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

Abstract

The present invention is directed stromal cell derived factor-1 peptides that have been mutated to make them resistant to digestion by the proteases dipeptidyl peptidase IV (DPPIV) and matrix metalloproteinase-2 (MMP-2) but which maintain the ability of native SDF-I to attract T cells. The mutants may be attached to membranes formed by self-assembling peptides and then implanted at sites of tissue damage to help promote repair.

What is claimed is: 
     
         1 - 20 . (canceled) 
     
     
         21 . A fusion protein comprising the formula: A—(L) n —(R) q , wherein:
 A is an isolated mutant form of stromal cell derived factor-1 (SDF-1) peptide comprising the formula mSDF-1 or X p -mSDF-1, wherein mSDF-1 comprises the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:52 and which is optionally extended at the C terminus of amino acids 1-8 of SEQ ID NO: 52 by all or any portion of the remaining sequence of SEQ ID NO:52, shown as amino acids 9-68 of the full-length of SEQ ID NO: 52 and the amino acids 1-8 comprises a mutation at the fourth and/or fifth amino acids from the N terminus of the amino acids 1-8 of SEQ ID NO:52, and wherein: 
 a) X is a proteinogenic amino acid or a protease protective organic group; 
 b) p is an integer from 1 to 4; and 
 c) the mutant form of SDF-1 peptide has chemoattractant activity for T cells, is inactivated by dipeptidyl peptidase IV (DPPIV) at a rate that is less than one-half of the rate at which native SDF-1 is inactivated, and is inactivated by MMP-2 at a rate that is less than one-half of the rate at which native SDF-1 is inactivated; 
 L is a linker sequence of 3-9 amino acids; 
 R is a self-assembling peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-51; 
 n is an integer from 0-3; and 
 q is an integer from 1-3. 
 
     
     
         22 . The fusion protein of  claim 21 , wherein A comprises:
 i) at least amino acids 1-8 of SEQ ID NO:53 and which is optionally extended at the C terminus of amino acids 1-8 of SEQ ID NO: 53 by all or any portion of the remaining sequence of SEQ ID NO:53, shown as amino acids 9-68 of the full-length of SEQ ID NO: 53;   ii) at least amino acids 1-8 of SEQ ID NO:54 and which is optionally extended at the C terminus of amino acids 1-8 of SEQ ID NO: 54 by all or any portion of the remaining sequence of SEQ ID NO:54, shown as amino acids 9-68 of the full-length of SEQ ID NO: 54;   iii) at least amino acids 1-8 of SEQ ID NO:55 and which is optionally extended at the C terminus of amino acids 1-8 of SEQ ID NO: 55 by all or any portion of the remaining sequence of SEQ ID NO:55, shown as amino acids 9-68 of the full-length of SEQ ID NO: 55; or   iv) at least amino acids 1-8 of SEQ ID NO:56 and which is optionally extended at the C terminus of amino acids 1-8 of SEQ ID NO: 56 by all or any portion of the remaining sequence of SEQ ID NO:56, shown as amino acids 9-68 of the full-length of SEQ ID NO: 56.   
     
     
         23 . The fusion protein of  claim 21 , wherein A comprises:
 i) at least amino acids 1-17 of SEQ ID NO:53 and which is optionally extended at the C terminus of amino acids 1-17 of SEQ ID NO: 53 by all or any portion of the remaining sequence of SEQ ID NO:53, shown as amino acids 18-68 of the full-length of SEQ ID NO: 53;   ii) at least amino acids 1-17 of SEQ ID NO:54 and which is optionally extended at the C terminus of amino acids 1-17 of SEQ ID NO: 54 by all or any portion of the remaining sequence of SEQ ID NO:54, shown as amino acids 18-68 of the full-length of SEQ ID NO: 54;   iii) at least amino acids 1-17 of SEQ ID NO:55 and which is optionally extended at the C terminus of amino acids 1-17 of SEQ ID NO: 55 by all or any portion of the remaining sequence of SEQ ID NO:55, shown as amino acids 18-68 of the full-length of SEQ ID NO: 55; or   iv) at least amino acids 1-17 of SEQ ID NO:56 and which is optionally extended at the C terminus of amino acids 1-17 of SEQ ID NO: 56 by all or any portion of the remaining sequence of SEQ ID NO:56, shown as amino acids 18-68 of the full-length of SEQ ID NO: 56.   
     
     
         24 . The fusion protein of  claim 21 , wherein A comprises the sequence of any one of SEQ ID NOs: 53-56. 
     
     
         25 . The fusion protein of  claim 21 , wherein X is serine. 
     
     
         26 . The fusion protein of  claim 25 , wherein p is 1. 
     
     
         27 . The fusion protein of  claim 21 , wherein R comprises the amino acid sequence of SEQ ID NO: 35. 
     
     
         28 . The fusion protein of  claim 27 , wherein q is 1. 
     
     
         29 , The fusion protein of  claim 21 , wherein L comprises the amino acid sequence of any one of SEQ ID NOs: 57-59. 
     
     
         30 . The fusion protein of  claim 29 , wherein n is 1. 
     
     
         31 . A method of treating a patient to promote the repair of damaged tissue, the method comprising administering to the patient the fusion protein of  claim 21 . 
     
     
         32 . The method of  claim 31 , wherein the patient is treated for a disease or condition selected from stroke, limb ischemia, tissue damage due to trauma, and diabetic ulcer. 
     
     
         33 . A biologically-compatible membrane comprising the fusion protein of  claim 21 . 
     
     
         34 . A method of treating a patient to promote the repair of damaged tissue, the method comprising administering to the patient the biologically-compatible membrane of  claim 33 . 
     
     
         35 . The method of  claim 34 , wherein the patient is treated for a disease or condition selected from stroke, limb ischemia, tissue damage due to trauma, and diabetic ulcer. 
     
     
         36 . The method of  claim 34 , wherein the biologically-compatible membrane is injected or implanted at the site of tissue damage. 
     
     
         37 . The method of  claim 34 , wherein the patient is treated for damage to cardiac tissue and the biologically-compatible membrane is injected or implanted into the myocardium of the patient.