WO2012027170A1 - Methods for repairing tissue damage using protease-resistant mutants of stromal cell derived factor-1 - Google Patents

Abstract

The disclosure provides various forms of mutant stromal cell derived factor-1 (SDF-1 ) peptides that have been mutated to increase their resistance to protease digestion, but chemoattractant activity of SDF-1 is retained. Further disclosed are methods of using the mutant SDF-1 peptides for treating or ameliorating tissue damage, which includes tissue damage associated with peripheral vascular disease, ulcers in the gastrointestinal tract or elsewhere, wounds resulted from accident, and cardiac tissue damage as a result of myocardial infarction, chronic heart failure, or other cardiovascular event.

Description

METHODS FOR REPAIRING TISSUE DAMAGE USING PROTEASE- RESISTANT MUTANTS OF STROMAL CELL DERIVED FACTOR-1 Cross-Reference to Related Applications

This application claims the benefit of the filing date of U.S. provisional application number 61/376,112, filed on August 23, 2010, which is herein

incorporated by reference.

Background of the Invention

In general, the invention relates to methods of repairing tissue damage using pro tease-resistant mutants of stromal cell derived factor- 1 (SDF-1).

SDF-1 (also known as CXCL12) is a 68 amino acid member of the chemokine family that is a chemoattractant for resting T-lymphocytes, monocytes, and CD34⁺ stem cells. SDF-1 is produced in multiple forms: SDF- la (CXCL12a), SDF-Ιβ (CXCL12b), and SDF-Ιγ, which are the result of differential mRNA splicing. The sequences of SDF- l and SDF-Ιβ are essentially the same, except that SDF-Ιβ is extended by four amino acids (Arg-Phe-Lys-Met) at the C-terminus. The first three exons of SDF-Ιγ are identical to those of SDF- la and SDF-Ιβ. The fourth exon of SDF-Ιγ is located 3200 base-pairs downstream from the third exon on the SDF-1 locus and lies between the third exon and the fourth exon of SDF- 1 β. SDF- 1 is initially made with a signal peptide (21 amino acids in length) that is cleaved to make the active peptide.

SDF-1 plays a key role in the homing of hematopoietic stem cells to bone marrow during embryonic development and after stem cell transplantation. In addition to its role in stem cell homing, SDF-1 is also important in cardiogenesis and

vasculo genesis. SDF-1 -deficient mice die perinatally and have defects in cardiac ventricular septal formation, bone marrow hematopoiesis, and organ- specific

vasculo genesis. It has also been reported that abnormally low levels of SDF-1 are at least partially responsible for impaired wound healing associated with diabetic patients and that impairment can be reversed by the administration of SDF-1 at the site of tissue damage.

In the normal adult heart, SDF-1 is expressed constitutively, but expression is upregulated within days after myocardial infarction. It has been shown previously that SDF-1 expression increased eight weeks after myocardial infarction by intramyocardial transplantation of stably transfected cardiac fibroblasts overexpressing SDF-1, in combination with G-CSF therapy. This procedure was associated with higher numbers of bone marrow stem cells (c-Kit or CD34⁺) and endothelial cells in the heart and resulted in an increase of vascular density and an improvement of left ventricular function. These studies suggest that the insufficiency of the naturally-occurring myocardial repair process may be, in part, due to inadequate SDF-1 availability. Hence, the delivery of SDF-1 in a controlled manner after myocardial infarction may attract more progenitor cells and thereby promote tissue repair.

There exists a need in the art for improved methods of promoting wound healing and tis sue repair.

Summary of the Invention

The present invention is directed to methods of treating tissue damage by intra- arterially administering compositions that include stromal cell derived factor- 1 (SDF-1) peptides that have been mutated in a manner that preserves their ability to function as chemoattractants, but renders them resistant to inactivation by proteases, particularly matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP- 9), dipeptidyl peptidase IV (DPPIV/CD26), leukocyte elastase, cathepsin G, carboxypeptidase M, and carboxypeptidase N. The methods of the present invention may be useful in the treatment of, for example, peripheral vascular disease (PVD; also known as peripheral artery disease (PAD) or peripheral artery occlusive disease (PAOD)); ulcers in the gastrointestinal tract or elsewhere; wounds resulting from accident, surgery, or disease; tissue damage; or cardiac tissue damaged as a result of myocardial infarction, chronic heart failure, or other cardiovascular event. The methods of the present invention may also be useful in treating or reducing the likelihood of tissue damage caused by wounds, ulcers, or lesions in diabetic patients.

Accordingly, in one aspect, the invention features a method of treating or ameliorating tissue damage (e.g., tissue damage resulting from a disease or condition) in a subject in need thereof by intra- arterially administering a composition that includes an isolated mutant form of SDF-1 peptide with the formula of: a mutant

SDF-1 (mSDF-1), mSDF-l-Y_z, X_p-mSDF-l, or X_p-mSDF-l-Y_z. SDF-1 is a peptide with the amino acid sequence of at least amino acids 1-8 of SEQ ID NO: l and which may be optionally extended at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO:l, and SEQ ID NO:l includes the amino acid sequence:

KPX₃X₄X₅X₆YRCPCRFFESHVARANVKHLKILNTPN CALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN

K (SEQ ID NO: 1), wherein X₃, X₄, X5, and X₆ are any amino acid, and 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) Y_z is a proteinogenic amino acid(s) or protease protective organic group and z is any integer from 1 to 4;

c) mSDF-1 or mSDF-l-Y_z 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;

d) X_p-mSDF-1 or X_p-mSDF-l-Y_z 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;

wherein isolated mutant form of SDF-1 is administered intra- arterially in an amount sufficient to treat or ameliorate tissue damage in a subject.

