment with a composition comprising human cardiomyocytes as described herein, any one or all of the symptoms or other clinically accepted symptoms or markers of disease, such as heart disease, heart failure, heart damage and / or heart disorder, are reduced by, for example, at least 10%.Methods for measuring these indicators are known to those skilled in the art and / or are described herein.In one embodiment, treatment is effective if, as described herein, transplanted cardiomyocytes engraft substantially without causing transplant arrhythmia. "Substantially without causing" in this context means that transplant arrhythmias do not occur or that rate or rhythm disturbances caused by the introduction of the cardiomyocytes described herein are at least 20% less in duration and / or severity, such as at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% less, compared to engraftment of similar cardiomyocytes that have not been treated or modified as described herein.

[0520] Indicators of cardiac disease or damage include functional indicators or parameters, such as cardiac output, heart rate, left ventricular ejection fraction, heart rate, cardiac rhythm, blood pressure, cardiac volume, regurgitation, etc., as well as biochemical indicators, such as decreases in markers of cardiac damage, such as serum lactate dehydrogenase or serum troponin. For example, myocardial ischemia and reperfusion are associated with decreased cardiac function. Subjects who have suffered an ischemic cardiac event and / or undergone reperfusion therapy have decreased cardiac function compared to their cardiac function before ischemia and / or reperfusion. Measures of cardiac function include, for example, ejection fraction and fractional shortening. The ejection fraction is the proportion of blood pumped out of the ventricle with each heartbeat. The term ejection fraction applies to both the right and left ventricles. LVEF refers to the left ventricular ejection fraction (LVEF). Fractional shortening refers to the difference between the end-diastolic dimension and the end-systolic dimension divided by the end-diastolic dimension.

[0521] Non-limiting examples of clinical tests that can be used to assess cardiac function parameters include echocardiography (with or without Doppler flow imaging), electrocardiogram (EKG), exercise stress testing, Holter ECG, or measurement of beta-natriuretic peptide.

[0522] If necessary or desired, animal models of injury or disease can be used to evaluate the effectiveness of certain compositions described herein.For example, isolated beating rabbit or rat heart model, or coronary artery ligation model in either dog or pig can be used.Animal models of cardiac function are useful for monitoring infarct location, coronary artery perfusion, electrical conduction, left ventricular end-diastolic pressure, left ventricular ejection fraction, heart rate, blood pressure, degree of hypertrophy, diastolic relaxation function, cardiac output, heart rate variability and ventricular wall thickness etc.The pig model described in the examples herein is particularly preferred.

[0523] In some embodiments, the composition comprising cardiomyocytes described herein is delivered at least 6 hours after the onset of reperfusion, for example, after myocardial infarction. During the ischemic attack and subsequent reperfusion, the cardiac microenvironment or the microenvironment of the infarcted site may be too "hostile" to allow the engraftment of administered cardiomyocytes into the heart. Thus, in some embodiments, the amount of erythrocyte sedimentation (ESH) in the blood is at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 31 hours, at least 32 hours, at least 33 hours, at least 34 hours, at least 35 hours, at least 36 hours, at least 37 hours, at least 38 hours, at least 39 hours, at least 40 hours, at least 41 hours, at least 42 hours, at least 43 hours, at least 44 hours, at least 45 hours, at least Also 46 hours, at least 47 hours, at least 48 hours, at least 49 hours, at least 50 hours, at least 51 hours, at least 52 hours, at least 53 hours, at least 54 hours, at least 55 hours, at least 56 hours, at least 57 hours, at least 58 hours, at least 59 hours, at least 60 hours, at least 61 hours, at least 62 hours, at least 63 hours, at least 64 hours, at least 65 hours, at least 66 hours, at least 67 hours, at least 68 hours, at least 69 hours, at least 70 hours, at least 71 hours, at least 72 hours, at least 73 hours, at least 74 hours, at least 75 hours, at least 76 hours, at least 77 hours, at least 78 hours, at least 79 hours, at least 80 hours, at least 81 hours, at least 82 hours, at least 83 hours, at least 84 hours, at least 85 hours, at least 86 hours, at least 87 hours, at least 88 hours,It is preferred to administer such compositions for at least 89 hours, at least 90 hours, at least 91 hours, at least 92 hours, at least 93 hours, at least 94 hours, at least 95 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or more. In some embodiments, the compositions comprising cardiomyocytes described herein can be administered to the infarct site, peri-infarct site, ischemic site, border zone, or border of the heart at any length of time after myocardial infarction (e.g., at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, or at least 30 years or more), although, as will be understood by one of skill in the art, successful engraftment after extended time intervals after infarction will depend on many factors, including, but not limited to, the amount of scar tissue deposited, the density of the scar tissue, the size of the infarct site, the degree of vascularization surrounding the infarct site, etc. Therefore, earlier intervention by administering a composition comprising cardiomyocytes may be more effective than, for example, administration one month or more after infarction.

