Engineered neuropeptide and extracellular matrix proteins

Abstract

The present disclosure relates to productive tissue repair and regeneration, and in particular fusion proteins and polypeptides, compositions including said fusion proteins and polypeptides, and methods of using said polypeptides or compositions for productive tissue repair and regeneration. In one aspect, there is provided a fusion protein comprising (i) a calcitonin gene-related peptide (CGRP) receptor agonist polypeptide; and (ii) an extracellular matrix (ECM) binding polypeptide. In another aspect, there is provided a fusion protein comprising: (a) a first polypeptide; and (b) a second polypeptide, wherein the first and second polypeptide are joined by a linker that comprises, consists essentially of or consists of the amino acid sequence KGYR (SEQ ID NO: 70).

Description

1005837019 1 Engineered neuropeptide and extracellular matrix proteins Cross-reference

[0001] This application claims priority from Australian provisional patent application no.2024900817, the entire contents of which is incorporated herein by reference. Field of the invention

[0002] The present disclosure relates to productive tissue repair and regeneration, and in particular fusion proteins and polypeptides, compositions including said fusion proteins and polypeptides, and methods of using said polypeptides or compositions for productive tissue repair and regeneration. Background of the invention

[0003] Regenerative medicine involves the repair, regeneration, maintenance, and replacement of tissues and organs using exogenous materials. Scaffolds may be seeded with cells, such as primary cells or stem cells, and various factors to encourage tissue growth. However, a number of challenges remain in the design of appropriate material for regenerative medicine and tissue engineering.

[0004] Millions of chronic wounds develop each year with estimated treatment costs reaching into the billions of dollars. Wounds can be conceptualized as defects in the protective covering of an individual organ or organ system. Without this physiological barrier, the tissue normally protected by the covering is subject to loss of biologic compartmentalization.

[0005] When tissue is no longer physiologically compartmentalized it is subject to fluid loss, invasion by microorganisms, electrolyte imbalances, and in some cases metabolic dysfunction. Fluids lost by non-compartmentalized tissue include but are not limited to: blood, plasma, lymph, enteric contents, bile, cerebral spinal fluid, mucus. These fluid losses lead to desiccation of the underlying tissue and enable invasion by microorganisms, leading to potential infection and, in many cases, progressive tissue loss. For example, the inability to heal a chronic skin wound on the lower extremity may lead to amputation of either a portion or all of the effected limb. There are several aetiologies for such chronic lower extremity skin wounds, including mechanical trauma, burns, radiation, arterial insufficiency, venous stasis, chronic infection, neuropathy, and1005837019 2 systemic diseases such as diabetes. Current methods for improving wound healing emphasize effective drainage, prevention of infection, reduction of inflammation and minimization of tissue and fluid loss.

[0006] Chronic cutaneous wounds pose significant health problems for patients with diverse medical conditions such as diabetes, burns, trauma such as wartime-sustained trauma, spinal cord injury, and vascular insufficiency. Some of these patients are at risk of developing chronic wounds as a consequence of immobility and pressure ulcers as well as chronic non-healing ulcers due to diabetes or peripheral vascular disease. The default response to injury in postnatal human skin is driven by the necessity of rapid wound closure and is destined to result in formation of a scar.

[0007] There is a need for new or improved treatments for tissue injury and / or for promoting tissue regeneration.

[0008] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art. Summary of the invention

[0009] In one aspect, there is provided a fusion protein comprising: (i) a calcitonin gene-related peptide (CGRP) receptor agonist polypeptide; and (ii) an extracellular matrix (ECM) binding polypeptide.

[0010] The CGRP receptor is a complex formed by calcitonin receptor-like receptor (CLR, also known as CALCRL) and Receptor Activity Modifying Protein 1 (Ramp1). In any embodiment, the CGRP receptor agonist polypeptide is, or is derived from, calcitonin gene-related peptide (CGRP), adrenomedullin (AM), adrenomedullin 2 / intermedin (AM2 / IMD), or amylin. Preferably, the CGRP receptor agonist polypeptide is, or is derived from, human CGRP, human AM, human AM2 / IMD), or human amylin, or an orthologue thereof.1005837019 3

[0011] The CGRP receptor agonist polypeptide and the ECM binding polypeptide may be joined directly or via a linker sequence.

[0012] In any aspect or embodiment, the calcitonin gene-related peptide (CGRP) receptor agonist polypeptide may be a CGRP polypeptide as described herein.

[0013] In any aspect or embodiment, the CGRP receptor agonist polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74. Alternatively, the CGRP polypeptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 74.

[0014] In any aspect or embodiment, the CGRP receptor agonist polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 75. Alternatively, the CGRP polypeptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 75 or an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 75.

[0015] In any aspect or embodiment, the CGRP receptor agonist polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1005837019 4 76. Alternatively, the CGRP polypeptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 76 or an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 76.

[0016] In one aspect, there is provided a fusion protein comprising: (i) a calcitonin gene-related peptide (CGRP) polypeptide; and (ii) an extracellular matrix (ECM) binding polypeptide.

[0017] The CGRP polypeptide and the ECM binding polypeptide may be joined directly or via a linker sequence.

[0018] In one embodiment, the CGRP receptor agonist polypeptide or the CGRP polypeptide is C-terminal to the ECM binding polypeptide. Preferably, the N-terminus of the CGRP receptor agonist polypeptide or the CGRP polypeptide is joined directly or via a linker sequence to the C-terminus of the ECM binding polypeptide.

[0019] In any aspect or embodiment, the linker sequence may be cleavable. The linker sequence may be any sequence that once cleaved allows release of the CGRP receptor agonist polypeptide or the CGRP polypeptide from the fusion protein where the released the CGRP receptor agonist polypeptide or CGRP polypeptide has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity of a wildtype CGRP receptor agonist polypeptide or CGRP polypeptide (in this context wildtype CGRP polypeptide is an unfused CGRP polypeptide, for example a polypeptide consisting of SEQ ID NO: 1 or SEQ ID NO: 80; and wildtype CGRP receptor agonist polypeptide, is an unfused CGRP receptor agonist polypeptide, for example a polypeptide consisting of SEQ ID NO: 74, 75, or 76). Activity may be the ability to inhibit neutrophil and macrophage migration and / or induce cAMP in neutrophils and macrophages via CLR / RAMP1 (the CGRP receptor complex).

[0020] Preferably, the linker sequence is cleavable by an enzyme. In this embodiment, the linker sequence may comprise or consist of a proteolytic cleavage site. The cleavage site may be cleavable by serine proteases, for example plasmin. The1005837019 5 linker may comprise, consist essentially of, or consist of an amino acid sequence that is cleavable by plasmin, for example the amino acid sequence of SEQ ID NO: 70.

[0021] In any aspect or embodiment, the CGRP polypeptide may be a human CGRP or an orthologue thereof.

[0022] In any embodiment of any aspect, the CGRP receptor agonist polypeptide comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ IDs NO: 77-79 or 81-82, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of any one of SEQ IDs NO: 77-79 or 81-82.

[0023] CGRP exists in two forms, αCGRP and βCGRP. The CGRP receptor agonist polypeptide or the CGRP polypeptide may be, or be derived from, a αCGRP and βCGRP, preferably αCGRP.

[0024] In any aspect or embodiment, the CGRP polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 80, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 80. Alternatively, the CGRP polypeptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 80, or an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 80.

[0025] In one embodiment, % identity or identical to a sequence means that the polypeptide has the same length, for example number of amino acids, but the amino acids across that length are only 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Typically, the only differences in amino acid identity is a result of conservative substitutions (for example those outlined in Table 2 below).1005837019 6

[0026] In one embodiment, the CGRP receptor agonist polypeptide or the CGRP polypeptide is equal to, or less than, about 55, about 54, about 53, about 52, about 51, about 50, about 49, about 48, about 47, about 46, about 45, about 44, about 43, about 42, about 41, about 40, about 39, about 38, or about 37 amino acids in length.

[0027] In one embodiment, the CGRP receptor agonist polypeptide or CGRP polypeptide is equal to, or less than, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, or 37 amino acids in length.

[0028] In any embodiment, the the CGRP receptor agonist polypeptide or the CGRP polypeptide, ECM binding polypeptide, or fusion protein comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7 or 8 conservative (for example those outlined in Table 2 below) or non-conservative amino acid substitutions, deletions or additions to the above sequences, and retains CGRP and / or ECM binding activity.

[0029] In any aspect or embodiment, the ECM binding polypeptide binds to any one or more of the following ECM molecules: collagen, α2-antiplasmin, vitronectin, fibronectin, tenascin C, osteopontin, fibrinogen, fibrin, heparan sulfate, and heparan sulfate proteoglycans (HSPG).

[0030] In any aspect or embodiment, the ECM binding polypeptide is derived from: placenta growth factor (PlGF), amphiregulin (AREG), neurturin (NRTN), collagenase (col), α2-antiplasmin inhibitor (alpha-2 antiplasmin inhibitor) or von Willebrand factor (vWF). Preferably, the PlGF, AREG, NRTN, col, α2-antiplasmin inhibitor or vWF is human.

[0031] In any aspect or embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of positively charged amino acid residues. Preferably, a contiguous sequence of positively charged amino acid residues. In one embodiment, the positively charged residues comprise, consists essentially of, or consist of RRRPK (SEQ ID NO: 84), RKKK(SEQ ID NO: 85), KRRR(SEQ ID NO: 86), NQEQVSPL (SEQ ID NO: 87) or any others described herein. In another embodiment, the ECM binding moiety comprises at least 2 contiguous sequences of positively charged amino acid residues.

[0032] In any embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID1005837019 7 NOs: 4 to 69. In another embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of any one of SEQ ID NOs: 4 to 69 wherein the ECM binding polypeptide binds to one or more ECM proteins with the same affinity, an affinity not significantly different, or an affinity of at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of an ECM binding polypeptide of any one of SEQ ID NOs: 4 to 69 from which it was derived.