In one particular embodiment, the isolated mutant form of SDF-1 peptide does not comprise the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:2,

In one embodiment, X₃ is valine, histidine, or cysteine. In another

embodiment, X₄ is serine or valine. In another embodiment, X₅ is leucine, proline, threonine, or valine. In another embodiment, X₆ is serine, cysteine, or glycine.

In certain embodiments of the methods of the present invention, the peptide is an X_p-mSDF-1 peptide or X_p-mSDF-l-Y_z peptide, wherein X is a serine and p is 1. In other embodiments, the peptide is an mSDF-l-Y_z peptide or X_p-mSDF-l-Y_z peptide, wherein Y is a serine and z is 1.

In certain embodiments, C-terminal modifications may be made to any of the

SDF-1 peptides described herein including, e.g., wild-type SDF-1.

The methods of the present invention may also feature an isolated mutant form of SDF-1, wherein SDF-1 is a fusion protein with the formula A-(L)_n-Fc, wherein: A is the isolated mutant form of SDF-1; n is an integer from 0-3 (e.g., 1); L is a linker sequence of 3-9 amino acids; and Fc is an Fc peptide from an Fc region of an immunoglobulin. In certain embodiments, L is GGGGS (SEQ ID NO:3).

In any embodiment of the present invention, the disease or condition being treated may be stroke, limb ischemia, tissue damage due to trauma, myocardial infarction, peripheral vascular disease, chronic heart failure or diabetes.

In any embodiment of the present invention, the damaged tissue is a cardiac tissue or a vascular tissue.

In any embodiment of the present invention, the composition is administered to a coronary artery (e.g., the aorta, the right coronary artery, the left coronary artery, the pulmonary artery, the circumflex artery, or the left anterior descending artery). In certain embodiments, the composition is administered to an artery of the leg (e.g., the iliac artery, the femoral artery, the popliteal artery, or the anterior and/or posterior tibial artery).

The SDF-1 or mutant SDF-1 protein composition may be administered one or more times to ameliorate one or more symptoms of the disease or condition. The SDF-1 or mutant SDF-1 composition may be administered one or more times until the tissue damage is reduced or the tissue is repaired.

In various embodiments, the disease or condition is tissue damage due to trauma, myocardial infarction, or peripheral vascular disease. In additional embodiments, the disease or condition is a cardiovascular disease or chronic heart failure.

By "an amount sufficient" is meant the amount of a therapeutic agent (e.g., an mSDF-1 peptide), alone or in combination with another therapeutic regimen, required to treat or ameliorate a disorder or condition in a clinically relevant manner. A sufficient amount of a therapeutic agent used to practice the present invention for therapeutic treatment of, e.g., tissue damage varies depending upon the manner of administration, age, and general health of the subject. Ultimately, the medical practitioner prescribing such treatment will decide the appropriate amount and dosage regimen.

By "fragment" is meant a portion of a nucleic acid or polypeptide that contains at least, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of the nucleic acid or polypeptide. A nucleic acid fragment may contain, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 or more nucleotides, up to the full length of the nucleic acid. A polypeptide fragment may contain, e.g., 10, 20, 30, 40, 50, or 60 or more amino acids, up to the full length of the polypeptide. Fragments can be modified as described herein and as known in the art.

By "intra- arterial administration" is meant the administration of a substance into an artery (e.g., a coronary artery (e.g., intra-coronary administration)). Intraarterial administration may include intra- arterial injection or infusion, or

administration via an intra-arterial catheter.

By "pharmaceutically acceptable carrier" is meant a carrier that is

physiologically acceptable to the treated subject while retaining the therapeutic properties of the composition with which it is administered. One exemplary pharmaceutically acceptable carrier substance is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20^th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA).

By "promoting wound healing" or "promoting tissue repair" is meant augmenting, improving, or increasing healing and repair of damaged tissue or inducing closure, healing, or repair of a wound. The wound or tissue damage may be the result of any disorder or condition (e.g., disease, injury, or surgery) and may be found in any location in the subject (e.g., an internal or external wound). For example, the wound or tissue damage may be the result of a cardiovascular condition such as, e.g., myocardial infarction, and the damaged tissue may be cardiac tissue. Alternatively, the wound or tissue damage may be the result of peripheral vascular disease or diabetes.

By "protein," "polypeptide," "polypeptide fragment," or "peptide" is meant any chain of two or more amino acid residues, regardless of posttranslational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide or peptide or constituting a non-naturally occurring polypeptide or peptide. A polypeptide or peptide may be said to be "isolated" or "substantially pure" when physical, mechanical, or chemical methods have been employed to remove the polypeptide from cellular constituents. An "isolated peptide," "substantially pure polypeptide," or "substantially pure and isolated polypeptide" is typically considered removed from cellular constituents and substantially pure when it is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. The polypeptide may be at least 75%, 80%, 85%, 90%, 95%, or 99% by weight pure. A substantially pure polypeptide may be obtained by standard techniques, for example, by extraction from a natural source (e.g., cell lines or biological fluids), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically

synthesizing the polypeptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or high pressure liquid chromatography (HPLC) analysis. Alternatively, a polypeptide is considered isolated if it has been altered by human intervention, placed in a location that is not its natural site, or if it is introduced into one or more cells.

