Muscle-specific expression cassettes
The present disclosure encompasses nucleic acid expression constructs that regulate the expression of a heterologous protein specifically in muscle tissues (e.g., skeletal muscle and cardiac muscle tissue). In particular embodiments of the disclosure, muscle-specific expression constructs are described that encode a heterologous protein. These heterologous proteins can be any protein that is desired to be expressed in a muscle cell. In particular embodiments of the disclosure, proteins are described for the treatment of Duchenne Muscular Dystrophy (e.g., engineered nucleases such as engineered meganucleases).
1 . A polynucleotide comprising a muscle-specific expression cassette comprising the nucleic acid sequence of SEQ ID NO: 86, wherein said polynucleotide is a recombinant adeno-associated virus 9 (AAV9) viral genome, wherein said muscle-specific expression cassette is flanked by a 5′ and inverted terminal repeat (ITR) sequence and a 3′ ITR sequence, and wherein said 5′ ITR sequence comprises a first D-sequence comprising the nucleic acid sequence of SEQ ID NO: 94 and said 3′ ITR sequence comprises a second D-sequence comprising the nucleic acid sequence of SEQ ID NO: 95.
2 . A recombinant AAV comprising said polynucleotide of claim 1 , wherein said AAV has an AAV9 capsid.
3 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said polynucleotide of claim 1 .
4 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said recombinant AAV of claim 2 .