Protease-resistant mutants of stromal cell derived factor-1 in the repair of tissue damage

1. An isolated mutant form of stromal cell derived factor-1 (SDF-1) peptide comprising the formula of a mutant SDF-1 (mSDF-1) or X p -mSDF-1, wherein said mSDF-1 is a peptide comprising the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:53 and which is optionally extended at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO:53, said SEQ ID NO:53 comprising the amino acid sequence:

K P X 3 X 4 X 5 X 6 Y R C P C R F F E S H V A R A N V K H L K I L N T P N C A L Q I V A R L K N N N R Q V C I D P K L K W I Q E Y L E K A L N K (SEQ ID NO:53), wherein

X 3 is any amino acid;

X 4 is serine or valine;

X 5 is leucine, proline, threonine, or valine; and

X 6 is any amino acid residue,

and wherein

a) X p is a proteinogenic amino acid(s) or a protease protective organic group and p is any integer from 1 to 4;

b) said mSDF-1 maintains chemoattractant activity for T cells and is inactivated by matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), leukocyte elastase, and/or cathepsin G at a rate that is at least 50% less than the rate of inactivation of native SDF-1;

c) said X p -mSDF-1 maintains chemoattractant activity for T cells, is inactivated by dipeptidyl peptidase IV (DPPIV) at a rate that is at least 50% less than the rate at which native SDF-1 is inactivated, and is inactivated by MMP-2, MMP-9, leukocyte elastase, and/or cathepsin G at a rate that is at least 50% less than the rate of inactivation of native SDF-1; and

d) said SDF-1 peptide does not comprise the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, or SEQ ID NOs:65-67.

2. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 , wherein said mSDF-1 peptide is SDF(V3H), consisting of SEQ ID NO:54.

3. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 2 , wherein said peptide is an X p mSDF-1 peptide and wherein X is a serine and p is 1.

4. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 , wherein said mSDF-1 peptide is SDF(V3C), consisting of SEQ ID NO:55.

5. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 4 , wherein said peptide is an X p -mSDF-1 peptide and wherein X is a serine and p is 1.

6. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 , wherein said mSDF-1 peptide is SDF(S6C), consisting of SEQ ID NO:61.

7. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 6 , wherein said peptide is an X p -mSDF-1 peptide and wherein X is a serine and p is 1.

8. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 , wherein said mSDF-1 peptide is SDF(S6G), consisting of SEQ ID NO:62.

9. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 8 , wherein said peptide is an X p -mSDF-1 peptide and wherein X is a serine and p is 1.

10. An isolated mutant form of stromal cell derived factor-1 (SDF-1) peptide comprising the formula of a mutant SDF-1 (mSDF-1) or X p -mSDF-1, wherein said mSDF-1 is a peptide comprising the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:68 and which is optionally extended at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO:68, said SEQ ID NO:68 comprising the amino acid sequence:

K P X 3 S L X 6 Y R C P C R F F E S H V A R A N V K H L K I L N T P N C A L Q I V A R L K N N N R Q V C I D P K L K W I Q E Y L E K A L N K (SEQ ID NO:68), wherein X 3 and X 6 are any amino acid, and wherein

a) X p is a proteinogenic amino acid(s) or a protease protective organic group and p is any integer from 1 to 4;

b) said mSDF-1 maintains chemoattractant activity for T cells and is inactivated by matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), leukocyte elastase, and/or cathepsin G at a rate that is at least 50% less than the rate of inactivation of native SDF-1;

c) said X p -mSDF-1 maintains chemoattractant activity for T cells, is inactivated by dipeptidyl peptidase IV (DPPIV) at a rate that is at least 50% less than the rate at which native SDF-1 is inactivated, and is inactivated by MMP-2, MMP-9, leukocyte elastase, and/or cathepsin G at a rate that is at least 50% less than the rate of inactivation of native SDF-1; and

d) said SDF-1 peptide does not comprise the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:52.

11. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 10 , wherein X 3 is valine and X 6 is any amino acid residue.

12. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 10 , wherein X 3 is any amino acid residue and X 6 is serine.

13. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 10 , wherein said peptide is an X p -mSDF-1 peptide and wherein X is a serine and p equals one.

14. A fusion protein comprising the formula: A-(L) n -(R) q , wherein: A is the isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 ; n is an integer from 0-3; q is an integer from 1-3; L is a linker sequence of 3-9 amino acids; and R is a self-assembling peptide selected from the group consisting of:

AKAKAEAEAKAKAEAE;

(SEQ ID NO: 1)

AKAEAKAEAKAEAKAE;

(SEQ ID NO: 2)

EAKAEAKAEAKAEAKA;

(SEQ ID NO: 3)

KAEAKAEAKAEAKAEA;

(SEQ ID NO: 4)

AEAKAEAKAEAKAEAK;

(SEQ ID NO: 5)

ADADARARADADARAR;

(SEQ ID NO: 6)

ARADARADARADARAD;

(SEQ ID NO: 7)

DARADARADARADARA;

(SEQ ID NO: 8)

RADARADARADARADA;

(SEQ ID NO: 9)

ADARADARADARADAR;

(SEQ ID NO: 10)

ARADAKAEARADAKAE;

(SEQ ID NO: 11)

AKAEARADAKAEARAD;

(SEQ ID NO: 12)

ARAKADAEARAKADAE;

(SEQ ID NO: 13)

AKARAEADAKARADAE;

(SEQ ID NO: 14)

AQAQAQAQAQAQAQAQ;

(SEQ ID NO: 15)

VQVQVQVQVQVQVQVQ;

(SEQ ID NO: 16)

YQYQYQYQYQYQYQYQ;

(SEQ ID NO: 17)

HQHQHQHQHQHQHQHQ;

(SEQ ID NO: 18)

ANANANANANANANAN;

(SEQ ID NO: 19)

VNVNVNVNVNVNVNVN;

(SEQ ID NO: 20)

YNYNYNYNYNYNYNYN;

(SEQ ID NO: 21)

HNHNHNHNHNHNHNHN;

(SEQ ID NO: 22)

ANAQANAQANAQANAQ;

(SEQ ID NO: 23)

AQANAQANAQANAQAN;

(SEQ ID NO: 24)

VNVQVNVQVNVQVNVQ;

(SEQ ID NO: 25)

VQVNVQVNVQVNVQVN;

(SEQ ID NO: 26)

YNYQYNYQYNYQYNYQ;

(SEQ ID NO: 27)

YQYNYQYNYQYNYQYN;

(SEQ ID NO: 28)

HNHQHNHQHNHQHNHQ;

(SEQ ID NO: 29)

HQHNHQHNHQHNHQHN;

(SEQ ID NO: 30)

AKAQADAKAQADAKAQAD;

(SEQ ID NO: 31)

VKVQVDVKVQVDVKVQVD;

(SEQ ID NO: 32)

YKYQYDYKYQYDYKYQYD;

(SEQ ID NO: 33)

HKHQHDHKHQHDHKHQHD;

(SEQ ID NO: 34)

RARADADARARADADA;

(SEQ ID NO: 35)

RADARGDARADARGDA;

(SEQ ID NO: 36)

RAEARAEARAEARAEA;

(SEQ ID NO: 37)

KADAKADAKADAKADA;

(SEQ ID NO: 38)

AEAEAHAHAHAHAHAH;

(SEQ ID NO: 39)

FEFEFKFKFEFEFKFK;

(SEQ ID NO: 40)

LELELKLKLELELKLK;

(SEQ ID NO: 41)

AEAEAKAKAEAEAKAK;

(SEQ ID NO: 42)

AEAEAEAEAKAK;

(SEQ ID NO: 43)

KAKAKAKAEAEAEAEA;

(SEQ ID NO: 44)

AEAEAEAEAKAKAKAK;

(SEQ ID NO: 45)

RARARARADADADADA;

(SEQ ID NO: 46)

ADADADADARARARAR;

(SEQ ID NO: 47)

DADADADARARARARA;

(SEQ ID NO: 48)

HEHEHKHKHEHEHKHK;

(SEQ ID NO: 49)

VEVEVEVEVEVEVEVEVEVE;

(SEQ ID NO: 50)

and

RFRFRFRFRFRFRFRFRFRF.