The peptides and polypeptides of the invention, as defined above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the peptides or polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogs of amino acids or may be a chimeric molecule of natural amino acids and non-natural analogs of amino acids. The mimetic can also incorporate any amount of conservative substitutions, as long as such substitutions do not substantially alter the mimetic' s structure or activity.

By "preventing" or "reducing the likelihood of is meant reducing the severity, the frequency, and/or the duration of a disease or disorder (e.g., myocardial infarction or peripheral vascular disease) or the symptoms thereof. For example, reducing the likelihood of or preventing tissue damage is synonymous with prophylaxis or the chronic treatment of tissue damage.

By "protease protective organic group" is meant an organic group, other than a proteinogenic amino acid, that, when added to the N-terminal amino acid of SDF- 1 or a mutated form of SDF-1 (mSDF-1), results in a modified peptide that maintains at least, for example, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of the chemoattractant activity of unmodified SDF-1 (as determined by, e.g., assays of

Jurkat T cell migration or other assays known in the art to measure chemotaxis) and that is inactivated by an enzyme (e.g., DPPIV) at a rate of less than, for example, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1% of the rate of inactivation of unmodified SDF- 1.

By "protease resistant" is meant a peptide or polypeptide that contains one or more modifications in its primary sequence of amino acids compared to a native or wild-type peptide or polypeptide (e.g., native or wild-type SDF-1) and exhibits increased resistance to proteolysis compared to the native or wild-type peptide or polypeptide without the one or more amino acid modifications. By "increased protease resistance" is meant an increase as assessed by in vitro or in vivo assays, as compared to the peptide or polypeptide absent the amino acid sequence changes. Increased resistance to proteases can be assessed by testing for activity or expression following exposure to particular proteases (e.g., MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, carboxypeptidase M, or carboxypeptidase N) using assays such as, for example, Jurkat T-lymphocyte migration assays, CXCR-4-cAMP receptor activation assays, and CXCR4- or CXCR7-P-arrestin assays. Typically, the increase in protease resistance is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more compared to the same peptide or polypeptide, absent the changes in amino acid sequence that confer the resistance.

By "proteinogenic" is meant that the amino acids of a polypeptide or peptide are the L-isomers of: alanine (A); arginine (R); asparagine (N); aspartic acid (D); cysteine (C); glutamic acid (E); glutamine (Q); glycine (G); histidine (H); isoleucine (I); leucine (L); lysine (K); methionine (M); phenylalanine (F); proline (P); serine (S); threonine (T); tryptophan (W); tyrosine (Y); or valine (V).

By "SDF" or "SDF-1" is meant a stromal cell derived factor peptide which can include the sequence of SEQ ID NO:2 or any of the multiple forms of SDF (e.g., SDF-la (CXCL12a), SDF-Ιβ (CXCL12b), and SDF-γ, produced by alternate splicing of the same gene). SDF-Ιβ includes an additional four amino acid residues at the C- terminus of SDF-l , Arg-Phe-Lys-Met. The first three exons of SDF-Ιγ are identical to those of SDF-la and SDF-Ιβ. The fourth exon of SDF-Ιγ is located 3200 base-pairs downstream from the third exon on the SDF-1 locus and lies between the third exon and the fourth exon of SDF-1 β. Although SEQ ID NO:2 shows the sequence of SDF-la, this sequence may be extended at the C-terminus to include additional amino acid residues. The invention includes mutations of SDF-1 a, SDF-1 β, and SDF-γ. For the purposes of the present invention, the term "SDF" or "SDF-1" refers to the active form of the peptide, i.e., after cleavage of the signal peptide.

By "mSDF-1," "mSDF," or "SDF(NqN')" (where N is the one letter designation of the amino acid originally present, q is its position from the N-terminus of the peptide, and N' is the amino acid that has replaced N) is meant a mutant SDF-1 peptide. Peptides that have been mutated by the addition of amino acids (e.g., one or more amino acids) at the N-terminus are abbreviated "X_p-R," where X is a proteinogenic amino acid or protease protective organic group, p is an integer, and R is the peptide prior to extension (e.g., SDF-1 or mSDF-1). For example, "S-SDF-1" or "S-mSDF-1" is an SDF-1 or mSDF-1 molecule, respectively, with a serine residue added at the N- terminus. Peptides that have been mutated by the addition of amino acids (e.g., one or more amino acids) at the C-terminus are abbreviated "R-Y_z," where Y is a proteinogenic amino acid or protease protective organic group, z is an integer, and R is the peptide prior to extension (e.g., SDF-1, mSDF-1, or X_p-mSDF-l). Unless otherwise indicated, all pharmaceutically acceptable forms of peptides may be used, including all pharmaceutically acceptable salts.

By "SDF-1 or mutant SDF-1 peptide of the invention" is meant any wild- type SDF-1 or mutant SDF-1 peptides described herein. Also included in the term are compositions (e.g., pharmaceutical compositions) that include the wild-type SDF-1 or mutant SDF-1 peptides described herein.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By "sustained release" or "controlled release" is meant that the therapeutically active component is released from the formulation at a controlled rate such that therapeutically beneficial levels (but below toxic levels) of the component are maintained over an extended period of time ranging from, e.g., about 12 hours to about 4 weeks (e.g., 12 hours, 24 hours, 48 hours, 1 week, 2 weeks, 3 weeks, or 4 weeks), thus providing, for example, a 12-hour to a 4-week dosage form.

