miRNA-Responsive mRNA for Cardiomyocyte Sorting
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Solution Overview
Problem
Current methods for sorting cardiomyocytes are inefficient in achieving high purity, often relying on surface markers and requiring complex processes like FACS, which can be cumbersome and unsafe for cell sorting.
Innovation Solution
The use of miRNA-responsive mRNA, specifically designed to target miRNAs like miR-1, miR-208a, and miR-499a-5p, which are uniquely expressed in cardiomyocytes, allows for the purification of cardiomyocytes by regulating gene expression and introducing drug resistance genes for selective sorting without immobilizing cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If surface marker-based sorting methods are used, then cardiomyocytes can be sorted, but the purity is insufficient and the process is complex
Solution Approach 1:
The invention extracts and utilizes miRNA-specific sequences that are uniquely expressed in cardiomyocytes as sorting markers. By designing mRNA molecules containing these specific miRNA recognition sequences, the method extracts the essential sorting function from complex surface marker systems and implements it through a simpler, more specific molecular mechanism that achieves high purity sorting.
Solution Approach 2:
The invention changes the sorting parameter from surface protein markers to intracellular miRNA expression patterns. By detecting and utilizing the specific miRNA expression profile unique to cardiomyocytes, the method achieves higher sorting precision while simplifying the overall process through a more fundamental cellular characteristic.
2Manufacturing precision
If FACS sorting is performed, then cardiomyocytes can be separated, but cell immobilization occurs and safety is compromised
Solution Approach 1:
The invention replaces the mechanical FACS sorting system with a biochemical selection mechanism. Instead of using mechanical forces and fluorescent detection, the method uses miRNA-specific mRNA molecules that selectively bind to cardiomyocytes through molecular recognition, enabling sorting without mechanical stress or cell immobilization.
Solution Approach 2:
The invention introduces miRNA-specific mRNA molecules as intermediary agents that mediate the sorting process. These mRNA molecules act as selective carriers that bind to cardiomyocytes through specific miRNA recognition, facilitating safe and effective sorting without direct mechanical intervention or cell immobilization.
3Manufacturing precision
If conventional sorting methods are used, then some purification is achieved, but the degree of purity is insufficient for therapeutic use
Solution Approach 1:
The invention applies excessive specificity by targeting unique miRNA sequences that are exclusively expressed in cardiomyocytes. This excessive action ensures that only true cardiomyocytes are sorted with extremely high purity, well beyond what conventional methods achieve, thereby ensuring reliability for therapeutic applications.
Solution Approach 2:
The invention performs preliminary enrichment by introducing miRNA-specific mRNA molecules before final sorting. This preliminary action pre-markes cardiomyocytes with high specificity, enabling subsequent sorting steps to achieve the extremely high purity required for safe transplantation therapy.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the safe and efficient purification of cardiomyocytes to a high degree of purity, facilitating their use in treating heart-related diseases by transiently introducing mRNA that is not incorporated into the genome, thus avoiding long-term genetic changes.
Implementation Method 1
miRNA-responsive mRNA consisting of a sequence comprising (i) a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes, and (ii) a nucleic acid corresponding to the coding region of a gene, wherein translation of (ii) is regulated by (i)
Data Source
AI summary
An object of the present invention is to provide a novel method for sorting cardiomyocytes. Another object of the present invention is to provide a method for producing high-purity cardiomyocytes and a kit used therefor. The present invention provides a method for sorting cardiomyocytes, comprising a step of introducing miRNA-responsive mRNA into a cell group, wherein the miRNA-responsive mRNA consists of a sequence comprising the following (i) and (ii):(i) a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes, and(ii) a nucleic acid corresponding to the coding region of a gene, wherein translation of (ii) the nucleic acid corresponding to the coding region of a gene into protein is regulated by the nucleic acid sequence in (i) above, thereby achieving the aforementioned objects.