Multicistronic miRNA delivery for cardiac reprogramming
Inventors
Hodgkinson, Conrad P. • Dzau, Victor J.
Assignees
Publication Number
US-12285444-B2
Publication Date
2025-04-29
Expiration Date
2041-12-03
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Abstract
The invention provides a method of promoting reprogramming of a cardiac fibroblast into a cardiomyocyte by contacting the cardiac fibroblast with an isolated polynucleotide molecule comprising at least two functional miRNA sequences using a multicistronic expression system for reprogramming of fibroblasts into functional mature cardiomyocytes.
Core Innovation
The invention provides a method for promoting the reprogramming of cardiac fibroblasts into cardiomyocytes by contacting the fibroblasts with an isolated polynucleotide molecule comprising at least two functional miRNA sequences selected from the group consisting of miR-1, miR-133, miR-208, and miR-499. This is achieved using a multicistronic expression system where the mature miRNA sequences of the endogenous miR-17-92 backbone are replaced by the desired miRNAs, enabling the delivery and expression of multiple reprogramming factors in a defined stoichiometry from a single construct.
The problem addressed by this invention is the inefficiency and unpredictability of previously used methods for fibroblast reprogramming, where individual lenti- or retro-viral vectors expressed single reprogramming factors, resulting in variable ratios and low conversion rates. Additionally, these viral vectors are not specific for cardiac fibroblasts, contributing to off-target effects and decreased reprogramming efficiency.
To address these challenges, the invention develops a multicistronic miR combo system that ensures each cardiac fibroblast receives all necessary miRNA reprogramming factors at optimized ratios. The multicistronic construct arranges the four miRNAs (miR-499, miR-1, miR-208, miR-133) in a defined order to achieve equivalent expression of each, which has been demonstrated to significantly enhance reprogramming efficiency. Furthermore, the construct is delivered by an adeno-associated virus serotype 1 (AAV1) that displays tropism toward cardiac fibroblasts, further improving targeting specificity and efficacy.
Claims Coverage
There is one independent claim describing the key inventive features relating to multicistronic miRNA delivery for cardiac reprogramming.
Method of reprogramming cardiac fibroblasts using an ordered multicistronic miRNA construct in a miR-17-92 backbone
The method involves contacting a cardiac fibroblast with an isolated polynucleotide containing functional miRNAs: miR-1, miR-133, miR-208, and miR-499. The miRNAs are arranged in a specific 5′ to 3′ orientation as an ordered combination (miR-499:miR-1:miR-208:miR-133:miR-1) within a miR-17-92 backbone. The mature miRNAs of the backbone are replaced by these functional miRNAs.
The claim defines a method of efficiently reprogramming cardiac fibroblasts by delivering a multicistronic polynucleotide comprising a specified arrangement of miRNAs within a miR-17-92 backbone, replacing native mature miRNAs with reprogramming miRNAs.
Stated Advantages
Balanced and precise delivery of all reprogramming miRNAs to each cell ensures defined stoichiometry, leading to significantly improved reprogramming efficiency compared to prior art.
Utilization of a multicistronic system allows expression of multiple miRNAs from a single construct, solving the problem of unequal or incomplete delivery associated with independent viral vectors.
Use of AAV1 for delivery achieves enhanced specificity for cardiac fibroblasts, decreasing off-target effects and further increasing conversion efficiency.
The invention provides clinical benefit for treating impaired cardiac muscle function and reversing cardiac scar tissue following myocardial infarction or injury.
Documented Applications
Treatment of human subjects diagnosed with impaired cardiac function or cardiac scar tissue, such as after myocardial infarction or other cardiac injuries.
In vivo reprogramming of cardiac fibroblasts to generate functional mature cardiomyocytes for improved cardiac function.
Use in cardiac gene therapy for regeneration of injured cardiac tissue and potential reversal of cardiac fibrosis.
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