By "treating" or "ameliorating" is meant administering a pharmaceutical composition for therapeutic purposes or administering treatment to a subject already suffering from a disorder to improve the subject's condition. By "treating a disorder" or "ameliorating a disorder" is meant that the disorder and the symptoms associated with the disorder are, e.g., alleviated, reduced, cured, or placed in a state of remission. As compared with an equivalent untreated control, such amelioration or degree of treatment is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as measured by any standard technique.

Other features and advantages of the invention will be apparent from the detailed description and from the claims.

Brief Description of the Drawings

Fig. 1 is a bar graph showing that intra-coronary delivery of SSDF-1(S4V) improves ejection fraction in an ischemia reperfusion model.

Fig. 2 is a bar graph showing that intra-coronary delivery of SSDF-1(S4V) improves end systolic elastance in an ischemia reperfusion model.

Detailed Description

The present invention is based upon the discovery that the recovery of damaged tissue, e.g., damaged cardiac tissue, is promoted by intra-arterial

administration of wild- type SDF-1 or SDF-1 that has been mutated to increase resistance to enzymatic cleavage (e.g., cleavage by one or more of MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, carboxypeptidase M, or carboxypeptidase N). Such peptides may be administered as isolated peptides, with or without a pharmaceutically acceptable carrier.

Intra-Arterial Administration

mSDF-1 peptide-containing compositions used in the methods of the present invention are administered intra-arterially, for example, by intra-arterial injection or using an implantable device (e.g., an intra-arterial stent or catheter).

Intra-arterial administration involves delivery of an mSDF-1 peptide- containing composition into at least one artery. The artery may be, for example, a coronary artery (e.g., the aorta, the right and left coronary arteries, the pulmonary artery, the circumflex artery, and the left anterior descending artery), a carotid artery, a anterior cerebral artery, anterior communicating artery, anterior tibial artery, axillary artery, basilar artery, brachial artery, brachiocephalic artery, Circle of Willis, common carotid artery, common hepatic artery, common iliac artery, contralateral iliac artery, deep brachial artery, deep femoral artery, external carotid artery, external iliac artery, facial artery, femoral artery, fibular artery, gastoduodenal artery, gonadal artery, hepatic artery proper, inferior mesenteric artery, internal carotid artery, internal iliac artery, internal thoracic artery, left gastric artery, left subclavian artery, middle cerebral artery, popliteal artery, posterior cerebral artery, posterior communicating artery, posterior tibial artery, radial artery, renal artery, right gastric artery, right subclavian artery, splenic artery, superficial palmar artery, superficial temporal artery, superior mesenteric artery, suprarenal artery, ulnar artery, or vertebral artery. In a preferred embodiment, the artery is a coronary artery.

An mSDF-1 peptide-containing composition may be administered into one artery or several arteries (e.g., the left and right coronary arteries). The mSDF-1 peptide-containing composition can be intra-arterially administered over a period of about 1 minute, 1 to 5 minutes, 10 to 20 minutes, or 20 to 30 minutes into, for example, one or more arteries. The administration can be repeated intermittently to achieve or sustain the predicted benefit. The timing for repeat administration is based on the subject's response, for example, by monitoring symptoms associated with tissue damage. A therapeutically effective dose or amount of an mSDF-1 peptide- containing composition that is to be given can be divided into two or more doses, and a dose may be administered into two or more arteries with a single puncture or multiple punctures.

Intra-arterial delivery has many clinical advantages compared to other routes of administration. For example, specialized formulations and instrumentation (e.g., catheters) are not required. In addition, intra-arterial delivery is more rapid than other forms of administration (e.g., ~2 minutes for intra-coronary delivery versus ~1 hour for multiple intramyocardial injections), and morbidity generally associated with other routes of administration (e.g., intra-myocardial administration) is eliminated. Intraarterial administration is also advantageous because the active component of the composition to be administered (e.g., an mSDF-1 peptide) can be uniformly distributed in the composition.

SDF-1 and Protease-Resistant Mutants

SDF- 1 is a small cytokine belonging to the chemokine family that is officially designated chemokine (C-X-C motif) ligand 12 (CXCL12). SDF-1 is produced in multiple forms, SDF-Ια (CXCL12a), SDF-Ιβ (CXCL12b), and SDF-Ιγ, by alternate splicing of the same gene.

Unmutated SDF-1 a has the following sequence:

KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO:2)

The SDF-1 peptides described herein include SDF-1 peptides with mutations to render the peptides resistant to, for example, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), dipeptidyl peptidase Γ (DPPrV), leukocyte elastase, cathepsin G, carboxypeptidase M, or carboxypeptidase N. In the methods of the present invention, unmutated SDF-1 may also be administered by intra-arterial delivery for treatment or amelioration of tissue damage.

The methods of the invention feature mutant forms of SDF-1 (mSDF-1), which are characterized by a change in the third, fourth, fifth, and/or sixth amino acid residue from the N-terminus of unmutated SDF-1. mSDF-1 peptides of the invention have at least amino acids 1-8 of SEQ ID NO:l and may be extended at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO:l, which has the following sequence:

KPX₃X₄X₅X₆YRCPCRFFESHVARANVKHLKILNTPN CALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN

K (SEQ ID NO:l), wherein X₃, X₄, X5, and X₆ are any amino acid residue.