[0033] In any embodiment, the ECM binding polypeptide is derived from PlGF, preferably human (hPIGF), more preferably human PlGF-2 or PlGF4. In one embodiment, human PlGF-2 comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2. In one embodiment, human PlGF-4 comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3.

[0034] In any embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence of any one of SEQ ID NOs: 4 to 44. In another embodiment, the ECM binding polypeptide comprises, consists essentially of or consist of an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of any one of SEQ ID Nos: 4 to 44 wherein the ECM binding polypeptide binds to one or more ECM proteins with the same affinity, an affinity not significantly different, or an affinity of at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of an ECM binding polypeptide of anyone of SEQ ID NOs: 4 to 44 from which it was derived.

[0035] In any embodiment, the ECM binding polypeptide is derived from AREG, preferably human AREG.

[0036] In any embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence of any one of SEQ ID NOs: 45 to 61. In another embodiment, the ECM binding polypeptide comprises, consists1005837019 8 essentially of, or consists of an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of any one of SEQ ID Nos: 45 to 61 wherein the ECM binding polypeptide binds to one or more ECM proteins with the same affinity, an affinity not significantly different, or an affinity of at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, an ECM binding polypeptide of anyone of SEQ ID Nos: 45 to 61 from which it was derived.

[0037] In any embodiment, the ECM binding polypeptide is derived from NRTN, preferably human NRTN (hNRTN).

[0038] In any embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence of any one of SEQ ID NOs: 62 to 66. In another embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of any one of SEQ ID NOs: 62 to 66 wherein the ECM binding polypeptide binds to one or more ECM proteins with the same affinity, an affinity not significantly different, or an affinity of at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of an ECM binding polypeptide of any one of SEQ ID NOs: 62 to 66 from which it was derived.

[0039] In any embodiment, the ECM binding polypeptide is derived from vWF, preferably human vWF(hWMF).

[0040] In any embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NOs: 67 or 68. In another embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of any one1005837019 9 of SEQ ID NOs: 67 or 68 wherein the ECM binding polypeptide binds to one or more ECM proteins with the same affinity, an affinity not significantly different, or an affinity of at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of an ECM binding polypeptide of anyone of SEQ ID NOs: 67 or 68 from which it was derived.

[0041] In any embodiment, the ECM binding polypeptide is derived from collagenase (col), preferably human collagenase.

[0042] In any embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 69. In another embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 69 wherein the ECM binding polypeptide binds to one or more ECM proteins with the same affinity, an affinity not significantly different, or an affinity of at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of an ECM binding polypeptide of SEQ ID NO: 69 from which it was derived.

[0043] In any embodiment, the ECM binding polypeptide is derived from α2- antiplasmin inhibitor (alpha-2 antiplasmin inhibitor), preferably human α2-antiplasmin inhibitor.

[0044] In one embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of the alphas 2 plasmin inhibitor 1-8 (a2PI1-8) sequence (NQEQVSPL; SEQ ID NO: 87), a substrate sequence for a transglutaminase (Factor XIII). This sequence allows the covalent linkage of the fusion to fibrin or other ECM proteins with a lysine (K) acceptor residue.

[0045] In any embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 87. In another embodiment, the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,1005837019 10 about 98%, about 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 89 wherein the ECM binding polypeptide binds to one or more ECM proteins with the same affinity, an affinity not significantly different, or an affinity of at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of an ECM binding polypeptide of SEQ ID NOs: 89 from which it was derived.

[0046] In one embodiment an extracellular matrix (ECM) binding polypeptide known in the art is included. Illustrative ECM binding peptides are described in US publication no.2014 / 0011978, US publication no.20140010832 and US 9,879,062, the contents of which are herein incorporated by reference in their entirety.

[0047] In any aspect or embodiment, the fusion protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 71. Alternatively, the fusion protein comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 71 or an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 71.

[0048] In any aspect or embodiment, the fusion protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 89 or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 89. Alternatively, the fusion protein comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 89 or an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:89.

[0049] In any embodiment of any aspect, the fusion polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 1 and 87, or SEQ ID NOs:1 and 88, or an amino acid sequence that is equal to, or at least, about 70%,1005837019 11 about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 1 and 87, or SEQ ID NOs:1 and 88.

[0050] In any aspect or embodiment, the fusion polypeptide comprises, consists essentially of, or consists of the amino acid sequence that is encoded by a nucleic acid sequence that comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 73, or a nucleic acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 73.

[0051] In any aspect or embodiment, the fusion polypeptide comprises, consists essentially of, or consists of the amino acid sequence that is encoded by a nucleic acid sequence that comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 90, or a nucleic acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 90.

[0052] In any embodiment, the fusion polypeptide is amidated at the C-terminus, preferably a C-terminal phenylalanine.

[0053] In any aspect or embodiment, the fusion polypeptide as described herein promotes greater retention of CGRP after delivery into tissues than the corresponding CGRP polypeptide not linked to an ECM binding polypeptide (for example a polypeptide consisting of SEQ ID NO: 1 or SEQ ID NO: 80). Assays to determine tissue retention may be any one known in the art or described herein, including in the Examples.

[0054] In any aspect or embodiment, the fusion polypeptide as described herein promotes greater wound closure, skin regeneration, and / or muscle regeneration than the corresponding CGRP polypeptide not linked to an ECM binding polypeptide (for example a polypeptide consisting of SEQ ID NO: 1 or SEQ ID NO: 80). Assays to1005837019 12 determine wound closure, skin regeneration and / or muscle regeneration may be any one known in the art or described herein, including in the Examples.

[0055] In any aspect or embodiment, the fusion polypeptide as described herein contributes to peripheral nociceptive sensitisation to a lesser extent than the corresponding CGRP polypeptide not linked to an ECM binding polypeptide (for example a polypeptide consisting of SEQ ID NO: 1 or SEQ ID NO: 80). Assays to determine peripheral nociceptive sensitisation may be any one known in the art or described herein, including in the Examples.

[0056] In any aspect or embodiment, a fusion protein as described herein may exhibit one or more, or all, of the following activities: • promote tissue healing; • promote wound closure; • promote skin regeneration; • promote muscle regeneration; • reduce peripheral nociceptive sensitisation in response to injury and / or inflammation; • reduce fibrosis in response to injury; • reduce neutrophil and monocyte / macrophage migration into injured tissues; • enhance neutrophil and pro-inflammatory macrophage death in the presence of inflammatory cytokines (e.g. IL-1 and TNF-α); • increase neutrophil clearance by stimulating macrophage efferocytosis; • increase macrophage polarisation from M1 to M2; • increase macrophage switching towards an anti-inflammatory / pro-repair phenotype; and / or • reduce release of cytokines IL-1, CCL2, CXCL2, MMP-2, and MMP-9.1005837019 13

[0057] Each one of the functions outlined above is directly supported by experimental data in the Examples and can be determined and / or measured by any assay known in the art or described herein, including in the Examples.

[0058] In another aspect, the present disclosure also provides isolated nucleic acids encoding a fusion protein as described herein, for example, one or more nucleic acids encoding a fusion protein comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO:89, SEQ ID NO:1 and SEQ ID NO: 87, or SEQ ID NO:1 and SEQ ID NO:88. An isolated nucleic acid may be recombinant or synthetic. An isolated nucleic acid may be purified. The nucleic acid may comprise, consist essentially of, or consist of the nucleic acid sequence set forth in SEQ ID NO: 73, SEQ ID NO: 90, or any functional variant thereof.

[0059] The nucleic acid molecule may be an RNA or DNA or RNA : DNA or a chemically modified form thereof. For example, the nucleic acid may be in the form of a viral or non-viral vector.

[0060] In another aspect, the present disclosure provides a vector comprising a nucleic acid encoding a fusion protein as described herein, for example, a fusion protein comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71 or SEQ ID NO:89 (or SEQ ID NO: 1 and 87, or SEQ ID NO: 1 and 88), optionally operably linked to control sequences.

[0061] In another aspect, the present disclosure provides a vector comprising a nucleic acid encoding a TSP-1 agonist as described herein.

[0062] In another aspect, the present disclosure provides a vector comprising a nucleic acid encoding a TSP-1 polypeptide as described herein, for example, a TSP-1 polypeptide may be human TSP-1 (hTSP-1) or a derivative or variant thereof. Human thrombospondin 1 (TSP-1; UniProt reference: P07996) is encoded by the TSHBS1 gene (NCBI Gene reference: 7057). A nucleic acid encoding a TSP-1 polypeptide may encode a TSP-1 polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 72, optionally operably linked to control sequences. The TSP- 1 polypeptide may comprise, consist essentially of, or consist of an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of1005837019 14 SEQ ID NO: 72; or an amino acid sequence that is encoded by a nucleic acid sequence that comprises or consists of SEQ ID NO:83. The nucleic acid encoding a TSP-1 polypeptide as described herein may comprise, consist essentially of, or consist of the nucleic acid sequence set forth in SEQ ID NO: 83, or a nucleic acid that is at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 83.

[0063] In another aspect, the present disclosure provides a vector comprising a nucleic acid encoding a TSP-1 agonist as described herein.

[0064] In another aspect, the present disclosure provides a host cell comprising a vector as described herein, and methods for producing and optionally recovering the fusion protein, TSP-1 agonist, or TSP-1 polypeptide described herein. Methods for producing a fusion protein, TSP-1 agonist polypeptide, or TSP-1 polypeptide described herein may comprise introducing into a host cell a vector or nucleic acid as described herein, under suitable conditions such that the fusion protein, TSP-1 agonist, or TSP-1 polypeptide is expressed.

[0065] In another aspect, the present disclosure provides a cell expressing a fusion protein, TSP-1 agonist, or TSP-1 polypeptide as described herein. The cell may be isolated. The cell may be in vitro, ex vivo, or in vivo. In some embodiments, the cell may be a sensory neuron, keratinocyte, fibroblast, endothelial cell, myoblast, myocyte, a myeloid cell such as a monocyte, macrophage, or neutrophil, or myeloid cell precursor such as myeloblast, myelocyte, multipotent common myeloid progenitor, haematopoietic stem cell. The disclosure further provides a population of cells comprising a plurality of a host cell or cell described herein, or progeny of said cell. In some embodiments, the cell, or a population of cells thereof, may provide a cell therapy wherein the cell or population of cells are administered (eg injected, grafted, implanted) to a tissue requiring injury repair and / or tissue regeneration.