(SEQ ID NO: 51)

15. The fusion protein of claim 14 , wherein A is either said mSDF-1 peptide or said X p -mSDF-1 peptide and comprises the amino acid sequence of any one of SEQ ID NOs: 53-55, 61, 62, and 68.

16. The fusion protein of claim 14 , wherein n=1 and L is selected from the group consisting of: GGGGGG (SEQ ID NO:57); GIVGPL (SEQ ID NO:58) and PVGLIG (SEQ ID NO:59).

17. The fusion protein of claim 14 , wherein q=1 and R is RARADADARARADADA (SEQ ID NO:35).

18. The fusion protein of claim 14 , wherein q=1 and R is RADARADARADARADA (SEQ ID NO:9).

19. A fusion protein comprising the formula: A-(L) n -Fc, wherein: A is the isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 ; n is an integer from 0-3; L is a linker sequence of 3-9 amino acids; and Fc is an Fc peptide from an Fc region of an immunoglobulin.

20. The fusion protein of claim 19 , wherein A is either said mSDF-1 peptide or said X p -mSDF-1 peptide and comprises the amino acid sequence of any one of SEQ ID NOs: 53-55, 61, 62, and 68.

21. The fusion protein of claim 19 , wherein n=1 and L is selected from the group consisting of: GGGGS (SEQ ID NO:84).

22. A biologically compatible peptide membrane comprising one or more self-assembling peptides having an amino acid sequence selected from the group consisting of SEQ ID NO:1-SEQ ID NO:51 and wherein between 0.1-10% of said self-assembling peptides are bound to the isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 .

23. A method of stimulating angiogenesis in damaged tissue resulting from a disease or condition in a subject in need thereof, said method comprising administering to said damaged tissue the isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 in an amount sufficient to stimulate angiogenesis in said damaged tissue in said subject.

24. The method of claim 23 , wherein said isolated mSDF-1 or X p -mSDF-1 peptide is attached to a biologically compatible membrane or is attached to a self-assembling peptide that forms a biologically compatible membrane after administration to said damaged tissue.

25. The method of claim 23 , wherein said disease or condition is selected from the group consisting of stroke, limb ischemia, tissue damage due to trauma, myocardial infarction, peripheral vascular disease, and diabetic ulcers.

26. The method of claim 25 , wherein said disease or condition is myocardial infarction.

27. The method of claim 25 , wherein said disease is peripheral vascular disease.

28. The method of claim 23 , wherein said subject is treated for damage to cardiac tissue.

29. The method of claim 23 , wherein said administration comprises injecting or implanting said isolated mSDF-1 or X p -mSDF-1 peptide into cardiac tissue of said subject.

30. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 1 or 10 , wherein said mSDF-1 is extended at the C-terminus by 10 or more amino acids of the remaining sequence of SEQ ID NO: 53.

31. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 30 , wherein said mSDF-1 is extended at the C-terminus by 30 or more amino acids of the remaining sequence of SEQ ID NO: 53.

32. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 31 , wherein said mSDF-1 is extended at the C-terminus by 50 or more amino acids of the remaining sequence of SEQ ID NO: 53.

33. The isolated mSDF-1 or X p -mSDF-1 peptide of claim 32 , wherein said mSDF-1 is a peptide comprising the amino acid sequence of SEQ ID NO: 53.