In certain embodiments, X₃ is valine, histidine, or cysteine.

In certain embodiments, X₄ is serine or valine.

In certain embodiments, X₅ is leucine, proline, threonine, or valine.

In certain embodiments, X₆ is serine, cysteine, or glycine.

For example, the mSDF-1 peptide may include a mutation at the fourth (e.g., Ser→Val) and/or fifth (e.g., Leu→Pro) amino acid position.

KPVVLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO:4) KPVSPSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO:5)

KPVVPSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO:6)

In another example, the mSDF-1 peptide may include a Val→His (SEQ ID NO:7) or Val→Cys (SEQ ID NO: 8) mutation at the third amino acid position.

KPHSLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO:7)

KPCSLSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO: 8)

In other embodiments, the mSDF-1 peptide may include a Leu→Thr (SEQ ID NO:9) or Leu→Val (SEQ ID NO: 10) mutation at the fifth amino acid position.

KPVSTSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO:9)

KPVSVSYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO: 10)

In other embodiments, the mSDF-1 peptide may include a Ser→Cys (SEQ ID NO: 11) or Ser→Gly (SEQ ID NO: 12) mutation at the sixth amino acid position.

KPVSLCYRCPCRFFESHVARANVKHLKILNTPNCA LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQ ID NO: 11) KPVSLGYRCPCRFFESHVARANVKHLKILNTPNCA

LQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

(SEQIDNO:12)

The methods of the invention may also include peptides that encompass any combination of the mutations described herein. For example, the mSDF-1 peptides may include a Val→Cys mutation at the third amino acid position of SEQ ID NO: 1 and a Ser→Cys mutation at the sixth amino acid position of SEQ ID NO:l.

Mutations made to the SDF-1 peptides to confer protease resistance may also include, for example, the addition of a moiety (e.g., a proteinogenic amino acid or protease protective organic group) to the N-terminus of, e.g., the mSDF-1 peptides (described above), yielding X_p-mSDF-l. For example, X may be: R¹-(CH₂)d-, where d is an integer from 0-3, and R¹ is selected from: hydrogen (with the caveat that when R¹ is hydrogen, d must be at least 1); a branched or straight Q-C3 alkyl; a straight or branched C₂-C₃ alkenyl; a halogen, CF₃; -CONR⁵R⁴; -COOR⁵; -COR⁵;

-(CH₂)_qNR⁵R⁴; -(CH₂)_qSOR⁵; -(CH₂)_qS0₂R⁵, -(CH₂)_qS0₂NR⁵R⁴; and OR⁵, where R⁴ and R⁵ are each independently hydrogen or a straight or branched CrC₃ alkyl. In instances where an organic group is used for X, p should be 1. X may also represent a proteinogenic amino acid, wherein, for example, 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1- 4, 1-3, 1-2, or 1) amino acid(s) is/are added to the N-terminus of SDF-1 (e.g., mSDF- 1), and one or more of these added amino acids may be substituted with a protease protective organic group. For example, a proteinogenic amino acid (e.g., serine) or protease protective organic group may be added to the N-terminus of SDF-1 (e.g., mSDF-1) to confer, for example, resistance to DPPrV cleavage without substantially changing the chemoattractant activity or resistance to other proteases (e.g., MMP-2).

Mutations made to the SDF-1 peptides to confer protease resistance may also include, for example, the addition of a moiety (e.g., a proteinogenic amino acid) to the C-terminus of, e.g., the mSDF-1 peptides (described above), yielding mSDF-l-Y_z or X_p-mSDF-l-Y_z. Y may represent a proteinogenic amino acid, wherein, for example, 1-10 (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1) amino acid(s) is/are added to the C-terminus of SDF-1 (e.g., mSDF-1 or X_p-mSDF-l). For example, a proteinogenic amino acid (e.g., serine) may be added to the C-terminus of SDF-1, mSDF-1, or X_p-mSDF-l to confer, for example, resistance to carboxypeptidase M or carboxypeptidase N cleavage without substantially changing the chemoattractant activity or resistance to other proteases (e.g., MMP-2). In one embodiment, the invention features an isolated mSDF-l-Y_z or X_p-mSDF-l-Y_z peptide, wherein SDF-1 includes the amino acid sequence of SEQ ID NO: l. However, C-terminal modifications may be made to SDF- 1 and any of the SDF- 1 peptides described herein. The mutated SDF-1 peptides described herein retain their ability to act as chemoattractants, but are resistant to enzymatic (e.g., proteolytic) digestion. The mSDF- 1 peptides maintain chemoattractant activity with a sensitivity (as determined by, e.g., the effective concentration needed to obtain 50% of maximal response in the assays of, e.g., Jurkat T cell migration or any other chemotaxis assay known in the art) of at least, for example, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of the sensitivity of unmutated SDF-1. Loss of chemoattractant activity may be due to cleavage by, for example, MMP-2, MMP-9, leukocyte elastase, DPPIV, cathepsin G, carboxypeptidase M, or carboxypeptidase N. The rate of inactivation of mSDF-1 may be less than, for example, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1% of the rate of inactivation of SDF- 1.

The mutated SDF-1 peptides may be resistant to cleavage by, for example, MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, carboxypeptidase M, or carboxypeptidase N. Thus, they are ideally suited for use at sites such as, e.g., damaged tissue (e.g., damaged cardiac tissue), where proteolytic enzymes are present at high concentrations, or delivery to the site via the blood or plasma. Accordingly, mutated SDF-1 peptides are suitable for intra- arterial (e.g., intra-coronary) administration due to the improved stability of such peptides.

