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Publication Number
US-11913012-B2
Publication Date
2024-02-27
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 invention provides vectors comprising expression cassettes that include a myocardin (MYOCD) polynucleotide encoding a MYOCD polypeptide and an Achaete-scute homolog 1 (ASCL1) polynucleotide encoding an ASCL1 polypeptide. The MYOCD polynucleotide and the ASCL1 polynucleotide are each operatively linked to at least one promoter, and the vector is an adeno-associated virus (AAV) vector or a lentiviral vector. The disclosure also includes recombinant vector embodiments for specific MYOCD/ASCL1 and 2A-linked constructs.
The expression cassettes further include MYOCD/ASCL1 constructs in which the coding polynucleotides are arranged in 5′ to 3′ order and are operatively linked to defined promoter–polynucleotide–polyadenylation arrangements. Bicistronic constructs are described that use a 2A linker to generate MYOCD/ASCL1 2A-linked polynucleotides, including MYOCD-2A-ASCL1 and related 2A-linked arrangements. Sequence-identity variants are described for the disclosed constructs and recombinant vector embodiments.
The invention further describes engineered MYOCD polynucleotides and engineered myocardin proteins defined by specific domain compositions, including an SRF interaction domain, an SAP domain, and a TAD domain, with an optional Mef2c interaction domain and an LZ domain. The engineered MYOCD polynucleotides and proteins are defined using quantitative sequence identity constraints to provided SEQ ID sequences, including internal deletion variants. The disclosure also includes treating myocardial infarction by administering a vector containing an expression cassette with MYOCD and ASCL1 polynucleotides by intramyocardial injection.
Claims Coverage
The consolidated claim coverage includes two independent claims and multiple dependent refinements. The inventive features center on a viral vector carrying a MYOCD/ASCL1 expression cassette and on intramyocardial treatment of myocardial infarction using that vector, with additional features including 2A-linked arrangement, AAV ITR architecture, sequence identity constraints, and decrease in ejection fraction.
Viral vector with MYOCD and ASCL1 expression cassette
A vector comprising an expression cassette having a myocardin (MYOCD) polynucleotide encoding a MYOCD polypeptide and an Achaete-scute homolog 1 (ASCL1) polynucleotide encoding an ASCL1 polypeptide, wherein each polynucleotide is operatively linked to at least one promoter, and wherein the vector is an adeno-associated virus (AAV) vector or a lentiviral vector.
Intramyocardial treatment of myocardial infarction using a MYOCD/ASCL1 vector
A method of treating myocardial infarction in a subject in need thereof, comprising administering, by intramyocardial injection, a vector comprising an expression cassette having a myocardin (MYOCD) polynucleotide and an Achaete-scute homolog 1 (ASCL1) polynucleotide, each operatively linked to at least one promoter, wherein the vector is an AAV vector or a lentiviral vector.
MYOCD polypeptide sequence identity constraint
The MYOCD polypeptide has at least 95% sequence identity to the polypeptide in SEQ ID NO: 16.
AAV vector construct with ITR-flanked expression cassette
The expression cassette is flanked by two AAV inverted terminal repeats (ITRs) within an AAV vector that includes an AAV capsid.
2A-linked MYOCD-ASCL1 polynucleotide arrangement
The MYOCD and ASCL1 polynucleotides are joined using a 2A sequence to create a MYOCD-2A-ASCL1 polynucleotide arranged 5' to 3' and operatively linked to a single promoter.
Decrease in ejection fraction
The one or more symptoms comprise a decrease in ejection fraction of the heart of the subject.
Lentiviral vector implementation
The vector is a lentiviral vector.
Overall, the claims cover viral delivery of a MYOCD/ASCL1 dual-expression cassette in AAV or lentiviral form and intramyocardial injection of that vector for treating myocardial infarction. The dependent features further specify 2A-linked construct arrangements, AAV ITR-flanked expression cassettes, sequence identity constraints, and decreased ejection fraction.
Stated Advantages
Improving cardiac phenotype markers.
Improving ejection fraction in MI models.
Documented Applications
Treating myocardial infarction in a subject in need thereof by administering an AAV or lentiviral vector intramyocardially.
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