# US 2024/0189481 A1 · Non-contractile cardiomyocytes for cardiac repair

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- Number: US 2024/0189481 A1
- Document ID: US20240189481A1
- Status: Published application
- Kind: A1
- Application number: 18/553,941
- Filed: 2022-04-06
- Published: 2024-06-13
- Earliest claimed: 2021-04-06
- Assignees: University of Washington
- Applicants: University of Washington
- Inventors: Charles E. MURRY; Michael REGNIER; Hans REINECKE; Xiulan YANG; Dasom YOO
- Web page: https://patentgrep.com/patent/US20240189481A1

## CPC

- A61L 27/3826: {Muscle cells, e.g. smooth muscle cells}
- A61K 35/34: Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44)
- A61L 27/52: Hydrogels or hydrocolloids
- A61P 9/00: Drugs for disorders of the cardiovascular system
- C12N 5/0657: {Cardiomyocytes; Heart cells}
- A61L 2400/06: Flowable or injectable implant compositions
- A61L 2430/20: for reconstruction of the heart, e.g. heart valves
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Described herein are compositions and methods relating to the improvement of cardiac function. Various embodiments relate to transplant compositions comprising cardiomyocytes which are engineered to be non-contractile, and to methods of using such cardiomyocytes or transplant compositions to improve cardiac function, e.g., by administering them to cardiac tissue.

## Claims

### Claim 1

A transplant composition comprising a non-contractile cardiomyocyte, wherein the non-contractile cardiomyocyte exhibits impaired excitation-contraction coupling.

### Claim 2 (depends on 1)

The transplant composition of claim 1, wherein the non-contractile cardiomyocyte is engineered to exhibit impaired excitation-contraction coupling.

### Claim 3 (depends on 1, 2)

The transplant composition of claim 1 or claim 2, wherein the non-contractile cardiomyocyte is a human non-contractile cardiomyocyte.

### Claim 4

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte exhibits normal action potentials.

### Claim 5

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte exhibits normal calcium transients.

### Claim 6

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte exhibits impaired function of a thin filament.

### Claim 7

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte is engineered to exhibit impaired function of a thin filament.

### Claim 8

The transplant composition of any preceding claim, wherein impaired function of a thin filament comprises impaired calcium binding to troponin.

### Claim 9

The transplant composition of any preceding claim, wherein impaired calcium binding to troponin is a result of knockout or mutation of at least one troponin isoform.

### Claim 10

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte comprises knockout of troponin I (TnI) and/or troponin T (TnT).

### Claim 11

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte comprises a mutation in troponin C that impairs calcium binding to troponin.

### Claim 12 (depends on 11)

The transplant composition of claim 11, wherein the mutation in troponin C comprises D65A.

### Claim 13 (depends on 11)

The transplant composition of claim 11, wherein the mutation in troponin C comprises I61Q, and/or L57Q.

### Claim 14

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte exhibits impaired actin-myosin binding.

### Claim 15

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte is engineered to have impaired actin-myosin binding.

### Claim 16 (depends on 14, 15)

The transplant composition of claim 14 or 15, wherein the impaired actin-myosin binding comprises a mutation in or deletion of myosin.

### Claim 17

The transplant composition of any preceding claim, further comprising a pharmaceutically acceptable carrier.

### Claim 18

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte is derived from in vitro differentiation of a pluripotent stem cell or a cardiac progenitor cell.

### Claim 19

The transplant composition of any preceding claim, wherein the non-contractile cardiomyocyte retains normal gap junction function when contacted with a cardiomyocyte.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)

A method for improving cardiac function in a subject in need thereof, the method comprising administering a cardiac transplant composition of any one of claim 1-19 to cardiac tissue of a subject in need thereof.

### Claim 21

A method for improving cardiac function in a subject in need thereof, the method comprising: administering a non-contractile cardiomyocyte or a composition thereof to a graft site in cardiac tissue of a recipient in need thereof, thereby improving cardiac function in the subject.

### Claim 22 (depends on 21)

The method of claim 21, wherein the non-contractile cardiomyocyte exhibits impaired excitation-contraction coupling.

### Claim 23 (depends on 21, 22)

The method of claim 21 or 22, wherein the non-contractile cardiomyocyte is engineered to exhibit impaired excitation-contraction coupling.

### Claim 24 (depends on 21, 22, 23)

The method of any one of claims 21-23, wherein the non-contractile cardiomyocyte exhibits normal action potentials.