[0066] In another aspect, the present disclosure provides a composition comprising a fusion protein, TSP-1 agonist, TSP-1 polypeptide, nucleic acid, vector, or cell as described herein and a pharmaceutically or physiologically acceptable carrier, diluent or excipient. The composition may be suitable for human or veterinary use. For human use, the composition may also be referred to as a pharmaceutical composition.1005837019 15

[0067] In another aspect, the present disclosure provides a composition comprising a fusion protein, TSP-1 agonist, TSP-1 polypeptide, nucleic acid, vector, or cell as described herein and further comprising a scaffold (semi-solid or solid support) or retentive material. Scaffolds can be derived from synthetic materials, such as poly(lactide-co-glycolide) (PGLA), polyurethane, poly(L-lactide-co-ε-caprolactone) (PLCL), polycaprolactone (PCL), polyethylene glycol (PEG), or acrylamide, or can be derived from biomaterials, including but not limited to: aliginate, chitosan, gelatin, collagen, hyaluronic acid, silk fibroin, or fibrin.

[0068] In one embodiment of this aspect, the scaffold or retentive material is a hydrogel, such as a hyaluronic acid, fibrin or acrylamide hydrogel. In one embodiment of this aspect, the scaffold or retentive material is a hyaluronic acid-based scaffold.

[0069] In another aspect, the present disclosure provides a method for promoting conversion from an inflammatory phase to a pro-repair phase in a subject in need thereof, the method comprising administering a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein. The subject may have an acute injury or a chronic injury. Preferably the method comprises administering the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell directly to the tissue requiring regeneration or wound requiring repair, for example, topically administering the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell to skin requiring regeneration or intramuscularly administering the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell to muscle requiring regeneration.

[0070] In another aspect, the present disclosure provides a fusion protein, TSP-1 agonist, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein for use in promoting conversion from an inflammatory phase to a pro- repair phase in a subject in need thereof.

[0071] In another aspect, the present disclosure provides use of a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein in the manufacture of a medicament for promoting conversion from an inflammatory phase to a pro-repair phase in a subject in need thereof. Preferably, the medicament is adapted or formulated for administration directly to the tissue requiring1005837019 16 regeneration or the wound requiring repair, for example, topically administering the fusion protein to skin requiring repair.

[0072] In another aspect, the present disclosure provides a method for promoting tissue regeneration in a subject in need thereof, the method comprising administering a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein. Preferably the method comprises administering the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell directly to the tissue requiring regeneration, for example, topically administering to skin requiring regeneration or intramuscularly administering to muscle requiring regeneration.

[0073] In another aspect, the present disclosure provides a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein for use in promoting tissue regeneration in a subject in need thereof. Preferably the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell is administered directly to the tissue requiring regeneration, for example, topically administering the fusion protein, TSP-1 polypeptide, composition, nucleic acid, vector, or cell to skin requiring regeneration or intramuscularly administering the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell to muscle requiring regeneration.

[0074] In another aspect, the present disclosure provides use of a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein in the manufacture of a medicament for promoting tissue regeneration in a subject in need thereof. Preferably the medicament is adapted or formulated for administration directly to the tissue requiring regeneration, for example, topical administration to skin requiring regeneration or intramuscular administration to muscle requiring regeneration.

[0075] In another aspect, the present disclosure provides a method for promoting wound repair in a subject in need thereof, the method comprising administering a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein. Preferably the method comprises administering the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell directly to1005837019 17 the wound requiring repair, for example, topically administering the fusion protein to skin requiring repair.

[0076] In another aspect, the present disclosure provides a fusion protein, TSP-1 agonist, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein for use in promoting wound repair in a subject in need thereof. Preferably, the fusion protein, TSP-1 polypeptide, composition, nucleic acid, vector, or cell is administered directly to the wound requiring repair, for example, topically administering the fusion protein to skin requiring repair.

[0077] In another aspect, the present disclosure provides use of a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein in the manufacture of a medicament for promoting wound repair in a subject in need thereof. Preferably, the medicament is adapted or formulated for administration directly to the wound requiring repair, for example, topically administering the fusion protein to skin requiring repair.

[0078] The tissue may be any described herein, in particular skin, muscle (such as cardiac, smooth or skeletal muscle, preferably skeletal muscle) or the corneal epithelium.

[0079] The wound may be any wound described herein, such as an acute injury or a chronic injury. The wound may be a chronic wound such as a diabetic wound.

[0080] Administration of a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein is particularly beneficial at an early-time point post-injury / wound formation, for example 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, 24 hours or less, 12 hours or less, 6 hours or less post-injury / wound formation. Preferably, the injury or wound is in, or predominantly in, an inflammatory phase. Preferably, for acute injury, treatment occurs at 6 days or less, no more than 6-days post-injury.

[0081] The subject in need thereof may have or be at risk of having a disorder, disease, or condition that impairs wound healing and / or tissue regeneration. Exemplary disorders, diseases or conditions include but are not limited to: diabetes, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, ischemia, vasculitis, primary antiphospholipid syndrome, gout, zinc deficiency, alcoholism, anemia, uremia,1005837019 18 malnutrition, and shock. In particular, the subject has an inflammatory or autoimmune disorder, disease, or condition that impairs wound healing and / or tissue regeneration due to a dysregulated inflammatory response.

[0082] A subject in need thereof may be any one described herein, including a subject suffering from a peripheral neuropathy, for example the subject may have diabetes.

[0083] A subject in need thereof may be of older age, wherein the ability to repair injured tissue, such as wounds, naturally decreases as the skin becomes thinner and the number of fibroblasts and amount of total skin collagen decrease. For example, a subject may be at least 40 years in age, at least 45 years in age, at least 50 years in age, at least 55 years in age, at least 60 years in age, at least 65 years of age, at least 70 years of age, at least 75 years of age, at least 80 years of age, or at least 85 years of age, or older.

[0084] A subject in need therefore may be taking a medication or medications that produce, or results in, impaired wound healing and / or tissue regeneration. For example, the subject may be undergoing, or have previously undertaken, chemotherapy or anti- CGRP medications. For example, the subject may be being treated with, or have been treated with an immunosuppressant drug such as steroids.

[0085] As described herein, in one embodiment, tissue regeneration stimulated by the method is associated with minimal fibrosis. Thus, in another aspect, the present disclosure provides fusion protein, TSP-1 agonist, TSP-1 polypeptide, nucleic acid, vector, or cell as described herein for use in reducing fibrosis development in a subject or biological tissue undergoing regenerative treatment. The regenerative treatment may be a method comprising administering a fusion protein, nucleic acid, vector or cell described herein.

[0086] In another aspect, the present disclosure provides a method reducing fibrosis in a subject undergoing regenerative treatment, the method comprising administering a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein. Preferably the method comprises administering the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell1005837019 19 directly to the tissue undergoing regenerative treatment, for example, administering the fusion protein to skin or muscle undergoing regenerative treatment.

[0087] In another aspect, the disclosure provides use of a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein in the manufacture of a medicament for reducing fibrosis in a subject undergoing regenerative treatment. The medicament may be adapted or formulated for administration in combination with a regenerative treatment. Preferably, the medicament is adapted or formulated for administration directly to the tissue requiring regenerative treatment, for example, topically administering the medicament to the skin or the muscle requiring regenerative treatment.

[0088] In another aspect, the present disclosure provides a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein for use in: • promoting tissue healing; • promoting wound closure; • promoting wound closure; • promoting skin regeneration; • promoting muscle regeneration; • reducing peripheral nociceptive sensitisation in response to injury and / or inflammation; • reducing fibrosis in response to injury; • reducing neutrophil and monocyte / macrophage migration into injured tissues; • enhancing neutrophil and pro-inflammatory macrophage death in the presence of inflammatory cytokines (e.g. IL-1 and TNF-α); • increasing neutrophil clearance by stimulating macrophage efferocytosis; • increasing macrophage polarisation from M1 to M2;1005837019 20 • increasing macrophage switching towards an anti-inflammatory / pro-repair phenotype; and / or • reducing release of cytokines IL-1, CCL2, CXCL2, MMP-2, and MMP-9; or any combination thereof.

[0089] In another aspect, the present disclosure provides use of a fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell as described herein in the manufacture of a medicament for • promoting tissue healing; • promoting wound closure; • promoting skin regeneration • promoting muscle regeneration; • reducing peripheral nociceptive sensitisation in response to injury and / or inflammation; • reducing fibrosis in response to injury; • reducing neutrophil and monocyte / macrophage migration into injured tissues; • enhancing neutrophil and pro-inflammatory macrophage death in the presence of inflammatory cytokines (e.g. IL-1 and TNF-α); • increasing neutrophil clearance by stimulating macrophage efferocytosis; • increasing macrophage polarisation from M1 to M2; • increasing macrophage switching towards an anti-inflammatory / pro-repair phenotype; and / or • reducing release of cytokines IL-1, CCL2, CXCL2, MMP-2, and MMP-9; or any combination thereof.

[0090] In another aspect, the present disclosure provides a fusion protein comprising:1005837019 21 (a) a first polypeptide; and (b) a second polypeptide, wherein the first and second polypeptide are joined by a linker that comprises, consists essentially of or consists of the amino acid sequence KGYR (SEQ ID NO: 70).

[0091] In any aspect or embodiment, the second polypeptide is a polypeptide that requires a free N-terminus, or N-terminal region, to perform its function. In any aspect or embodiment, the second polypeptide is a polypeptide that when its N-terminus is covalently linked to another molecule, for example via a peptide bond to one or more additional amino acids, its ability to perform its function is reduced or inhibited.

