Cell and gene based methods to improve cardiac function

一种心脏功能、心肌细胞的技术，应用在化学仪器和方法、生物化学设备和方法、引入外来遗传物质而修饰的细胞等方向，能够解决cTnC非特异性、有害等问题

Inactive Publication Date: 2014-07-23
UNIV OF WASHINGTON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some of the disadvantages associated with such compounds include their nonspecificity for cTnC and their 2+ protein involved in processing 21,22 and other aspects of the excitation-contraction coupling pathway 5 harmful effects of

Method used

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  • Cell and gene based methods to improve cardiac function
  • Cell and gene based methods to improve cardiac function
  • Cell and gene based methods to improve cardiac function

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Experimental program
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Embodiment approach

[0138] In one aspect, the invention provides a method for treating cardiac function comprising administering an engineered cTnC variant that is not clinically identified as a HCM / DCM mutation, but elicits contractile Ca 2+ Similar increase or decrease in sensitivity.

[0139] In one aspect, the invention provides a method for improving cardiac function in an individual in need thereof comprising administering to the cardiac tissue of the individual 2+ A vector of cTnC variants with binding affinity. In a specific embodiment, the cTnC variant comprises the L48Q amino acid substitution.

[0140] In a certain embodiment, the individual has a cardiac condition that results in decreased contraction. In a specific embodiment, the individual has an infarcted heart.

[0141] In one aspect, the invention provides a method for improving cardiac function in an individual in need thereof comprising administering to the cardiac tissue of the individual 2+ A vector of cTnC variants with...

Embodiment 1

[0173] Isolation and culture of adult and neonatal cells. These studies were approved by the University of Washington (UW) Animal Care Committee and were conducted in accordance with federal guidelines. The Department of Comparative Medicine at UW cares for animals in accordance with National Institutes of Health regulations for the humane use, care and management of laboratory animals. Isolation of Adult Rat (Fischer344) Cardiomyocytes (ARC) from the Heart Using Aortic Retrograde Perfusion by Enzymatic (Collagenase / Protease) Dispersion of Cells 10 . as previously stated 11 , Neonatal rat cardiomyocytes (NRC) were isolated from 1-3 day old neonatal Fischer344 rats by enzymatic dispersion.

Embodiment 2

[0175] Plasmid design and virus production. We generated adenoviral vectors generated from HEK293 expressing histidine-tagged (N-terminal 6-His) L48Q cTnC or WT cTnC from the CMV promoter. Both vectors contain a secondary expression cassette for green fluorescent protein (GFP) as a transduction reporter protein, we also express GFP-only vectors. Virus was introduced into cardiomyocytes at -250 particles per cell.

[0176] Recombinant adenoviruses containing appropriate cDNA constructs driven by CMV promoters were used to induce overexpression of L48Q and WT cTnC in transfected cultured adult and neonatal rat cardiomyocytes. A second expression cassette for green fluorescent protein (GFP) was included in each adenovirus as a reporter for successful transduction. Cardiomyocytes were infected with adenovirus containing the gene for [L48Q cTnC+GFP] or [WT cTnC+GFP] for 2 days. We obtained almost 100% transfection efficiency and gene transfer, as generally shown by green fluores...

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Abstract

Compositions and methods for improving cardiac function, myocardial contractility and relaxation in a mammal are provided. Cardiomyocytes transfected with one or more expression vectors comprising a ribonucleotide reductase subunit Rl -encoding nucleic acid sequence and a ribonucleotide reductase subunit R2-encoding nucleic acid sequence operably linked to a promoter are grafted to a mammalian myocardium. Alternatively, viral vector(s) having the Rl and R2-encoding construct(s) are administered to the mammal directly. Overexpression of Rl and R2 subunits leads to formation of the RR complex, which in turn generates dATP. Improvement of cardiac function can also be effected by administration of vectors comprising a nucleic acid sequence encoding a L48Q-substituted, I61Q-substituted, or L57Q-substituted cTnC variant.; Also provided are compositions and methods for delivering dATP to a myocardium through grafting of donor cells overexpressing Rl and R2. dATP is thereby produced in situ and delivered through gap junctions established between donor cells and host cardiomyocytes.

Description

[0001] Statement of Rights in Inventions Obtained Under Federally Sponsored Research or Development [0002] This invention was made with government support under Grant Nos. HL61683, R21HL091368, HL07828, HL65497, R01HL64387, R01HL084642, and P01HL004374 awarded by the National Institutes of Health. The Government has certain rights in this invention. [0003] Cross References to Related Applications [0004] This application claims the benefit of U.S. Provisional Application No. 61 / 490,450, filed May 26, 2011, which is hereby incorporated by reference in its entirety, under 35 U.S.C. § 119(e). Background of the invention [0005] Heart disease is the leading cause of mortality and morbidity in the United States and has risen dramatically worldwide . Sarcomeric heart disease such as HCM and DCM involves amino acid mutations in one of several myofilament proteins that often lead to heart failure and, in some cases, sudden cardiac death . As the number of mutants identifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12P21/04
CPCC12N9/0093A01K67/0275A61K48/005C07K14/4716C12Y117/04002C12N15/8509A01K2217/052A01K2227/105A01K2267/0375C12N2799/022C12N2799/025C12Y117/04001A61P9/00A61P9/04A61P9/10C12N15/85C12N15/864C12N5/10A61K48/00A61K9/0019A61K35/34C12N5/0657C12N7/00C12N15/86C12N2750/14132C12N2750/14143
Inventor M·雷尼尔M·拉弗拉梅C·默里F·S·科尔特S·伦迪S·D·豪施卡J·S·张伯伦
Owner UNIV OF WASHINGTON
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