Protease-resistant SDF- 1 peptides described herein may include amino acids or sequences modified either by natural processes, such as posttranslational processing, or by chemical modification using techniques known in the art.

Modifications may occur anywhere in a polypeptide, including the polypeptide backbone, the amino acid side-chains, and the amino- or carboxy- terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. Modifications include, e.g., PEGylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., a fluorescent or radioactive marker), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross -linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins (e.g., arginylation), and ubiquitination. Posttranslational modifications also include the addition of polymers to stabilize the peptide or to improve

pharmacokinetics or pharmacodynamics. Exemplary polymers include, e.g., poly(2- hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), polyglutamic acid (PGA), and polyorthoesters.

Fusion Proteins

The methods of the invention may also utilize fusion proteins in which any of the SDF-1, mSDF- 1 , X_p-mSDF- 1 , mSDF- 1 - Y_z, or X_p-mSDF- 1 - Y_z peptide sequences described herein are linked to the Fc region of IgG (e.g., human IgGl). Alternatively, the Fc region may be derived from IgA, IgM, IgE, or IgD of humans or other animals, including swine, mice, rabbits, hamsters, goats, rats, and guinea pigs. The Fc region of IgG includes the CH2 and CH3 domains of the IgG heavy chain and the hinge region. The hinge serves as a flexible spacer between the two parts of the Fc fusion protein, allowing each part of the molecule to function independently. The Fc region used in the present invention can be prepared in, for example, monomeric and dimeric form.

An exemplary Fc fusion peptide is S-SDF-l(S4V)-Fc with the following amino acid sequence. The GGGGS linker (SEQ ID NO:3) is indicated in bold and the Fc peptide is underlined.

SKPVVLSYRCPCRFFESHVARANVKHLKILNTPNC ALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK GGGGSVDKTHTCPPCPAPELLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGOPREPQVYTLP PSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)

Other non-limiting examples of Fc fusion peptides include, e.g., SDF-1 (S4V)- Fc, SDF-1(L5P)-Fc, SDF-l(S6C)-Fc, SDF-1(V3H)-Fc, SDF-l-Fc, S-SDF-l-Fc, and SDF-l-Fc.

All of the above proteins are included in the terms "SDF-1 and mSDF-1 proteins of the invention" or "peptides of the invention."

Peptide Synthesis

The pro tease-resistant SDF-1 peptides used in the methods of the present invention can be made by solid-phase peptide synthesis using, for example, standard N-tert-butyoxycarbonyl (t-Boc) chemistry and cycles using n-methylpyrolidone chemistry. Exemplary methods for synthesizing peptides are found, for example, in U.S. Patent Nos.4,192,798; 4,507,230; 4,749,742; 4,879,371; 4,965,343; 5,175,254; 5,373,053; 5,763,284; and 5,849,954, hereby incorporated by reference. These peptides may also be made using recombinant DNA techniques.

Once peptides have been synthesized, they can be purified using procedures such as, for example, HPLC on reverse-phase columns. Purity may also be assessed by HPLC, and the presence of a correct composition can be determined by amino acid analysis. A purification procedure suitable for mSDF-1 peptides is described, for example, in U.S. Patent Application Publication No.2008/0095758, hereby incorporated by reference.

Fusion proteins may either be chemically synthesized or made using recombinant DNA techniques. Other non-limiting examples of Fc fusion peptides include, e.g., SDF-1 (S4V)-Fc, SDF-1 (L5P)-Fc, SDF-1 (S6C)-Fc, SDF-1 (V3H)-Fc, SDF-l-Fc, S-SDF-l-Fc, and SDF-l-Fc. Pharmaceutical Compositions and Dosages

Any of the peptides employed in the methods of the present invention may be contained in any appropriate amount in any suitable carrier substance, and the pro tease-resistant peptides or fusion proteins are generally present in an amount of 1- 95% by weight of the total weight of the composition, e.g., 5%, 10%, 20%, or 50%. The protease-resistant SDF-1 peptides or fusion proteins described herein may be incorporated into a pharmaceutical composition containing a carrier such as, e.g., saline, water, Ringer's solution, and other agents or excipients. The composition is designed for intra- arterial injection. Thus, the composition may be in the form of, e.g., suspensions, emulsions, solutions, or injectables. All compositions may be prepared using methods that are standard in the art (see, e.g., Remington's

Pharmaceutical Sciences, 16th ed., A. Oslo, ed., Easton, PA (1980)).

The peptides of the invention can be delivered in a controlled-release or sustained-release system. For example, polymeric materials can be used to achieve controlled or sustained release of the peptides (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; PCT Publication Nos. WO 99/15154 and WO 99/20253, hereby incorporated by reference). Examples of polymers used in sustained-release formulations include, e.g., poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co- glycolides) (PLGA), polyglutamic acid (PGA), and polyorthoesters.

It is expected that the skilled practitioner can adjust dosages of the peptide on a case by case basis using methods well established in clinical medicine. The optimal dosage may be determined by methods known in the art and may be influenced by factors such as the age of the subject being treated, disease state, and other clinically relevant factors. Generally, when administered to a human, the dosage of any of the therapeutic agents (e.g., protease-resistant SDF-1 peptides) described herein will depend on the nature of the agent and can readily be determined by one skilled in the art. Typically, such a dosage is normally about 0.001 μg to 2000 mg per day, desirably about 1 mg to 1000 mg per day, and more desirably about 5 mg to 500 mg per day.