[0524] The compositions comprising cardiomyocytes described herein can be administered to any desired region of the heart, including, but not limited to, the infarct region, peri-infarct region, ischemic region, border, border zone, area of ​​wall thinning, area of ​​non-compaction, or within an area at risk for maladaptive cardiac remodeling.

[0525] The invention may be as described in any one of the numbered paragraphs below. 1. In vitro differentiated human cardiomyocytes in which HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited and KCNJ2 activity is at least partially stimulated. 2. In vitro differentiated human cardiomyocytes with reduced expression of HCN4, CACNA1H, and SLC8A1 and expression of a transgene encoding KCNJ2 regulated by endogenous HCN4 regulatory sequences. 3. The in vitro differentiated human cardiomyocytes of paragraph 2, wherein HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited compared to cardiomyocytes or other control cells. 4. The in vitro differentiated human cardiomyocytes of paragraph 2 or paragraph 3, wherein KCNJ2 activity is at least partially stimulated compared to cardiomyocytes or other control cells. 5. The in vitro differentiated human cardiomyocytes of any one of claims 1 to 4, wherein at least partial inhibition of HCN4, CACNA1H, and SLC8A1 comprises inhibition via contacting the cardiomyocytes with one or more inhibitor drugs and / or comprises genetic manipulation. 6. The in vitro differentiated human cardiomyocytes of any one of claims 1 to 5, wherein at least partial stimulation of KCNJ2 activity comprises contacting the cardiomyocytes with one or more activating drugs and / or comprises genetic manipulation. 7. An in vitro differentiated human cardiomyocyte according to any one of claims 2 to 6, wherein the reduced expression of HCN4, CACNA1H, or SLC8A1 is due to genetic manipulation. 8. The in vitro differentiated human cardiomyocyte of any one of claims 2 to 8, wherein the at least partially stimulated activity of KCNJ2 is due to genetic manipulation. 9. The in vitro differentiated human cardiomyocyte of any one of claims 1 to 8, wherein one or more of the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated. 10. The in vitro differentiated human cardiomyocyte of any one of claims 1 to 9, wherein each of the genes encoding HCN4, CACNA1H, and SLC8A1 is inactivated. 11. The in vitro differentiated human cardiomyocyte of any one of claims 1 to 10, wherein HCN4, CACNA1H, and SLC8A1 activity is completely inhibited and KCNJ2 activity is at least partially stimulated compared to control cells. 12. The in vitro differentiated human cardiomyocytes of any one of claims 5 to 11, wherein the one or more inhibitor drugs, activator drugs and / or genetic manipulations do not alter the expression of HCN4, CACNA1H, SLC8A1 and / or KCNJ2. 13. The in vitro differentiated human cardiomyocyte of any one of claims 1 to 12, wherein HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000% or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, compared to cardiomyocytes or other control cells that have not been manipulated by one or more inhibitor drugs and / or genetic manipulation. 14. The in vitro differentiated human cardiomyocyte of any one of claims 1 to 13, wherein HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% to at least 1000%, or 1-fold to 100-fold, and KCNJ2 activity is stimulated by at least 10% or 1-fold, compared to cardiomyocytes not manipulated by genetic engineering or other control cells. 15. The in vitro differentiated human cardiomyocyte of any one of claims 1 to 14, wherein HCN4, CACNA1H, and SLC8A1 activity is completely inhibited by genetic manipulation and KCNJ2 activity is at least partially stimulated compared to control cells. 16. The in vitro differentiated human cardiomyocyte of any one of claims 11 to 15, wherein the cardiomyocyte or other control cell is a wild-type cardiomyocyte, a primary cardiomyocyte, an in vitro differentiated cardiomyocyte derived from a PSC or ESC, or starting material. 17. The in vitro differentiated human cardiomyocyte of any one of claims 11 to 16, wherein the control cells have not been manipulated by one or more inhibitor drugs, activator drugs and / or genetic manipulation. 18. The in vitro differentiated human cardiomyocytes of paragraph 17, wherein the one or more inhibitor drugs, activator drugs and / or genetic manipulations do not alter expression of HCN4, CACNA1H, SLC8A1 and / or KCNJ2. 19. The in vitro differentiated human cardiomyocyte of any one of claims 11-18, wherein the control cell is an in vitro differentiated cardiomyocyte derived from a PSC or ESC, wherein the control cell, PSC or ESC, comprises one, two or three but not all four edits, wherein the HCN4, CACNA1H, and SLC8A1 activity is at least partially inhibited and KCNJ2 activity is at least partially stimulated, and wherein the PSC is optionally an iPSC. 20. The in vitro differentiated human cardiomyocyte of any one of claims 1 to 19, wherein HCN4, CACNA1H, and SLC8A1 activity is inhibited by at least 10% or 1-fold and KCNJ2 activity is stimulate...