[0092] Performing function in this context may be, but not limited to, binding to its binding partner, enzymatic activity, catalysing a reaction.

[0093] In any aspect or embodiment, the amino acid C-terminal to KGYR (SEQ ID NO: 70) is the N-terminal amino acid of the second polypeptide.

[0094] In another aspect, the present disclosure provides a method of increasing the activity of a second polypeptide or reducing the inhibition of the activity of a second polypeptide, the method comprising contacting a fusion protein comprising the second polypeptide with plasmin, wherein the fusion protein comprises: (a) a first polypeptide; and (b) a second polypeptide, wherein the first and second polypeptide are joined by a linker that comprises, consists essentially of or consists of the amino acid sequence KGYR (SEQ ID NO: 70). In one embodiment, the first polypeptide comprises ^2PI1-8 (SEQ ID NO: 87). In one embodiment, the fusion protein comprises, consists essentially of, or consists of an amino acid sequence or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO:89.1005837019 22

[0095] Preferably, the second polypeptide is a polypeptide that when its N-terminus is covalently linked to the first polypeptide via the linker, its ability to perform its function is reduced or inhibited.

[0096] In any embodiment, the TSP-1 agonist may be a known TSP-1 mimetic or synthetic analog, such as ABT-510, ABT-898, PKT16, or PKHB1. The skilled person will be able to (i) identify suitable TSP-1 agonists; (ii) test its agonistic activity; and (iii) determine the effectiveness of the TSP-1 agonist on tissue regeneration and wound repair using methods as described and exemplified herein.

[0097] In any embodiment, the TSP agonist is a molecule which increases the activity and / or expression of TSP-1. The TSP-1 agonist may act directly or indirectly to increase TSP-1 activity and / or expression. TSP-1 activity and / or expression can be assessed using methods known to those in the art, including methods exemplified in the Examples included herein. Preferably, the TSP-1 agonist promotes TSP-1 expression in myeloid cells, in particular neutrophils and pro-inflammatory macrophages. The TSP-1 agonist may comprise or consist of a fusion protein described herein. The TSP-1 agonist may be a small molecule, such as adenosine. Alternatively, the TSP-1 agonist may comprise or consist of a polypeptide. For example, macrophage colony stimulating factor (M-CSF), HIF-1α, or angiotensin II, may stimulate TSP-1 expression in macrophages.

[0098] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0099] By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0100] By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 additional amino acid residues at the N-terminus or C-terminus of a polypeptide sequence) are optional and may or1005837019 23 may not be present depending upon whether or not they affect the activity or action of the listed elements. Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings

[0101] Figure 1: Nav1.8+ nociceptors expressing CGRP mediate tissue healing via myeloid cells. a-b) Full-thickness skin wounds were created in denervated mice (Nav1.8Cre / Rosa26DTA) and littermate controls (Rosa26DTA). Wound closure evaluated by histomorphometric analysis D6 and D10 post-injury (a) (Rosa26DTA D6, n = 14; Nav1.8Cre / Rosa26DTA D6, n = 8; D10, n = 12). Representative histology D6 post-injury (b). Black arrows indicate wound edges. Red arrows indicate tips of epithelium tongue. Scale bar = 1 mm. c-d) Volumetric muscle loss was performed on quadriceps of Nav1.8Cre / Rosa26DTA and Rosa26DTA littermate controls. Extent of muscle regeneration evaluated by histomorphometric analysis D8 and D12 post-injury (c) (D8, n = 7; D12, n = 10). Representative histology D12 post-injury (fibrotic tissue is stained blue; muscle tissue is stained red) (d). Scale bar = 500 μm. e) Nav1.8+ sensory neuron (red) distribution in skin and muscle of Nav1.8Cre / Rosa26tdT mice before and after injury (GT indicates granulation tissue area within orange lines, white lines indicate keratinocyte layers, nuclei in blue). Scale bars = 500 μm. Repeated independently 4 times. f) Expression of CGRP in skin and muscle before and after injury detected by immunohistochemistry. Scale bar = 500 μm in skin, 100 μm in muscle. Repeated independently 4 times. g-j) Ramp1 was deleted in myeloid cells using LysMCre+ / - / Ramp1fl / fl mice. LysMCre+ / - mice were controls. g) Wound closure quantification D6 post-injury (n = 10). h) Representative skin histology. i) Muscle regeneration quantification D12 post-injury (n = 10). j) Representative muscle histology. All data are plotted in box plots showing median (centre line) and IQR (bounds). Whiskers show min. to max. range. Dots represent individual injuries. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons in a, c. Two-tailed Student’s t-test in g, i. P values are indicated.

[0102] Figure 2: CGRP regulates myeloid cell function during tissue healing. a) Analysis of neutrophil and monocyte / macrophage (Mo / Mϕ) populations by flow cytometry during tissue healing. Geometric-mean of fluorescence intensity (MFI) of1005837019 24 CD206 in macrophages used to assess M2-like polarisation. Data are plotted in kinetic line plots showing mean ± SEM (Skin D0, D6, D10, n = 20; D3, n = 22; D14, n = 12. Muscle D0, n = 16; D3, n = 20; D6, D10, n = 18; D14, n = 10). b-e) Neutrophils and macrophages were treated with saline (PBS, 0 nM CGRP) or CGRP (1 or 20 nM). Results are expressed as fold change over the PBS / 0 nM CGRP group. Transwell migration towards CXCL1 or CCL2 with or without CGRP (b) (n = 6-8). Cell death in response to CGRP and TNF-α / IL-1 (c) (neutrophils, n = 4; macrophages, n = 6). Macrophage efferocytosis of neutrophils after CGRP treatment with or without TNF-α / IL- 1 (d) (n = 4). Macrophage polarisation determined via CD206 and arginase-1 protein expression following CGRP and IL-4 / IL-13 or IL-10 treatment (e) (n = 6). f, Tdtomato+ bone marrow cells were administered systemically D2 post-injury. Fold change of Tdtomato+ neutrophils and Mo / Mϕ detected in injured tissues D3 post-injury was assessed by flow cytometry. For efferocytosis, dead / dying Tdtomato+ neutrophils were injected in skin wound borders D3 post-injury. Fold change of Tdtomato+ endogenous Mo / Mϕ was assessed by flow cytometry 30 minutes post-injection (n = 6). g, CD11b+ cell death 3D post-injury assessed by TUNEL assay on injured tissue sections (n = 6). All data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent independent experiments. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons in a-e. Two-tailed Student’s t-test in f, g. P values are indicated; n.s., non-significant.

[0103] Figure 3: CGRP upregulates TSP-1 on neutrophils and macrophages to mediate its activity. a-b) RNA-seq analysis of CGRP-treated neutrophils and macrophages (1 nM or saline for 4 hours). GO enrichment analysis of significantly upregulated (red) and downregulated (blue) genes (FDR < 0.05) in CGRP-treated and saline-treated groups (a). Volcano plots in (b) showing differentially expressed genes (FDR < 0.05) with a fold change > |1.5|, between CGRP-treated and saline-treated groups. Significantly up- (red) and downregulated- (blue) genes in CGRP-treated neutrophils and macrophages are presented (n = 3). Thbs1 is annotated. c-f) Neutrophils and macrophages were treated with saline (PBS, 0 nM TSP-1) or TSP-1 (1, 10, or 100 nM). Results are expressed as fold change over the PBS / 0 nM TSP-1 control group. Data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent independent experiments. Transwell migration towards a chemoattractant (CXCL1 or CCL2) after TSP-1 treatment (c) (n = 3 for neutrophils, n = 4 for macrophages). Cell death in response to TSP-1 with or without1005837019 25 TNF-α / IL-1 (d) (n = 4). Macrophage efferocytosis of neutrophils after TSP-1 treatment (e) (n = 4). Macrophage M2-like polarisation determined via CD206 and arginase-1 expression in response to TSP-1 and IL-4 / IL-13 or IL-10 treatments (f) (CD206, n = 8; arginase-1, n = 4). Two-tailed Student’s t-test in b. One-way ANOVA with Tukey post hoc test for pair-wise comparisons in c-f. P values are indicated; n.s., non-significant.

[0104] Figure 4: Delivering eCGRP promotes tissue healing in diabetic mice. a) CGRP expression in skin and muscle of Nav1.8Cre / Rosa26DTA and diabetic (Leprdb / db) mice detected by immunostaining. CGRP, green; nuclei, blue. White lines indicate keratinocyte layer. Scale bar = 300 μm. Repeated independently 6 times. b-c) Saline, CGRP (500 ng) or equimolar eCGRP was delivered on Leprdb / dbskin wounds D1 and D3 post-injury. Wound closure evaluated by histomorphometric analysis D10 post-injury (b) (saline, eCGRP n = 14; CGRP, n = 12). Data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Representative skin histology (c). Black arrows indicate wound edges and red arrows point to tips of epithelium tongue. Scale bar = 1 mm. d-e) Saline, CGRP (1 μg) or equimolar eCGRP was delivered in Leprdb / db quadricep volumetric muscle loss defect via a fibrin hydrogel. Muscle regeneration (represented by the percentage of fibrotic tissue and muscle area) evaluated by histomorphometric analysis D8 post-injury (d). Data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range (n = 10). Representative histology (fibrotic tissue is stained blue; muscle tissue is stained red) (e). Scale bar = 500 μm. f) Numbers of neutrophils (CD11b+, Ly6G+, F4 / 80–) and monocytes / macrophages (CD11b+, F4 / 80+, Ly6G–), and expression of Ly6C and CD206 in macrophages (represented by MFI) in Leprdb / db wounds and muscle injuries treated saline or eCGRP were quantified by flow cytometry (skin, n = 6; muscle D4, n = 8, muscle D7, n = 9). Data are plotted in kinetic line plots showing mean ± SEM. One-way ANOVA with Tukey post hoc test for pair-wise comparisons in b, d. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons in f. P values are indicated; n.s., non-significant.