The peptides of the invention may be administered once, twice, three times, four times, or five times each day; once per week, twice per week, three times per week, four times per week, five times per week, or six times per week; once per month, once every two months, once every three months, or once every six months; or once per year. Alternatively, the peptides of the invention may be administered one or two times and repeated administration may not be needed. Administration of the peptides described herein can continue until tissue damage (e.g., tissue damage resulting from myocardial infarction or peripheral vascular disease) has healed or has been ameliorated. The duration of therapy can be, e.g., one week to one month;

alternatively, the peptides of the invention can be administered for a shorter or a longer duration. Continuous daily dosing with the peptides may not be required. A therapeutic regimen may require cycles, during which time a composition is not administered, or therapy may be provided on an as-needed basis.

The SDF-1 or mutant SDF-1 peptides of the invention may be delivered once over the duration of therapy or multiple times over the duration of therapy. Depending on the severity of the tissue damage, the SDF-1 or mutant SDF-1 peptides of the invention may be delivered repeatedly over time to ensure repair or recovery of the damaged tissue.

Appropriate dosages of the peptides used in the methods of the invention depend on several factors, including the administration method, the severity of the disorder, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic information (e.g., the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic) about a particular subject may affect the dosage used.

Diagnosis and Treatment

The methods of the present invention are useful for treating any subject that has been diagnosed with or has suffered from tissue damage (e.g., damage to cardiac tissue due to myocardial infarction or tissue damage resulting from peripheral vascular disease) or wounds (e.g., diabetic wounds). Tissue damage may be the result of, for example, a cardiovascular condition (e.g., myocardial infarction or chronic heart failuire); peripheral vascular disease (PVD); peripheral artery disease (PAD); ulcers (e.g., skin wound ulcers); surgery; or diabetes. The methods of the present invention may be used to promote wound healing or tissue repair. One skilled in the art will understand that subjects of the invention may have been subjected to standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors. Diagnosis of these disorders may be performed using any standard method known in the art.

The methods described herein may also be used to treat any disease or condition characterized by a high concentration of protease (e.g., MMP-2, MMP-9, DPPIV, leukocyte elastase, cathepsin G, carboxypeptidase M, and/or

carboxypeptidase N), where the attraction of stem cells upon the administration of a protease-resistant SDF-1 peptide may induce regeneration or healing. Exemplary disorders to be treated by compositions of the present invention include inflammatory and ischemic diseases (e.g., myocardial infarction, stroke or limb ischemia), wound healing, and diabetic ulcers.

The efficacy of treatment can be monitored using methods known to one of skill in the art including, e.g., assessing symptoms of the disease or disorder, physical examination, histopathological examination, blood chemistry analysis, computed tomography, cytological examination, and magnetic resonance imaging. In certain embodiments, hemodynamic data is collected to determine the efficacy of treatment. Hemodynamic tests may include, e.g., determining an ejection fraction (e.g., fraction of blood pumped out of ventricles with each heart beat), determining end diastolic pressure, and determining end systolic elastance (e.g., volume of blood present in the left ventricle). In one example, hemodynamic tests may be used to monitor cardiac function in a subject that has suffered tissue damage resulting from myocardial infarction or other form of cardiac ischemia.

The methods of the present invention may be used in combination with additional therapies to promote wound healing or tissue repair. Treatment therapies that can be used in combination with the methods of the invention include, but are not limited to, heparin, β-blockers (e.g., atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, or timolol), angiotensin-converting enzyme (ACE) inhibitors (e.g., captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, trandolapril, or benazepril), angiotensin II receptor blockers (e.g., candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, or valsartan), diuretics, aspirin, cholesterol-lowering drugs (e.g., HMG-CoA reductase inhibitors (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin)), cell therapy, anti-platelet drugs (e.g., clopidogrel, prasugrel, ticlopidine, cilostazol, abciximab, eptifibatide, tirofiban, or dipyridamole), anti-hypertensive drugs, anti- arrhythmic drugs (e.g., quinidine, procainamide, disopyramide, lidocaine, mexiletine, tocainide, phenytoin, moricizine, flecainide, sotalol, ibutilide, amiodarone, bretylium, dofetilide, diltiazem, or verapamil), angiogenic drugs, wound dressings, PDGF, and/or negative pressure devices and therapies.

Examples

The present invention is illustrated by the following example, which is in no way intended to be limiting of the invention.

Example 1. Hemodynamic function analysis in an ischemia reperfusion model

In the following experiments, we describe experiments demonstrating that intra- arterial delivery (e.g., intra-coronary delivery) of an mSDF-1 peptide-containing composition improves cardiac function in an ischemia reperfusion model compared to direct injection into tissue (e.g., via intra-myocardial injection).