### Claim 25 (depends on 21, 22, 23, 24)

The method of claim any one of claims 21-24, wherein the non-contractile cardiomyocyte exhibits normal calcium transients.

### Claim 26 (depends on 21, 22, 23, 24, 25)

The method of any one of claims 21-25, wherein the non-contractile cardiomyocyte exhibits impaired function of a thin filament.

### Claim 27 (depends on 21, 22, 23, 24, 25, 26)

The method of any one of claims 21-26, wherein the non-contractile cardiomyocyte is engineered to exhibit impaired function of a thin filament.

### Claim 28 (depends on 26, 27)

The method of claim 26 or 27, wherein the impaired function of a thin filament comprises impaired calcium binding to troponin.

### Claim 29 (depends on 28)

The method of claim 28, wherein the impaired calcium binding to troponin is a result of knockout or mutation of at least one troponin isoform.

### Claim 30 (depends on 22, 23, 24, 25, 26, 27, 28, 29)

The method of any one of claims 22-29, wherein the non-contractile cardiomyocyte comprises knockout of troponin I (TnI) and/or troponin T (TnT).

### Claim 31 (depends on 28, 29)

The method of claim 28 or 29, wherein the non-contractile cardiomyocyte comprises a mutation in troponin C that impairs calcium binding to troponin.

### Claim 32 (depends on 31)

The method of claim 31, wherein the mutation in troponin C comprises D65A.

### Claim 33 (depends on 22, 23, 24, 25, 26, 27)

The method of any one of claims 22-27, wherein the non-contractile cardiomyocyte exhibits impaired actin-myosin binding.

### Claim 34 (depends on 22, 23, 24, 25, 26, 27)

The method of any one of claims 22-27, wherein the non-contractile cardiomyocyte is engineered to exhibit impaired actin-myosin binding.

### Claim 35 (depends on 33, 34)

The method of claim 33 or 34, wherein the impaired actin-myosin binding comprises a mutation in or deletion of myosin.

### Claim 36 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)

The method of any one of claims 20-35, wherein the improvement in cardiac function comprises an increase in regional wall motion, fractional shortening, or ejection fraction.

### Claim 37 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)

The method of any one of claims 20-36, wherein the subject in need thereof comprises a cardiac disease or disorder.

### Claim 38 (depends on 37)

The method of claim 37, wherein the cardiac disease or disorder comprises impaired contractility.

### Claim 39 (depends on 37, 38)

The method of claim 37 or 38, wherein the cardiac disease or disorder comprises a myocardial infarction, an ischemia/reperfusion injury, a cardiomyopathy or heart failure.

### Claim 40 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The method of any one of claims 20-39, wherein the risk or incidence of engraftment arrhythmia is reduced compared to a graft comprising wild-type cardiomyocytes.

### Claim 41 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The method of any one of claims 20-40, further comprising administering a wild-type cardiomyocyte in combination with the non-contractile cardiomyocyte.

### Claim 42

A transplant composition comprising a cardiomyocyte that exhibits attenuated contractility and impaired excitation-contraction coupling.

### Claim 43

A transplant composition comprising a cardiomyocyte engineered to have attenuated contractility and/or impaired excitation-contraction coupling.

### Claim 44 (depends on 42, 43)

The transplant composition of claim 42 or 43, wherein the cardiomyocyte is human.

### Claim 45 (depends on 42, 43, 44)

The transplant composition of any one of claims 42-44, wherein the cardiomyocyte engineered to have attenuated contractility exhibits normal action potentials.

### Claim 46 (depends on 42, 43, 44, 45)

The transplant composition of any one of claims 42-45, wherein the cardiomyocyte engineered to have attenuated contractility exhibits normal calcium transients.

### Claim 47 (depends on 42, 43, 44, 45, 46)

The transplant composition of any one of claims 42-46, wherein the non-contractile cardiomyocyte exhibits reduced function of a thin filament.

### Claim 48 (depends on 42, 43, 44, 45, 46, 47)

The transplant composition of any one of claims 42-47, wherein the non-contractile cardiomyocyte is engineered to exhibit reduced function of a thin filament.

### Claim 49 (depends on 47, 48)

The transplant composition of claim 47 or 48, wherein the reduced function of a thin filament comprises reduced calcium binding to troponin.

### Claim 50 (depends on 49)

The transplant composition of claim 49, wherein the reduced calcium binding to troponin is a result of a mutation in troponin C that reduces calcium binding to troponin C by at least 20%.

