Cardiac-specific nucleic acid regulatory elements and methods and use thereof
The present invention relates to nucleic acid regulatory elements that are able to enhance cardiac-specific expression of genes, methods employing these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. The present invention is particularly useful for applications using gene therapy.
1. A nucleic acid expression cassette comprising at least one nucleic acid regulatory element of 300 nucleotides or less for enhancing cardiac-specific gene expression, wherein the nucleic acid regulatory element is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8 and a sequence having 95% identity to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8, and wherein the nucleic acid regulatory element is operably linked to a promoter and a transgene.
2. The nucleic acid expression cassette of claim 1 , wherein the nucleic acid regulatory element is SEQ ID NO: 1 or a sequence having 95% identity thereto.
3. The nucleic acid expression cassette of claim 1 , wherein the nucleic acid regulatory element is 250 nucleotides or less.
4. A nucleic acid expression cassette comprising two or more nucleic acid regulatory elements each of 300 nucleotides or less for enhancing cardiac-specific gene expression, wherein the nucleic acid regulatory elements are selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8 and a sequence having 95% identity to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8, and wherein each of the nucleic acid regulatory element is operably linked to a promoter and a transgene.
5. The nucleic acid expression cassette of claim 4 , wherein the two or more nucleic acid regulatory elements are identical.
6. The nucleic acid expression cassette of claim 1 , wherein the promoter is a cardiac-specific promoter.
7. The nucleic acid expression cassette of claim 6 , wherein the promoter is from the myosin heavy chain gene.
8. The nucleic acid expression cassette of claim 1 , additionally comprising a β-globin intron.
9. The nucleic acid expression cassette of claim 1 , wherein the transgene encodes a therapeutic protein.
10. A vector comprising the nucleic acid expression cassette of claim 1 .
11. A vector comprising: a nucleic acid regulatory element of 300 nucleotides or less selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8 and a sequence having 95% identity to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8, and wherein the nucleic acid regulatory element is operably linked to a promoter and a transgene.
12. The vector according to claim 11 , which is a viral vector.
13. The vector according to claim 12 , which is an AAV9 vector or an AAV2/9 vector.
14. A method for expressing a protein in a heart cell, the method comprising: introducing into at least one heart cell the nucleic acid expression cassette of claim 1 ; and expressing the transgene protein product thereof in the heart cell.
15. The method according to claim 14 , wherein the protein is expressed in vivo.
16. The nucleic acid expression cassette of claim 7 , wherein the promoter is from the myosin heavy chain alpha (αMHC) gene.
17. The nucleic acid expression cassette of claim 9 , wherein the therapeutic protein is selected from the group consisting of an angiogenic factor, VEGF, PlGF, an ATPase, SERCA2a, an ion channel, a cytokine, and a growth factor.
18. The vector of claim 12 , wherein the vector is a lentiviral or an AAV vector.
19. A vector comprising: a nucleic acid expression cassette comprising at least one nucleic acid regulatory element of 300 nucleotides or less, wherein the nucleic acid regulatory element is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8 and sequence having 95% identity to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8, and wherein the nucleic acid regulatory element is operably linked to a promoter and a transgene.
20. An isolated nucleic acid regulatory element of 300 nucleotides or less for enhancing cardiac-specific gene expression, wherein the nucleic acid regulatory element is selected from a sequence having between 95% and 99% identity to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8, and wherein the nucleic acid regulatory element is operably linked to a promoter and a transgene.
21. A pharmaceutical composition comprising the nucleic acid expression cassette of claim 9 , and a pharmaceutically acceptable carrier.