[0105] Figure 5: The lack of Nav1.8+ sensory neurons impairs tissue healing. a-g) Full-thickness skin wounds were created in Nav1.8Cre / Rosa26DTA and littermate control Rosa26DTA mice. Wound closure was evaluated by histomorphometric analysis of tissue sections at D6 post-injury. Representative histology at the edge and centre of the wounds (a). Blue lines indicate wound edges. Purple lines indicate the epithelial1005837019 26 tongue. GT indicates granulation tissue. Scale bar = 500 μm. Wound closure was evaluated by macroscopic evaluation (b) to calculate the wound healing rate constant κ (c) (n = 14). Representative macroscopic images of the wounds (d). Scale bar = 5 mm. Measurement of the length of the migrating epithelial tongue (e) (n = 12). Quantification of the number of proliferating keratinocytes (Ki-67 positive) in tissue sections (f) (n = 12). Representative image of immunostaining for keratin 14 (K14, purple) detecting the epithelial tongue and Ki-67 positive cells (green) (g). Blue lines indicate wound edges. Scale bar = 500 μm. In b) data are plotted in kinetic line plots showing mean ± SEM mean. In c, e, f, data are plotted in box plots showing median (centre line) and IQR (bounds). Whiskers show min. to max. range. Dots represent individual wounds. Two- way ANOVA with Bonferroni post hoc test for pair-wise comparisons in b. Two-tailed Student’s t-test in c, e, f. P values are indicated; n.s., non-significant. h, Volumetric muscle loss was performed on the quadriceps of Nav1.8Cre / Rosa26DTA and Rosa26DTA littermate control mice. Representative histology is shown (fibrotic tissue is stained blue; muscle tissue is stained red). Scale bars = 500 μm. Repeated independently 7 times for D8 and 10 times for D12.

[0106] Figure 6: Nav1.8+ sensory neurons mainly express CGRP during skin and muscle healing. a) Quantification of Nav1.8+ sensory neurons in skin and muscle before and after injury. Results are expressed as the percentage of tissue area positive for tdTomato in healthy tissue at D0 (uninjured) and in the granulation tissue at D3 and D6 post-injury (n = 4). b-d) Expression of neuropeptides in skin and muscle were detected by immunohistochemistry (neuropeptides, green; Nav1.8, red; nuclei, blue). Scale bars = 500 μm in skin and 100 μm in muscle. Quantification of neuropeptide signal in Nav1.8+ sensory neurons (d) (n = 4). e) Neuropeptide expression in DRGs before and on D3 and D6 after skin and muscle injury (neuropeptides, green; Nav1.8, red; nuclei, blue). Scale bars = 100 μm. f) Percentage of Nav1.8+ cell bodies expressing a neuropeptide type (n = 3). g) CGRP localisation in skin and muscle before and after tissue injury in Nav1.8Cre / Rosa26DTA and littermate controls Rosa26DTA. White lines indicate the separation between the granulation tissue (GT) and the top of the wound in skin and the separation between the granulation tissue and the muscle tissue in muscle. Scale bars = 100 μm. h) Quantification of CGRP signal (n = 4). In a, d, h, data are plotted in box plots showing median (centre line) and IQR (bounds). Whiskers show min. to max. range. Dots represent independent injuries. In f, data are plotted as bar graph showing mean ± SD. Dots represent individual DRGs. One-way1005837019 27 ANOVA with Tukey post hoc test for pair-wise comparisons in a, d. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons in f, h. P values are indicated.

[0107] Figure 7: CGRP signalling in immune cells mediates skin and muscle healing. a-d) Wildtype (wt) or Ramp1- / -mice were γ-irradiated and received a bone marrow transplant from wt or Ramp1- / -donor mice. Full-thickness skin wounds or quadriceps volumetric muscle loss were performed. Wound closure was evaluated by histomorphometric analysis of tissue sections at D6 post-injury (a) (n = 8). Representative histology (b). Black arrows indicate wound edges. Red arrows indicate tips of epithelium tongue. Scale bar = 1 mm. The extent of muscle regeneration (represented by the percentage of fibrotic tissue and muscle area) was evaluated by histomorphometric analysis of tissue sections at D12 post-injury (c) (n = 7). Representative histology D12 post-injury (fibrotic tissue is stained blue; muscle tissue is stained red) (d). Scale bar = 500 µm. e, Cell proliferation in response to CGRP treatment (n = 4 for 2% FBS; n = 5 for 10% FBS). Foetal bovine serum (FBS, 10%-20%) was used as a positive control and results are expressed as fold increase over saline control (0 nM CGRP with 2% or 10% FBS). For gel source data, see Supplementary Figure 1. f) Calcrl and Ramp1 expression in fibroblasts, keratinocytes, myoblasts, and endothelial cells, detected by RT-PCR. Gapdh was used as the housekeeping gene. Repeated independently 3 times. g) Representative images showing the close proximity of CD11b+ cells with Nav1.8+ sensory neurons in granulation tissue 6D after skin and muscle injury (CD11b, green; Nav1.8, red; nuclei, blue). Scale bar = 50 µm. Repeated independently 4 times. All data are plotted in box plots showing median (centre line) and IQR (bounds). Whiskers show min. to max. range. Dots represent individual injuries or experiments. Two-tailed Student’s t-test in a, c. Two-tailed one-sample t-test over fold increase of 1 in e. P values are indicated; n.s., non-significant.

[0108] Figure 8: Analysis of wound immune cell dynamics in injured skin and muscle. a) Gating strategy to analyse myeloid cells in skin and muscle post-injury. Flow cytometry dot plots representing the step by step (1-6) gating strategy to identify neutrophil (CD11b+, Ly6G+, F4 / 80-) monocytes / macrophages (Mo / Mϕ; CD11b+, F4 / 80+, Ly6G-), and dendritic cells (CD11c+, MHC-II+). MFI is the geometric-mean of fluorescence intensity. b) Gating strategy to analyse T cells in skin and muscle post- injury. Flow cytometry dot plots representing the step by step (1-6) gating strategy to identify CD4 T cells (CD3+, CD4+), cytotoxic T cells (CD3+, CD8+), and γδ T cells1005837019 28 (CD3+, TCRβ-, TCRγ+). c) Numbers of dendritic and T cells in skin and muscle injuries analysis measured by flow cytometry. Data are plotted in kinetic line plots showing mean ± SEM (n = 8 for D0, n = 10 for the other time points). Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. P values are indicated; n.s., non- significant. d) Bar graph representation of the average number of neutrophils, Mo / Mϕ, dendritic cells, and T cells in injured tissue at D3 post-injury in Rosa26DTA and Nav1.8Cre / Rosa26DTAmice. e) Representative images of TUNEL assay at D3 post-injury in in Rosa26DTAand Nav1.8Cre / Rosa26DTAmice (CD11b, green; TUNEL, red; nuclei, blue). Scale bar = 100 µm. Repeated independently 6 times.

[0109] Figure 9: CGRP expression in neutrophils and macrophages, and its effects on M2-Like macrophages, Ly6C expression, cells. a) Calcrl andRamp1 expression in bone marrow-derived neutrophils and macrophages detected by RT-PCR. Gapdh was used as the housekeeping gene. Repeated independently 3 times. b) CLR and RAMP1 expression was detected in bone marrow-derived neutrophils and macrophages using immunostaining. CLR, green; RAMP1, red; nuclei, blue. Scale bars = 25 μm. Repeated independently 3 times. c, d, Bone marrow-derived macrophages were treated with saline (0 nM CGRP) or CGRP (1 or 20 nM). Cell death in response to CGRP when macrophages were cultured with anti-inflammatory cytokines (IL-4 / IL-13, or IL-10) (c) (n = 6). Ly6C expression in response to CGRP treatment after macrophage culture in inflammatory (TNF-α / IL-1) or anti-inflammatory conditions (IL-10) (d) (n = 6). Results are expressed as fold increase over treatment without CGRP and without cytokines. e, Neutrophils and macrophages derived from mouse bone marrow of LysMCre+ / - / Ramp1fl / flwere treated with saline (PBS, 0 nM CGRP) or CGRP (1 nM) and transwell migration towards a chemoattractant (CXCL1 or CCL2) was tested (n = 4). Results are expressed as fold change over the saline PBS / 0 nM CGRP control group. All data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent independent experiments. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. n.s., non-significant.

[0110] Figure 10: TSP-1 mediates the activity of CGRP on neutrophils and macrophages. a) Differentially expressed genes contributing to GO enrichment analysis of CGRP-treated neutrophils and macrophages. Neutrophils and monocytes / macrophages were treated with CGRP (1 nM) or saline for 4 hours.1005837019 29 Transcriptomic profiling was performed by bulk RNA sequencing. Heatmap of selected significantly upregulated and downregulated genes depicting standardised gene expression values (z-scores) in CGRP-treated cells compared to PBS-treated cells individual biological replicates are shown (n = 3). Genes are displayed in alphabetical order and further classified according to known functions indicated by the coloured circles next to the heat map. b) Thbs1 expression after CGRP (1 nM) stimulation for 4 hours in neutrophils and macrophages isolated form LysMCre+ / -and LysMCre+ / - / Ramp1fl / flmice (n = 3). Dots represent independent experiments. Horizontal bars show mean. Whiskers show min. to max. range. c) TSP-1 concentration is skin and muscle before (uninjured) and at D3 post-injury quantified by ELISA (n = 4). d) Knockdown (KD) efficiency of TSP-1 by siRNA verified by qPCR analysis in macrophages. Results are expressed as relative expression over cells transfected with scramble siRNA (n = 5). e- h) Macrophages derived from mouse bone marrow were transfected with scramble siRNA or Thbs1 siRNA and treated with saline (PBS, 0 nM CGRP) or CGRP (1 nM). Results are expressed as fold change over the PBS / 0 nM CGRP control group. Transwell migration towards CCL2 in the presence of CGRP (e) (n = 9). Cell death in response to CGRP with or without TNF-α / IL-1 (f) (n = 7). Macrophage efferocytosis of neutrophils after CGRP treatment (g). (n = 5). Macrophage M2-like polarisation determined via CD206 expression in response to CGRP and IL-4 / IL-13 (h) (n = 10). i) Saline or TSP-1 (total 10 μg) was injected in mouse skin wound borders at D and D3 post-injury or delivered in mouse quadriceps volumetric muscle loss defect via a fibrin hydrogel right after injury. Neutrophil and monocyte / macrophage (Mo / Mϕ) populations in injured tissues were analysed by flow cytometry at D3 post-delivery. CD206 level measurements were performed at D14 post-delivery (n = 6). MFI is the geometric-mean of fluorescence intensity. All data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent independent experiments. Two-tailed Student’s t-test in b, c, d, i. One-way ANOVA with Tukey post hoc test for pair-wise comparisons in e-h. P values are indicated; n.s., non-significant.