### Claim 51 (depends on 50)

The transplant composition of claim 50, wherein the mutation in troponin C comprises I61Q and/or L57Q.

### Claim 52 (depends on 42, 43, 44, 45, 46, 47, 48)

The transplant composition of any one of claims 42-48, wherein the cardiomyocyte comprises reduced actin-myosin binding relative to wild-type.

### Claim 53 (depends on 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)

The transplant composition of any one of claims 42-52, further comprising a pharmaceutically acceptable carrier.

### Claim 54 (depends on 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53)

The transplant composition of any one of claims 42-53, wherein the cardiomyocyte that has reduced contractility is derived from in vitro differentiation of a pluripotent stem cell or a cardiac progenitor cell.

### Claim 55 (depends on 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)

The transplant composition of any one of claims 42-54, wherein the cardiomyocyte having reduced contractility retains normal gap junction function when contacted with a cardiomyocyte.

### Claim 56 (depends on 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55)

A method for improving cardiac function in a subject in need thereof, the method comprising administering a transplant composition of any one of claims 42-55 to cardiac tissue of a subject in need thereof.

### Claim 57

A method for improving cardiac function in a subject in need thereof, the method comprising: administering a cardiomyocyte that exhibits attenuated contractility or a composition thereof to a graft site in cardiac tissue of the subject in need thereof, thereby improving cardiac function in the subject.

### Claim 58 (depends on 56, 57)

The method of claim 56 or 57, wherein the improvement in cardiac function comprises an increase in regional wall motion, fractional shortening, or ejection fraction.

### Claim 59 (depends on 56, 57, 58)

The method of claim any one of claims 56-58, wherein the subject in need thereof comprises a cardiac disease or disorder.

### Claim 60 (depends on 59)

The method of claim 59, wherein the cardiac disease or disorder comprises reduced contractility.

### Claim 61 (depends on 60)

The method of claim 60, wherein the cardiac disease or disorder comprises a myocardial infarction, an ischemia/reperfusion injury, a cardiomyopathy or heart failure.

### Claim 62 (depends on 56, 57, 58, 59, 60, 61)

The method of any one of claims 56-61, wherein the risk or incidence of engraftment arrhythmia is reduced compared to a graft comprising wild-type cardiomyocytes.

### Claim 63 (depends on 56, 57, 58, 59, 60, 61)

The method of any one of claims 56-61, further comprising administering a wild-type cardiomyocyte in combination with the cardiomyocyte engineered to have attenuated contractility.

### Claim 64

A unit dosage formulation comprising non-contractile cardiomyocytes for administration to cardiac tissue of a subject in need thereof, the unit dosage formulation comprising 1×103 to 5×109 non-contractile cardiomyocytes in a gel or matrix.

### Claim 65 (depends on 64)

The unit dosage formulation of claim 64, wherein the non-contractile cardiomyocyte is engineered to exhibit impaired excitation-contraction coupling.

### Claim 66 (depends on 64, 65)

The unit dosage formulation of claim 64 or claim 65, wherein the non-contractile cardiomyocyte is a human non-contractile cardiomyocyte.

### Claim 67 (depends on 64, 65, 66)

The unit dosage formulation of any one of claims 64-66, wherein the non-contractile cardiomyocyte exhibits normal action potentials.

### Claim 68 (depends on 64, 65, 66, 67)

The unit dosage formulation of any one of claims 64-67, wherein the non-contractile cardiomyocyte exhibits normal calcium transients.

### Claim 69 (depends on 64, 65, 66, 67, 68)

The unit dosage formulation of any one of claims 64-68, wherein the non-contractile cardiomyocyte exhibits impaired function of a thin filament.

### Claim 70 (depends on 69)

The unit dosage formulation of claim 69, wherein the non-contractile cardiomyocyte is engineered to exhibit impaired function of a thin filament.

### Claim 71 (depends on 69, 70)

The unit dosage formulation of claim 69 or 70, wherein impaired function of a thin filament comprises impaired calcium binding to troponin.

### Claim 72 (depends on 71)

The unit dosage formulation of claim 71, wherein calcium binding to troponin is a result of knockout or mutation of at least one troponin isoform.

### Claim 73 (depends on 72)

The unit dosage formulation of claim 72, wherein the non-contractile cardiomyocyte comprises knockout of troponin I (TnI) and/or troponin T (TnT).

