Compositions and methods for genetically modifying myosin phosphatase target subunit (Mypt1) gene for lowering blood pressure

1. A therapeutically effective method of lowering blood pressure in a subject, comprising genetically modifying a myosin phosphatase target subunit (Mypt1) gene in vascular smooth muscle cells of the subject, whereby the genetic modification of Mypt1 results in a deletion or inactivation of exon 24, the modification being accomplished by administering to the cells a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system comprising:

(a) a guide RNA (gRNA) molecule comprising a targeting domain which is complementary with a target domain sequence of the Mypt1 gene, the targeting domain being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and combinations thereof; and

(b) a CRISPR associated protein 9 (Cas9) molecule or a nucleotide sequence encoding a Cas9 molecule.

2. The method of claim 1 , wherein the method comprises administering a nucleic acid composition that comprises: (a) a first nucleotide sequence encoding the gRNA molecule and (b) a second nucleotide sequence encoding the Cas9 molecule.

3. The method of claim 1 , wherein the Cas9 molecule is from Staphylococcus aureus.

4. The method of claim 3 , wherein the target domain sequence is present immediately upstream of a sequence comprising NNGRRT.

5. The method of claim 2 , wherein the CRISPR/Cas system is delivered to the cells by a virus.

6. The method of claim 5 , wherein the virus is an adeno-associated virus (AAV), a lentivirus, a retrovirus or a combination thereof.

7. The method of claim 6 , wherein the virus is an adeno-associated virus (AAV).

8. The method of claim 7 , wherein the AAV is AAV9.

9. The method of claim 1 , wherein only one allele of Mypt1 is genetically modified in the cells to delete or inactivate exon 24.

10. The method of claim 1 , wherein two alleles of Mypt1 are genetically modified in the cells to delete or inactivate exon 24.

11. The method of claim 1 , wherein the genetically modified gene produces a Mypt1 isoform that is sensitive to nitric oxide.

12. The method of claim 1 , wherein the subject is a human.

13. The method of claim 1 , wherein the targeting domain is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and combinations thereof.

14. A vector comprising a CRISPR/Cas system for genetically modifying a Mypt1 gene, wherein the genetic modification of Mypt1 results in a deletion or inactivation of exon 24, and wherein the vector comprises

(a) a nucleotide sequence encoding one or more gRNA molecules comprising a targeting domain which is complementary with a target domain sequence of the Mypt1 gene, the targeting sequence domain being selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and combinations thereof; and

(b) a nucleotide sequence encoding a Cas9 molecule.

15. The vector of claim 14 , wherein the vector is a viral vector.

16. The vector of claim 15 , wherein the viral vector is an adeno-associated virus (AAV), a lentivirus, a retrovirus or a combination thereof.

17. The vector of claim 14 , wherein the virus is an adeno-associated virus (AAV), a lentivirus, a retrovirus or a combination thereof.

18. The vector of claim 17 , wherein the virus is an adeno-associated virus (AAV).

19. The method of claim 18 , wherein the AAV is AAV9.

20. The vector of claim 14 , wherein the Cas9 molecule is from Staphylococcus aureus.