TAT-utrophin as a protein therapy for dystrophinopathies
Disclosed is a fusion protein including a full-length TAT-utrophin or an anti-dystrophinopathic fragment thereof, a method of treating dystrophinopathies (including Duchenne muscular dystrophy) using the fusion protein, a pharmaceutical composition for treating dystrophinopathies in mammals comprising the fusion protein, and nucleic acid constructs for expressing the fusion protein.
1. An isolated fusion protein comprising:
a first protein region which is effective to transduce the fusion protein into mammalian muscle cells, operationally linked to;
a second protein region comprising a full-length utrophin protein or an anti-dystrophinopathic fragment thereof.
2. The isolated fusion protein of claim 1 , further comprising an affinity tag operationally linked to the fusion protein.
3. The isolated fusion protein of claim 2 , wherein the affinity tag comprises an amino acid sequence DYKDDDDK (SEQ. ID. NO: 1) or a fragment thereof.
4. The isolated fusion protein of claim 2 , wherein the affinity tag comprises an amino acid sequence DYKD (SEQ. ID. NO: 28).
5. The isolated fusion protein of claim 1 , wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
6. The isolated fusion protein of claim 1 , wherein the second protein region is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
7. The isolated fusion protein of claim 1 , which is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 11, 13, 15, 17, 19, 21, 23, and 25.
8. The isolated fusion protein of claim 1 , wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
9. The isolated fusion protein of claim 1 , wherein the second protein region is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
10. The isolated fusion protein of claim 1 , wherein the first protein region is an amino acid sequence as shown in SEQ. ID. NO: 2.
11. The isolated fusion protein of claim 10 , wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
12. The isolated fusion protein of claim 10 , wherein the second protein region is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
13. The isolated fusion protein of claim 1 , wherein the first protein region is an amino acid sequence as shown in SEQ. ID. NO: 5: YGRKKRRQRRR.
14. The isolated fusion protein of claim 13 , wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
15. The isolated fusion protein of claim 13 , wherein the second protein region is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
16. The isolated fusion protein of claim 1 , wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising no more than 75% of the mass of the full-length utrophin protein.
17. The isolated fusion protein of claim 1 , wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising no more than 50% of the mass of the full-length utrophin protein.
18. The isolated fusion protein of claim 1 , wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising no more than 25% of the mass of the full-length utrophin protein.
19. Pharmaceutically suitable salts of the isolated fusion protein recited in claim 1 .
20. A pharmaceutical composition for treating dystrophinopathies in mammals, including humans, comprising:
an isolated fusion protein or a pharmaceutically suitable salt thereof as recited in claim 1 , in combination with a pharmaceutically suitable carrier.
21. A method of treating dystrophinopathies in mammals, the method comprising administering to a mammalian subject in need thereof an anti-dystrophinopathic amount of an isolated fusion protein or a pharmaceutically suitable salt thereof as recited in claim 1 .
22. An isolated nucleic acid expression construct encoding a fusion protein, the nucleic acid expression construct comprising:
a first nucleic acid region that encodes a first protein region of the fusion protein, wherein the first protein region is effective to transduce the fusion protein into mammalian muscle cells, operationally linked to;
a second nucleic acid region that encodes a second protein region of the fusion protein, wherein the second protein region comprises a full-length utrophin protein or an anti-dystrophinopathic fragment thereof;
wherein the expression construct drives expression of the fusion protein when transformed into a suitable host cell or disposed into a suitable cell-free expression system.
23. The isolated nucleic acid expression construct of claim 22 , further comprising a third nucleic acid region that encodes an affinity tag that is operationally linked to the fusion protein.
24. The isolated nucleic acid expression construct of claim 22 , wherein the third nucleic acid region encodes an amino acid sequence DYKDDDDK (SEQ. ID. NO: 1) or a fragment thereof.
25. The isolated nucleic acid expression construct of claim 22 , wherein the third nucleic acid region encodes an amino acid sequence DYKD (SEQ. ID. NO: 28).
26. The isolated nucleic acid expression construct of claim 22 , wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
27. The isolated nucleic acid expression construct of claim 22 , wherein the second nucleic acid region encodes an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
28. The isolated nucleic acid expression construct of claim 22 , which is a nucleic acid sequence selected from the group consisting of SEQ. ID. NOS: 10, 12, 14, 16, 18, 20, 22, and 24.
29. The isolated nucleic acid expression construct of claim 22 , wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
30. The isolated nucleic acid expression construct of claim 22 , wherein the second nucleic acid region encodes an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
31. The isolated nucleic acid expression construct of claim 22 , wherein the first nucleic acid region encodes an amino acid sequence as shown in SEQ. ID. NO: 2.
32. The isolated nucleic acid expression construct of claim 31 , wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
33. The isolated nucleic acid expression construct of claim 31 , wherein the second nucleic acid region encodes an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
34. The isolated nucleic acid expression construct of claim 22 , wherein the first nucleic acid region encodes an amino acid sequence as shown in SEQ. ID. NO: 5: YGRKKRRQRRR.
35. The isolated nucleic acid expression construct of claim 34 , wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain plus 4, 7, 10, or 11 spectrin-like repeats.
36. The isolated nucleic acid expression construct of claim 34 , wherein the second nucleic acid region encodes an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
37. The isolated nucleic acid expression construct of claim 22 , wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising no more than 75% of the mass of the full-length utrophin protein.
38. The isolated nucleic acid expression construct of claim 22 , wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising no more than 50% of the mass of the full-length utrophin protein.
39. The isolated nucleic acid expression construct of claim 22 , wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising no more than 25% of the mass of the full-length utrophin protein.