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Publication Number
US-12168778-B2
Publication Date
2024-12-17
Expiration Date
Abstract
The present disclosure provides methods for generating induced cardiomyocytes and/or inducing a cardiomyocyte phenotype in cells in vivo or in vitro, such as by expression of ASCL1 or MYF6 and MYOCD. The present disclosure further provides gene-delivery vectors comprising one or more polynucleotides selected from ASCL1, MYF6, MYOCD, MEF2C, and TBX5. It further provides compositions comprising induced cardiomyocytes and provides methods of treating a heart condition, such as myocardial infarction. The disclosure also provides engineered myocardin proteins with an internal deletion, vectors encoding such engineered mycocardins, and methods of use thereof.
Core Innovation
The disclosure provides an engineered human MYOCD polynucleotide encoding an engineered myocardin protein comprising a deletion of at least 200 amino acids in the region corresponding to amino acids 414-764 of a human myocardin protein of SEQ ID NO: 3. The engineered myocardin protein retains myocardin functionality and includes an Mef2c interaction domain sharing at least 85% identity to SEQ ID NO: 17, with domain definitions for SRF, SAP, and TAD, and optional LZ domain inclusion in certain embodiments.
The engineered myocardin protein is configured for use in cardiac cell reprogramming with transcription factors ASCL1 and/or MYF6 and myocardin (MYOCD). The disclosure also includes 2A-linked polycistronic formats such as MYOCD-2A-ASCL1 and ASCL1-2A-MYOCD, along with related cardiac reprogramming factor polynucleotides including MYOCD-2A-MYF6, My43-2A-ASCL1, My43-2A-MYF6, ASCL1-2A-My43, and MYF6-2A-My43.
Recombinant viral vector formats are described, including recombinant adeno-associated virus (rAAV) and recombinant lentivirus (rLV), with expression cassettes containing promoter and polyadenylation sequence elements in a 5′ to 3′ order arrangement. For rAAV, embodiments include 5′/3′ ITRs, a CAG promoter, an SV40 intron, and a polyadenylation sequence, with an ordered 5′→3′ sequence of the engineered human MYOCD polynucleotide, a polynucleotide encoding a 2A peptide linker, and a polynucleotide encoding ASCL1.
Claims Coverage
The independent claim coverage centers on an engineered human MYOCD polynucleotide encoding an engineered myocardin protein with a specified internal deletion region. Across the consolidated items, six inventive features are identified, including deletion refinements, an interaction-domain identity constraint, a relative expression-level constraint, 2A-linked multigene arrangements, and an rAAV expression-cassette ordering.
Engineered human myocardin polynucleotide with internal deletion in the 414-764 region
An engineered human myocardin (MYOCD) polynucleotide encoding an engineered myocardin protein, wherein the engineered myocardin protein comprises a deletion of at least 200 amino acids in the region corresponding to amino acids 414-764 of a human myocardin protein of SEQ ID NO: 3.
Engineered myocardin deletion interval refinement
The engineered myocardin protein includes a deletion of amino acids from about 560 to about 763 of the human myocardin protein.
Mef2c interaction domain identity constraint for the engineered myocardin protein
The polynucleotide encodes an engineered myocardin protein that includes a Mef2c interaction domain sharing at least 85% identity to SEQ ID NO: 17.
Relative expression level constraint of the engineered myocardin protein
The engineered myocardin protein is expressed at at least 10% of the level of the human myocardin protein in a same expression system.
2A-linked ASCL1 and engineered MYOCD multigene polynucleotide arrangement
The ASCL1 polynucleotide is linked to the engineered human MYOCD polynucleotide via a 2A polynucleotide encoding a 2A peptide linker to form either an MYOCD-2A-ASCL1 or an ASCL1-2A-MYOCD polynucleotide.
Ordered rAAV expression cassette integrating engineered MYOCD, 2A linker, and ASCL1
A recombinant adeno-associated virus (rAAV) has an expression cassette with 5′/3′ ITRs, a CAG promoter, an SV40 intron, a polyadenylation sequence, and a 5′→3′ order of (i) the engineered human MYOCD polynucleotide, (ii) a polynucleotide encoding a 2A peptide linker, and (iii) a polynucleotide encoding ASCL1.
Overall, the claim coverage focuses on an engineered human MYOCD polynucleotide encoding an engineered myocardin protein with an internal deletion in the 414-764 region, together with refinements specifying a tighter deletion interval, maintaining a Mef2c interaction domain with defined identity, achieving a minimum relative expression level, and implementing 2A-linked multigene polynucleotide formats, particularly via an rAAV expression cassette with a defined 5′→3′ arrangement including engineered MYOCD and ASCL1.
Stated Advantages
Increased reprogramming marker expression in cardiac fibroblast reprogramming contexts.
Enhancement of calcium transients (CaT) in the reprogramming context.
Functional improvement in vivo in MI models as assessed by echocardiographic ejection fraction.
Engineered myocardin variants with internal deletions retain function and can enhance cardiomyocyte reprogramming.
Documented Applications
Cardiac fibroblast reprogramming, with reprogramming marker induction and calcium transient enhancement.
In vivo MI treatment in MI models, with echocardiographic ejection fraction improvement.
Use of engineered constructs in induced cardiomyocytes (iCM) contexts.
Cardiac cell reprogramming to induced cardiomyocytes (iCM) using transcription factors including ASCL1 and/or MYF6 with myocardin (MYOCD).
Therapeutic uses for heart conditions, including myocardial infarction (MI), dilated cardiomyopathy, and heart failure.
AAV delivery of My43 and/or ASCL1 for myocardial infarction (MI) and heart failure repair.
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