Rats were anesthetized with 0.05 mg/kg of buprenorphine and 2-3% of isoflurane. After intubation, the chest was opened between ribs 4 and 5, and the left anterior descending (LAD) coronary artery was ligated for 45 minutes. After 45 minutes, the suture was removed from the LAD to initiate reperfusion in the infarct zone. mSDF-1 peptide was administered intra-arterially (>15 rats per group). For intra-coronary injection, 100 μΐ of S-SDF-1 (S4V) (at a concentration of 2 μg or 20 μg S-SDF-1 (S4V) per 100 μΐ) in PBS were injected into the rats in the ventricular cavity of the heart while clamping the aorta. Clamping the aorta forced the injected sample first through the coronary arteries. The chest and skin of the rats were closed after injection of the protein. S-SDF-1 (S4V) was also administered intra-arterially to rats at multiple dosages (i.e., 0.001, 0.01, 0.1, or 1 mg/kg) and compared to a PBS only control. In each of the experiments described above, hemodynamic function in the rats was analyzed in a randomized and blinded study four weeks after the ischemia reperfusion injury. Rats were anesthetized with 0.05 mg/kg of buprenorphine and 2- 3% of isoflurane. A 16G endotracheal tube was inserted into the rats and mechanical ventilation was started. The left jugular vein was cannulated with PE 10 to deliver hyperosmotic saline (50 μΐ of a 25% NaCl solution in water). Hyperosmotic saline was used to measure parallel conductance of the volume measurements.

To determine the ejection fraction and intra-ventricular pressure, the right carotid artery was cannulated. A pressure- volume catheter was inserted and passed into the left ventricle. A baseline pressure- volume measurement was obtained. A hyperosmotic saline solution (described above) was injected into the jugular vein, and a pressure-volume measurement was then obtained.

To determine end systolic elastance, lateral incisions were made below the diaphragm. The diaphragm was cauterized to expose the inferior vena cava and thoracic artery. The inferior vena cava was transiently occluded for 4-5 seconds, and a pressure tracing was obtained.

Our results showed that intra-coronary injection of S-SDF-1(S4V) resulted in an 8% improvement in the measured ejection fraction in rats compared to the PBS control (Fig. 1) and a 60% improvement in end systolic elastance compared to the PBS control (Fig. 2).

Other Embodiments

From the foregoing description, it is apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

All publications, patent applications, and patents, including, for example, U.S. Patent Application Publication No. 2008/0095758 and U.S. Provisional Application No. 61/376,112, filed August 23, 2010, mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

What is claimed is:

Claims

Claims

1. A method of treating or ameliorating tissue damage in a subject in need thereof, said tissue damage resulting from a disease or condition, wherein said method comprises intra- arterially administering a composition comprising an isolated mutant form of stromal cell derived factor- 1 (SDF-1) peptide comprising the formula of a mutant SDF-1 (mSDF-1), mSDF-l-Y_z, X_p-mSDF-1, or X_p-mSDF-l-Y_z, wherein said SDF-1 is a peptide comprising the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:l and which is optionally extended at the C-terminus by all or any portion of the remaining sequence of SEQ ID NO:l, said SEQ ID NO: 1 comprising the amino acid sequence:

KPX₃X₄X₅X₆YRCPCRFFESHVARANVKHLKILNTPNCAL QIVARLKNNNPvQVCIDPKLKWIQEYLEKALNK (SEQ ID NO:l), wherein X₃, X₄, X5, and X₆ 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) Y_z is a proteinogenic amino acid(s) or protease protective organic group and z is any integer from 1 to 4;

c) said mSDF-1 or said mSDF-l-Y_z 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 ;

d) said X_p-mSDF-1 or said X_p-mSDF-l-Y_z 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;

wherein said isolated mutant form of SDF-1 is administered intra-arterially in an amount sufficient to treat or ameliorate said tissue damage in said subject.

2. The method of claim 1, wherein said isolated mutant form of SDF-1 peptide does not comprise the amino acid sequence of at least amino acids 1-8 of SEQ ID NO:2.

3. The method of claim 1, wherein said X₃ is valine, histidine, or cysteine.

4. The method of any one of claims 1-2, wherein said X₄ is serine or valine.

5. The method of any one of claims 1-4, wherein said X₅ is leucine, proline, threonine, or valine.

6. The method of any one of claims 1-5, wherein said X₆ is serine, cysteine, or glycine.

7. The method of any one of claims 1-6, wherein said peptide is an X_p-mSDF-l peptide or X_p-mSDF-l-Y_z peptide and wherein X is a serine and p is 1.

8. The method of any one of claims 1-6, wherein said peptide is an mSDF-l-Y_z peptide or X_p-mSDF-l-Y_z peptide and wherein Y is a serine and z is 1.

9. The method of any one of claims 1-8, wherein said isolated mutant form of SDF-1 is a fusion protein comprising the formula A-(L)_n-Fc, wherein: A is the isolated mutant form of SDF-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.

10. The method of claim 9, wherein n=l and L is GGGGS (SEQ ID NO:3).

11. The method of any one of claims 1-10, 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, chronic heart failure, and diabetes.

12. The method of claim 11, wherein said disease or condition is myocardial infarction.

13. The method of claim 11, wherein said disease or condition is peripheral vascular disease.

14. The method of claim 11, wherein said disease or condition is diabetes.

15. The method of claim 11, wherein said disease or condition is diabetic wound healing.

16. The method of any one of claims 1-15, wherein said composition is administered to a coronary artery.

17. The method of claim 16, wherein said coronary artery is the aorta, the right coronary artery, the left coronary artery, the pulmonary artery, the circumflex artery, or the left anterior descending artery.

18. The method of claim 13, where said composition is administered to an artery of the leg.

19. The method of claim 18, wherein said artery of the leg is the iliac artery, the femoral artery, the popliteal artery, or the anterior and/or posterior tibial artery.