Methods for maturing cardiomyocytes and uses thereof

• US 9,624,471 B2
• Filed: 06/12/2014
• Issued: 04/18/2017
• Est. Priority Date: 06/12/2013
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Kuppusamy, Kavitha, Ruohola-Baker, Hannele, Sperber, Henrik
• Primary Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US14/897,403
• Publication Number:
  US 20160130555A1
• Time in Patent Office: 1,041 Days
• Field of Search: None
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

Described herein are methods and compositions useful for inducing maturation of a cardiomyocyte to a mature (e.g., adult) phenotype, such that the function and morphology of the mature cardiomyocyte matches or more closely mimics that of the adult heart. The methods and compositions use Let-7 miRNAs and modified forms thereof. Such methods and compositions permit the study and treatment of adult-onset cardiac diseases, disorders or injuries with mature cardiomyocytes that mimic the heart function of an adult. Methods of using cardiomyocytes matured in this manner for drug identification and drug cardiotoxicity testing are also provided.

Claims

1. A method for inducing maturation of a cultured cardiomyocyte comprising contacting in vitro a cultured cardiomyocyte derived from a pluripotent stem cell or an induced pluripotent stem cell (iPS cell) with an isolated microRNA from the let-7 family, or with a vector that expresses said miRNA of the let-7 family, thereby inducing maturation of the cultured cardiomyocyte.

2. The method of claim 1, wherein the miRNA from the let-7 family is selected from the group consisting of:

let-7a-1, let-7a-2, let-7b, let-7c, let-7d, let-7e, let-7f-1, let-7f-2, let-7g, and let-7i.

3. The method of claim 2, wherein the miRNA is let-7g or let-7i.

4. The method of claim 1, wherein the miRNA from the let-7 family is resistant to inhibition by Lin28.

5. The method of claim 1, wherein the cardiomyocyte expresses the fetal isoform of myosin heavy chain (MHC), troponin 1, or carnitine palmitoyl transferase 1 (CPT-1) prior to contacting with the miRNA.

6. The method of claim 1, wherein the maturation of the cardiomyocyte comprises a shift in the primary source of energy from glycolysis to fatty acid oxidation.

7. The method of claim 1, wherein the resulting mature cardiomyocyte expresses at least one of:

cardiac troponin (cTnT), myosin heavy chain-7 (MYH7), sacrcoendoplasmic reticulum ATPAse (SERCA2a), gap junction protein alpha 1 (GJA1), or ryanodine receptor 2 (RYR2).

8. The method of claim 1, further comprising a step of administering the resulting mature cardiomyocyte to a subject in need thereof, wherein the subject has cardiac tissue damage as a result of an acute myocardial infarction, ischemia/reperfusion injury, autophagy, cardiomyopathy, dilated cardiomyopathy, heart failure, restenosis, apoptosis, or necrosis.

9. The method of claim 1, wherein the cultured cardiomyocyte is a human cardiomyocyte.