Claims

1. In vitro differentiated human cardiomyocytes containing reduced expression of HCN4, CACNA1H, and SLC8A1, and expression of a transgene encoding KCNJ2 regulated by endogenous HCN4 regulatory sequences.

2. The in vitro differentiated human cardiomyocyte of claim 1, wherein HCN4, CACNA1H, and SLC8A1 activity are partially or completely inhibited by genetic manipulation and KCNJ2 activity is at least partially stimulated compared to control cells.

3. 3. The in vitro differentiated human cardiomyocyte of claim 2, wherein the genetic manipulation method is gene knockout or gene inactivation, and optionally, the gene knockout is by inducing an insertion or deletion in a gene using a gene editing system, wherein the gene editing system is optionally selected from the group consisting of ZFN, TALEN, meganuclease, transposase, CRISPR / Cas system, nickase system, base editing system, prime editing system, and gene writing system.

4. The in vitro differentiated human cardiomyocyte of claim 1, wherein the KCNJ2 coding sequence replaces the coding sequence of HCN4 or CACNA1H such that the replaced HCN4 or CACNA1H coding sequence is not expressed and the replaced KCNJ2 is expressed under the regulation of the endogenous HCN4 or CACNA1H regulatory sequence.

5. The in vitro differentiated human cardiomyocyte of claim 1, wherein the genes encoding HCN4, CACNA1H, and SLC8A1 contain an indel in at least one allele.

6. The in vitro differentiated human cardiomyocyte of claim 1, wherein the genes encoding HCN4, CACNA1H, and SLC8A1 are inactivated using the CRISPR / Cas system, and the KCNJ2 polypeptide is overexpressed under the control of an endogenous HCN4 regulatory sequence or under the control of an endogenous CACNA1H regulatory sequence.

7. 2. The in vitro differentiated human cardiomyocyte of claim 1, wherein said cardiomyocyte is differentiated in vitro from pluripotent stem cells or from a starting material.

8. The in vitro differentiated human cardiomyocyte of claim 1, wherein the cardiomyocytes are differentiated in vitro from starting material or from iPSCs derived from a subject into which the in vitro differentiated human cardiomyocytes are to be transplanted or derived from a healthy subject.

9. The starting material is primary cells collected from a donor, which are optionally stem cells; Stem cell lines; iPSCs; or pluripotent stem cells The in vitro differentiated human cardiomyocyte of claim 8, selected from any one of the following:

10. Pluripotent stem cells genetically engineered for reduced gene expression of HCN4, CACNA1H, and SLC8A1 and increased expression of a transgene encoding KCNJ2 under the regulation of HCN4 or CACNA1H regulatory sequences.

11. A cardiomyocyte differentiated in vitro from the pluripotent stem cell of claim 10.

12. A pharmaceutical composition comprising in vitro differentiated human cardiomyocytes according to any one of claims 1 to 11 or derived from any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

13. An in vitro differentiated cardiomyocyte as defined in any one of claims 1 to 9 for use in a method of transplantation into a subject in need thereof, the transplantation comprising the step of contacting or delivering to cardiac tissue of the subject a pharmaceutical composition comprising the in vitro differentiated cardiomyocyte as defined in any one of claims 1 to 11 or derived from any one of claims 1 to 11, or the in vitro differentiated human cardiomyocyte as defined in any one of claims 1 to 11 or derived from any one of claims 1 to 11, and a pharma- ceutically acceptable carrier.

14. 10. The in vitro differentiated cardiomyocyte according to any one of claims 1 to 9 for use in a method for treating a disease or disorder involving damage or dysfunction of cardiac tissue in a subject in need thereof, the method comprising delivering to cardiac tissue of a subject in need thereof a pharmaceutical composition comprising the in vitro differentiated cardiomyocyte according to or derived from any one of claims 1 to 11 or the in vitro differentiated human cardiomyocyte according to or derived from any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

15. The in vitro differentiated cardiomyocyte of claim 13, wherein the contacting or delivering step results in reduced transplant arrhythmias compared to transplantation of in vitro differentiated cardiomyocytes lacking inhibition of HCN4, CACNA1H, and SLC8A1 and lacking stimulation of KCNJ2.

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