### Claim 74 (depends on 72)

The unit dosage formulation of claim 72, wherein the non-contractile cardiomyocyte comprises a mutation in troponin C that impairs calcium binding to troponin.

### Claim 75 (depends on 72, 74)

The unit dosage formulation of claim 72 or 74 wherein the mutation comprises D65A mutation of troponin C.

### Claim 76 (depends on 72, 74)

The unit dosage formulation of claim 72 or 74, wherein the mutation comprises I61Q and/or L57Q mutation of troponin C.

### Claim 77 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76)

The unit dosage formulation of any one of claims 64-76, wherein the non-contractile cardiomyocyte exhibits impaired actin-myosin binding.

### Claim 78 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77)

The unit dosage formulation of any one of claims 64-77, wherein the non-contractile cardiomyocyte is engineered to have impaired actin-myosin binding.

### Claim 79 (depends on 77, 78)

The unit dosage formulation of claim 77 or 78, wherein the impaired actin-myosin binding comprises a mutation in or deletion of myosin.

### Claim 80 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)

The unit dosage formulation of any one of claims 64-79, further comprising a pharmaceutically acceptable carrier.

### Claim 81 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80)

The unit dosage formulation of any one of claims 64-80, wherein the non-contractile cardiomyocyte is derived from in vitro differentiation of a pluripotent stem cell or a cardiac progenitor cell.

### Claim 82 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81)

The unit dosage formulation of any one of claims 64-81, wherein the non-contractile cardiomyocyte retains normal gap junction function when contacted with a cardiomyocyte.

### Claim 83 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The unit dosage formulation of any one of claims 64-82, wherein the gel or matrix comprises a solubilized basement membrane protein or preparation thereof.

### Claim 84 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83)

The unit dosage formulation of any one of claims 64-83, wherein the formulation further comprises one or more of an immunosuppressive agent, a pan-caspase inhibitor, an anti-apoptotic agent, IGF-1 and a KATP channel opening agent.

### Claim 85 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84)

The unit dosage formulation of any one of claims 64-84, wherein the unit dosage formulation comprises 1×103 to 5×109 non-contractile cardiomyocytes.

### Claim 86 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85)

The unit dosage formulation of any one of claims 64-85, wherein the unit dosage formulation comprises 1×104 to 5×109 non-contractile cardiomyocytes.

### Claim 87 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86)

The unit dosage formulation of any one of claims 64-86, wherein the unit dosage formulation comprises 1×105 to 5×109 non-contractile cardiomyocytes.

### Claim 88 (depends on 64, 65, 66, 67, 68)

The unit dosage formulation of any one of claims 64-68, wherein the unit dosage formulation comprises 1×106 to 5×109 non-contractile cardiomyocytes.

### Claim 89 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88)

The unit dosage formulation of any one of claims 64-88, wherein the unit dosage formulation comprises 1×107 to 5×109 non-contractile cardiomyocytes.

### Claim 90 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89)

The unit dosage formulation of any one of claims 64-89, wherein the unit dosage formulation comprises 1×108 to 5×109 non-contractile cardiomyocytes.

### Claim 91 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90)

The unit dosage formulation of any one of claims 64-90, wherein the unit dosage formulation comprises 1×105 to 1×109 non-contractile cardiomyocytes.

### Claim 92 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91)

The unit dosage formulation of any one of claims 64-91, wherein the unit dosage formulation comprises 1×105 to 5×108 non-contractile cardiomyocytes.

### Claim 93 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92)

The unit dosage formulation of any one of claims 64-92, wherein the unit dosage formulation comprises 1×105 to 1×108 non-contractile cardiomyocytes.

### Claim 94 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93)

The unit dosage formulation of any one of claims 64-93, wherein the unit dosage formulation comprises 1×105 to 5×107 non-contractile cardiomyocytes.

### Claim 95 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94)

The unit dosage formulation of any one of claims 64-94, wherein the unit dosage formulation comprises 1×105 to 1×107 non-contractile cardiomyocytes.

### Claim 96 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95)

The unit dosage formulation of any one of claims 64-95, wherein the unit dosage formulation comprises 1×105 to 5×106 non-contractile cardiomyocytes.

### Claim 97 (depends on 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96)

The unit dosage formulation of any one of claims 66-96, wherein the unit dosage formulation comprises 1×105 to 1×106 non-contractile cardiomyocytes.