[0111] Figure 11: Rescue of tissue healing in Nav1.8Cre / Rosa26DTAmice by local delivery of CGRP variants. a) Design and amino acid sequences of wild-type mouse αCGRP (SEQ ID NO:77) and eCGRP (SEQ ID NO:71). Disulphide bonds are indicated in yellow. Amid indicates amidation of the C-terminus phenylalanine. ECM-binding sequence from PlGF is in red. Plasmin-sensitive site from vitronectin is in grey. b) CGRP and eCGRP were incubated with or without plasmin and analysed by SDS-1005837019 30 PAGE. The gels show cleavage of the ECM-binding sequence in eCGRP by plasmin. Repeated independently 3 times. c) eCGRP activity was assessed using neutrophil and macrophage migration. The graphs show migration towards a chemoattractant (CXCL-1 or CCL2) in the presence of saline (PBS) or CGRP variants (20 nM). Results are expressed as fold change over saline control. Data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent individual experiments (n = 4). d) Neutrophils and macrophages were isolated from LysMCre+ / -and LysMCre+ / - / Ramp1fl / flmice. Cells were stimulated with CGRP (1 nM) or equimolar eCGRP for 30 minutes. cAMP concentration in cell lysates was measured by competitive ELISA (n = 4). e-f) CGRP (1 μg) or eCGRP was delivered intradermally or intramuscularly in Nav1.8Cre / Rosa26tdTmice. One day post-delivery, tissues were harvested and CGRP variants were detected by immunostaining. Representative skin and muscle tissue sections. CGRP signal coming from exogenous CGRP variants appears in green. Arrows indicate CGRP signal coming from Nav1.8+ sensory neurons (in red). Nuclei are in blue. Scale bar = 50 μm. Quantification of CGRP-positive area and signal intensity expressed as integrated density (f) (n = 5 for skin, n = 4 for muscle). g, h, Saline, low dose of CGRP (250 ng), high dose of CGRP (500 ng), or equimolar eCGRP was delivered on Nav1.8Cre / Rosa26DTAmouse skin wounds (D1 post-injury for low dose and D1 and D3 post-injury for high dose). Skin wound closure D6 post-injury evaluated by histomorphometric analysis (g) (n = 8 for saline; n = 8 for low; n = 10 for high). Representative skin histology (h). Black arrows indicate wound edges and red arrows indicate tips of epithelium tongue. Scale bar = 1 mm. i-j) Saline, low dose of CGRP (250 ng), high dose of CGRP (1 μg), or equimolar eCGRP was delivered in Nav1.8Cre / Rosa26DTAmouse quadriceps volumetric muscle loss defect via a fibrin hydrogel. The extent of muscle regeneration (represented by the percentage of fibrotic tissue and muscle area) was evaluated by histomorphometric analysis of tissue sections at D8 (low dose) and D12 (high dose) post-injury (i). (n = 6). Representative histology (fibrotic tissue is stained dark blue; muscle tissue is stained red) (j). Scale bar = 1 mm. k) Mice received one injection of saline, CGRP (1 μg), equimolar eCGRP, or capsaicin (positive control) in the right hind paw. Graphs show duration and frequency of nocifensive behaviours recorded during 5 minutes at various time points (n = 8). l, Mice received one injection of saline, CGRP (1 μg), or equimolar eCGRP in the right hind paw. Graph shows thermal withdrawal latency at various time points post-injection (n = 8). All data are plotted in box plots showing median (central line) and IQR (bounds).1005837019 31 Whiskers show min. to max. range. Dots represent independent experiments or injuries. One-way ANOVA with Tukey post hoc test for pair-wise comparisons in c, d, f, g, i. Two- way ANOVA with Bonferroni post hoc test for pair-wise comparisons in k, l. P values are indicated; n.s., non-significant.

[0112] Figure 12: CGRP levels in Leprdb / dbmice and TSP-1 deposition in response to eCGRP delivery. a) CGRP expression in uninjured skin and muscle of wildtype (Lepr+ / +) and diabetic (Leprdb / db) mice was detected by immunostaining of tissue sections. The graphs show quantification of CGRP-positive area and signal intensity expressed as integrated density as well as the CGRP-positive area in CD11b+ cells (n = 6). b) Skin wounds and muscle defects in Leprdb / dbmice were treated with eCGRP. Expression of TSP-1 in granulation tissue was detected at D4 via immunostaining of tissue sections. TSP-1 in green, myeloid cells (CD11b) in red, nuclei in blue. Scale bar = 200 μm. Graphs show quantification of TSP-1–positive area (n = 6). All data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent individual tissue sections. Two-tailed Student’s t-test in a, b. P values are indicated.

[0113] Figure 13: Gating strategy to analyse wound immune cell dynamics in diabetic mice as well as cytokine and protease levels after eCGRP treatment. a) Gating strategy to analyse myeloid cells skin and muscle post-injury. Flow cytometry dot plots representing the step by step (1-6) gating strategy to identify neutrophil (CD11b+, Ly6G+, F4 / 80–) and macrophage populations (CD11b+, F4 / 80+, Ly6G–). MFI is the geometric-mean of fluorescence intensity. b-c) Saline or eCGRP was delivered on Leprdb / dbskin wounds or in quadricep volumetric muscle loss defect via a fibrin hydrogel. The levels of CCL2, IL-1β, CXCL2 (b), MMP-2, and MMP-9 (c) in injured tissues were quantified by ELISA (n = 8 for skin, n = 4 for muscle). Data are plotted in box plots showing median (central line) and IQR (bounds). Whiskers show min. to max. range. Dots represent individual injuries. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. P values are indicated.

[0114] Figure 14: Schematic of the neuro-immune-regenerative axis after acute injury in skin and muscle. The schematic shows the proposed mechanisms by which nociceptors promote tissue healing via controlling neutrophils and monocytes / macrophages (Mo / Mϕ) in injured tissues. Following tissue injury, CGRP- expressing nociceptor endings grow into the granulation tissue. CGRP signalling in1005837019 32 neutrophils and macrophages induces the release of the ECM protein TSP-1. TSP-1 is deposited in the injured tissue milieu inhibiting neutrophil and monocytes / macrophage migration and eventually accelerating the cell death response of neutrophils and macrophages to inflammatory cytokines. In addition, CGRP promotes efferocytosis and macrophage polarisation into a M2-like phenotype via an autocrine or paracrine effect of TSP-1. Overall, nociceptors are critical for the transition of the injured tissue microenvironment towards a tissue healing phase. Blue line indicates inhibition, red line indicates induction. Dashed grey lines indicate that CGRP and TSP-1 may also promote tissue healing by acting on non-immune cells.

[0115] Figure 15: Immunoregulation on neutrophils and macrophages by CGRP variants. a. CGRP and CGRP variants were incubated with or without plasmin and analysed by SDS-PAGE. The SDS-PAGE show cleavage of the ECM-binding sequence in CGRP variants by plasmin (repeated independently 3 times). b, c. Bioactivity of CGRP variants was assessed using neutrophil and macrophage migration and polarisation assays. Cells were treated with saline (PBS, 0 nM CGRP) or CGRP / CGRP variants (20 nM). Results are expressed as fold change over the PBS (0 nM CGRP) group. b. Transwell migration towards CXCL1 or CCL-2. c. Macrophage polarisation determined by CD206 expression, an anti-inflammatory indicator, following CGRP and IL-4 / IL-13 treatment. In b, boxes show median (centre line) and interquartile range (edges), whiskers show the range of values. Dots represent independent experiments. In c, data is presented as a mean ^ SEM. For a–c, Two-way ANOVA with Bonferroni post hoc test for pairwise comparisons. p-values are indicated. Figure 16: Skin wounds and muscle defects in db / db mice were treated with saline or TSP-1 (20µg). a. Wound closure quantification. b. Representative skin histology. Black arrows, wound edges; red arrows, tip of the keratinocyte layer. c. Fibrotic and muscle area quantification. d. Representative muscle histology. Muscle tissue in red; fibrotic tissue in blue. Student's t-test. p-values are indicated.1005837019 33 Summary of sequence listing Table 1. Sequence information. SEQ ID NO: DESCRIPTION SEQUENCE SEQ ID NO: 1 Human alpha calcitonin ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKA gene-related peptide F (αCGRP) SEQ ID NO: 2 Human placenta growth MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGS factor-2 (PlGF-2) SEVEVVPFQEVWGRSYCRALERLVDVVSEYPSEVE HMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQ LLKIRSGDRPSYVELTFSQHVRCECRPLREKMKPER RRPKGRGKRRREKQRPTDCHLCGDAVPRR SEQ ID NO: 3 Human placenta growth MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGS factor-4 (PlGF-4) SEVEVVPFQEVWGRSYCRALERLVDVVSEYPSEVE HMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQ LLKIRSGDRPSYVELTFSQHVRCECRHSPGRQSPDM PGDFRADAPSFLPPRRSLPMLFRMEWGCALTGSQS AVWPSSPVPEEIPRMHPGRNGKKQQRKPLREKMKP ERRRPKGRGKRRREKQRPTDCHLCGDAVPRR SEQ ID NO: 4 Human placental growth RRRPKGRGKRRREKQRPTD factor 123-141 (PlGF123-141) SEQ ID NO: 5 Human PlGF123-152 RRRPKGRGKRRREKQRPTDCHLCGDAVPRR (heparin binding domain) SEQ ID NO: 6 Mouse equivalent of RRKTKGKRKRSRNSQTEE human PlGF123-141SEQ ID NO:4 SEQ ID NO: 7 Placenta growth factor RRRPKGRGKRKREKQRPTD isoform X7 [Canis lupus familiaris] SEQ ID NO: 8 Placenta growth factor RRRPKGRGKRRREKQKPTD isoform X1 [Hylobates moloch] SEQ ID NO: 9 Growth factor isoform RRRPKGRGKRRREKQRPKD X1 [Callithrix jacchus]1005837019 34 SEQ ID NO: 10 Placenta growth factor RRRPKGRGKRKRDKQRPTD isoform X1 [Galeopterus variegatus] SEQ ID NO: 11 Placenta growth factor RRRPKGRGKRKREKQKPTD isoform X1 [Carlito syrichta] SEQ ID NO: 12 Placenta growth factor RRRPKGRGKRKREKQRHTD isoform X3 [Lagenorhynchus obliquidens] SEQ ID NO: 13 Placenta growth factor RRRPKGRGKRKREKQRPRD isoform X3 [Suricata suricatta] SEQ ID NO: 14 Placenta growth factor RRRSRGRGKRKREKQRPTD isoform X1 [Trichechus manatus latirostris] SEQ ID NO: 15 Placenta growth factor RRRPKGQGKRRREKQRP isoform X1 [Pelodiscus sinensis] SEQ ID NO: 16 Placenta growth factor RRRPKGSGKRKKEKQRPTD isoform X1 [Eptesicus fuscus] SEQ ID NO: 17 Placenta growth factor RRRPKGQGKRKREKQRP isoform X1 [Chrysemys picta bellii] SEQ ID NO: 18 Placenta growth factor RRRHKGRRKRKREKQRPTD isoform X1 [Ailuropoda melanoleuca] SEQ ID NO: 19 Placenta growth factor RRRPKVRGKRKREKQKPT isoform X1 [Fukomys damarensis] SEQ ID NO: 20 Placenta growth factor RRRPKGRSKRKRAKQRPKD [Podarcis muralis] SEQ ID NO: 21 Placenta growth factor RRRPKGRSKRKRAKQRPKD [Lacerta agilis] SEQ ID NO: 22 Placenta growth factor RRRLKGRGKRKKEKQRSTD isoform X1 [Ictidomys tridecemlineatus]1005837019 35 SEQ ID NO: 23 Placenta growth factor RRRPKVRGKRKRENQKPT isoform X1 [Cavia porcellus] SEQ ID NO: 24 Placenta growth factor RRRYRGRGKRKREKQRATD [Echinops telfairi] SEQ ID NO: 25 Placenta growth factor- RRRPKGRGRKRKEKQR like [Electrophorus electricus] SEQ ID NO: 26 Placenta growth factor RRRNKGSGKRKKEKQRPT [Phyllostomus discolor] SEQ ID NO: 27 Placenta growth factor RRRPKGRGKRRGEKKRRKD [Anas platyrhynchos] SEQ ID NO: 28 Placenta growth factor RRRQKGRGRKRKDKQRPKD [Paramormyrops kingsleyae] SEQ ID NO: 29 Placenta growth factor RRRPKGRGKRRQDKMR [Strigops habroptila] SEQ ID NO: 30 Placenta growth factor RRRSKDRGKRKRERPRPT [Ornithorhynchus anatinus] SEQ ID NO: 31 Placenta growth factor RRRPKGRGKRRQERTR [Amazona aestiva] SEQ ID NO: 32 Vascular endothelial RRRPRGRGRKRKEKQR growth factor A-like [Pygocentrus nattereri] SEQ ID NO: 33 Placenta growth factor RRRFKGRGKRKRDKQRTKD [Sarcophilus harrisii] SEQ ID NO: 34 Consensus placental RRRPKGRGKRKREKQRPTD growth factor sequence (differs by only one amino acid from the human sequence) SEQ ID NO: 35 Human PlGF123-142 RRRPKGRGKRRREKQRPTDC SEQ ID NO: 36 Human PlGF123-143 RRRPKGRGKRRREKQRPTDCH SEQ ID NO: 37 Human PlGF123-145 RRRPKGRGKRRREKQRPTDCHLC SEQ ID NO: 38 Human PlGF123-146 RRRPKGRGKRRREKQRPTDCHLCG SEQ ID NO: 39 Human PlGF123-147 RRRPKGRGKRRREKQRPTDCHLCGD SEQ ID NO: 40 Human PlGF123-148 RRRPKGRGKRRREKQRPTDCHLCGDA SEQ ID NO: 41 Human PlGF123-149 RRRPKGRGKRRREKQRPTDCHLCGDAV1005837019 36 SEQ ID NO: 42 Human PlGF123-150 RRRPKGRGKRRREKQRPTDCHLCGDAVP SEQ ID NO: 43 Human PlGF123-151RRRPKGRGKRRREKQRPTDCHLCGDAVPR SEQ ID NO: 44 Human PIGF123-138 RRRPKGRGKRRREKQR SEQ ID NO: 45 Human amphiregulin RKKKGGKNGKNRR 126-138 (AREG126-138) SEQ ID NO: 46 Human AREG126-139RKKKGGKNGKNRRN SEQ ID NO: 47HumanAREG126-140 RKKKGGKNGKNRRNR SEQ ID NO: 48 Human AREG126-141 RKKKGGKNGKNRRNRKSEQ ID NO: 49 HumanAREG126-142RKKKGGKNGKNRRNRKKSEQ ID NO: 50 Human AREG126-143 RKKKGGKNGKNRRNRKKK SEQ ID NO: 51 Human AREG126-144RKKKGGKNGKNRRNRKKKN SEQ ID NO: 52 Human AREG126-145RKKKGGKNGKNRRNRKKKNP SEQ ID NO: 53 Human AREG126-146 RKKKGGKNGKNRRNRKKKNPC SEQ ID NO: 54 Human AREG126-147RKKKGGKNGKNRRNRKKKNPCN SEQ ID NO: 55HumanAREG126-148 RKKKGGKNGKNRRNRKKKNPCNA SEQ ID NO: 56 Human AREG123-145 KPKRKKKGGKNGKNRRNRKKKNP SEQ ID NO: 57 Human AREG124-145PKRKKKGGKNGKNRRNRKKKNP SEQ ID NO: 58 Human AREG125-145KRKKKGGKNGKNRRNRKKKNP SEQ ID NO: 59HumanAREG123-148 KPKRKKKGGKNGKNRRNRKKKNPCNA SEQ ID NO: 60 Human AREG130-141 GGKNGKNRRNRK SEQ ID NO: 61HumanAREG126-135 RKKKGGKNGK SEQ ID NO: 62 Human neurturin 146- RRLRQRRRLRRE 157 (NRTN146-157)SEQ ID NO: 63Human NRTN146-163RRLRQRRRLRRERVRAQPSEQ ID NO: 64 Human NRTN146-160RRLRQRRRLRRERVR SEQ ID NO: 65HumanNRTN146-154 RRLRQRRRL SEQ ID NO: 66 Human NRTN149- 154RQRRRL SEQ ID NO: 67 Human von Willebrand WREPSFMALS factor (vWF) ECM binding moiety 1 SEQ ID NO: 68 Human von Willebrand CSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANI factor (vWF) ECM GPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDV binding moiety 2 MQREGGPSQIGDALGFAVRYLTSEMHGARPGASKA VVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYD AAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLH KLCSGFVRICTG SEQ ID NO: 69 Human Collagenase TKKTLRT ECM binding moiety1005837019 37 SEQ ID NO: 70 Plasmin sensitive site KGYR SEQ ID NO: 71 Illustrative fusion protein RRRPKGRGKRRREKQRPTDKGYRSCNTATCVTHRL of the disclosure, AGLLSRSGGVVKDNFVPTNVGSEAF eCGRP (linker bold and underlined) SEQ ID NO: 72 Human TSP-1 NRIPESGGDNSVFDIFELTGAARKGSGRRLVKGPDP SSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKGFLLL ASLRQMKKTRGTLLALERKDHSGQVFSVVSNGKAG TLDLSLTVQGKQHVVSVEEALLATGQWKSITLFVQE DRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIA KGGVNDNFQGVLQNVRFVFGTTPEDILRNKGCSSST SVLLTLDNNVVNGSSPAIRTNYIGHKTKDLQAICGISC DELSSMVLELRGLRTIVTTLQDSIRKVTEENKELANEL RRPPLCYHNGVQYRNNEEWTVDSCTECHCQNSVTI CKKVSCPIMPCSNATVPDGECCPRCWPSDSADDG WSPWSEWTSCSTSCGNGIQQRGRSCDSLNNRCEG SSVQTRTCHIQECDKRFKQDGGWSHWSPWSSCSV TCGDGVITRIRLCNSPSPQMNGKPCEGEARETKACK KDACPINGGWGPWSPWDICSVTCGGGVQKRSRLC NNPTPQFGGKDCVGDVTENQICNKQDCPIDGCLSN PCFAGVKCTSYPDGSWKCGACPPGYSGNGIQCTDV DECKEVPDACFNHNGEHRCENTDPGYNCLPCPPRF TGSQPFGQGVEHATANKQVCKPRNPCTDGTHDCN KNAKCNYLGHYSDPMYRCECKPGYAGNGIICGEDT DLDGWPNENLVCVANATYHCKKDNCPNLPNSGQED YDKDGIGDACDDDDDNDKIPDDRDNCPFHYNPAQY DYDRDDVGDRCDNCPYNHNPDQADTDNNGEGDAC AADIDGDGILNERDNCQYVYNVDQRDTDMDGVGDQ CDNCPLEHNPDQLDSDSDRIGDTCDNNQDIDEDGH QNNLDNCPYVPNANQADHDKDGKGDACDHDDDND GIPDDKDNCRLVPNPDQKDSDGDGRGDACKDDFDH DSVPDIDDICPENVDISETDFRRFQMIPLDPKGTSQN DPNWVVRHQGKELVQTVNCDPGLAVGYDEFNAVDF SGTFFINTERDDDYAGFVFGYQSSSRFYVVMWKQV TQSYWDTNPTRAQGYSGLSVKVVNSTTGPGEHLRN ALWHTGNTPGQVRTLWHDPRHIGWKDFTAYRWRL SHRPKTGFIRVVMYEGKKIMADSGPIYDKTYAGGRL GLFVFSQEMVFFSDLKYECRDP1005837019 38 SEQ ID NO: 73 Nucleic acid of cgccgccgcccgaaaggccgcggcaaacgccgccgcgaaaaacag illustrative fusion protein cgcccgaccgataaaggctatcgcagctgcaacaccgcgacctgcgtg of the disclosure, acccatcgcctggcgggcctgctgagccgcagcggcggcgtggtgaaa eCGRP gataactttgtgccgaccaacgtgggcagcgaagcgttt SEQ ID NO: 74 Human adrenomedullin YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTD 1-2 KDKDNVAPRSKISPQGY SEQ ID NO: 75 Human adrenomedullin TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQ 2 DSAPVDPSSPHSY SEQ ID NO: 76 Human amylin KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNT Y SEQ ID NO: 77 Mouse alpha calcitonin SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEA gene-related peptide F (αCGRP) SEQ ID NO: 78 Rat alpha calcitonin SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEA gene-related peptide F (αCGRP) SEQ ID NO: 79 Pig alpha calcitonin SCNTATCVTHRLAGLLSRSGGMVKSNFVPTDVGSE gene-related peptide AF (αCGRP) SEQ ID NO: 80 Human beta calcitonin ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSK gene-related peptide AF (βCGRP) SEQ ID NO: 81 Mouse beta calcitonin SCNTATCVTHRLAGLLSRSGGVLKDNFVPTDVGSEA gene-related peptide F (βCGRP) SEQ ID NO: 82 Rat beta calcitonin SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSKA gene-related peptide F (βCGRP) SEQ ID NO: 83 Nucleic acid sequence aaccgcattccggaaagcggcggcgataacagcgtgtttgatatttttgaa encoding human TSP-1 ctgaccggcgcggcgcgcaaaggcagcggccgccgcctggtgaaagg cccggatccgagcagcccggcgtttcgcattgaagatgcgaacctgattc cgccggtgccggatgataaatttcaggatctggtggatgcggtgcgcgcg gaaaaaggctttctgctgctggcgagcctgcgccagatgaaaaaaaccc gcggcaccctgctggcgctggaacgcaaagatcatagcggccaggtgtt tagcgtggtgagcaacggcaaagcgggcaccctggatctgagcctgac cgtgcagggcaaacagcatgtggtgagcgtggaagaagcgctgctggc gaccggccagtggaaaagcattaccctgtttgtgcaggaagatcgcgcg cagctgtatattgattgcgaaaaaatggaaaacgcggaactggatgtgcc gattcagagcgtgtttacccgcgatctggcgagcattgcgcgcctgcgcat1005837019 39 tgcgaaaggcggcgtgaacgataactttcagggcgtgctgcagaacgtg cgctttgtgtttggcaccaccccggaagatattctgcgcaacaaaggctgc agcagcagcaccagcgtgctgctgaccctggataacaacgtggtgaac ggcagcagcccggcgattcgcaccaactatattggccataaaaccaaa gatctgcaggcgatttgcggcattagctgcgatgaactgagcagcatggt gctggaactgcgcggcctgcgcaccattgtgaccaccctgcaggatagc attcgcaaagtgaccgaagaaaacaaagaactggcgaacgaactgcg ccgcccgccgctgtgctatcataacggcgtgcagtatcgcaacaacgaa gaatggaccgtggatagctgcaccgaatgccattgccagaacagcgtga ccatttgcaaaaaagtgagctgcccgattatgccgtgcagcaacgcgac cgtgccggatggcgaatgctgcccgcgctgctggccgagcgatagcgcg gatgatggctggagcccgtggagcgaatggaccagctgcagcaccagc tgcggcaacggcattcagcagcgcggccgcagctgcgatagcctgaac aaccgctgcgaaggcagcagcgtgcagacccgcacctgccatattcag gaatgcgataaacgctttaaacaggatggcggctggagccattggagcc cgtggagcagctgcagcgtgacctgcggcgatggcgtgattacccgcatt cgcctgtgcaacagcccgagcccgcagatgaacggcaaaccgtgcga aggcgaagcgcgcgaaaccaaagcgtgcaaaaaagatgcgtgcccg attaacggcggctggggcccgtggagcccgtgggatatttgcagcgtgac ctgcggcggcggcgtgcagaaacgcagccgcctgtgcaacaacccga ccccgcagtttggcggcaaagattgcgtgggcgatgtgaccgaaaacca gatttgcaacaaacaggattgcccgattgatggctgcctgagcaacccgt gctttgcgggcgtgaaatgcaccagctatccggatggcagctggaaatgc ggcgcgtgcccgccgggctatagcggcaacggcattcagtgcaccgatg tggatgaatgcaaagaagtgccggatgcgtgctttaaccataacggcga acatcgctgcgaaaacaccgatccgggctataactgcctgccgtgcccg ccgcgctttaccggcagccagccgtttggccagggcgtggaacatgcga ccgcgaacaaacaggtgtgcaaaccgcgcaacccgtgcaccgatggc acccatgattgcaacaaaaacgcgaaatgcaactatctgggccattatag cgatccgatgtatcgctgcgaatgcaaaccgggctatgcgggcaacggc attatttgcggcgaagataccgatctggatggctggccgaacgaaaacct ggtgtgcgtggcgaacgcgacctatcattgcaaaaaagataactgcccg aacctgccgaacagcggccaggaagattatgataaagatggcattggc gatgcgtgcgatgatgatgatgataacgataaaattccggatgatcgcgat aactgcccgtttcattataacccggcgcagtatgattatgatcgcgatgatgt gggcgatcgctgcgataactgcccgtataaccataacccggatcaggcg gataccgataacaacggcgaaggcgatgcgtgcgcggcggatattgat ggcgatggcattctgaacgaacgcgataactgccagtatgtgtataacgt ggatcagcgcgataccgatatggatggcgtgggcgatcagtgcgataac1005837019 40 tgcccgctggaacataacccggatcagctggatagcgatagcgatcgca ttggcgatacctgcgataacaaccaggatattgatgaagatggccatcag aacaacctggataactgcccgtatgtgccgaacgcgaaccaggcggat catgataaagatggcaaaggcgatgcgtgcgatcatgatgatgataacg atggcattccggatgataaagataactgccgcctggtgccgaacccggat cagaaagatagcgatggcgatggccgcggcgatgcgtgcaaagatgat tttgatcatgatagcgtgccggatattgatgatatttgcccggaaaacgtgg atattagcgaaaccgattttcgccgctttcagatgattccgctggatccgaa aggcaccagccagaacgatccgaactgggtggtgcgccatcagggca aagaactggtgcagaccgtgaactgcgatccgggcctggcggtgggcta tgatgaatttaacgcggtggattttagcggcaccttttttattaacaccgaac gcgatgatgattatgcgggctttgtgtttggctatcagagcagcagccgcttt tatgtggtgatgtggaaacaggtgacccagagctattgggataccaaccc gacccgcgcgcagggctatagcggcctgagcgtgaaagtggtgaacag caccaccggcccgggcgaacatctgcgcaacgcgctgtggcataccgg caacaccccgggccaggtgcgcaccctgtggcatgatccgcgccatatt ggctggaaagattttaccgcgtatcgctggcgcctgagccatcgcccgaa aaccggctttattcgcgtggtgatgtatgaaggcaaaaaaattatggcgga tagcggcccgatttatgataaaacctatgcgggcggccgcctgggcctgtt tgtgtttagccaggaaatggtgttttttagcgatctgaaatatgaatgccgcg atccg SEQ ID NO:84 Exemplary ECM RRRPK binding moiety SEQ ID NO: 85 Exemplary ECM RKKK binding moiety SEQ ID NO: 86 Exemplary ECM KRRR binding moiety SEQ ID NO: 87 α2PI1-8 sequence NQEQVSPL SEQ ID NO: 88 α2PI1-8 sequence with NQEQVSPLKGYR cleavable linker KGYR SEQ ID NO: 89 Exemplary fusion NQEQVSPLKGYRSCNTATCVTHRLAGLLSRSGGVV protein: KDNFVPTNVGSEAF ^2PI1-8 -KGYR-mouse αCGRP SEQ ID NO: 90 Nucleic acid encoding aaccaggaacaggtgagcccgctgaaaggctatcgcagctgcaacac ^2PI1-8 -KGYR- mouse cgcgacctgcgtgacccatcgcctggcgggcctgctgagccgcagcgg αCGRP cggcgtggtgaaagataactttgtgccgaccaacgtgggcagcgaagcg ttt1005837019 41 SEQ ID NO: 91 Human_Calcrl primer CATGCACATCCTTATGCAC SEQ ID NO: 92 Human_Calcrl primer CCATCACTGATTGTTGACAC SEQ ID NO: 93 Human_Ramp1 GCCAGGAGGCTAACTACG primer SEQ ID NO: 94 Human_Ramp1 GAAGAACCTGTCCACCTCTG primer SEQ ID NO: 95 Mouse_Calcrl primer GGTACCACTACTTGGCATTG SEQ ID NO: 96Mouse_Calcrl primer GTCACTGATTGTTGACACTG SEQ ID NO: 97 Mouse_Ramp1 primer GACGCTATGGTGTGACT SEQ ID NO: 98 Mouse_Ramp1 primer GAGTGCAGTCATGAGCAG SEQ ID NO: 99 dT primer CAAGCAGAAGACGGCATACGAGAT Detailed description of the embodiments

[0116] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0117] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present disclosure as defined by the claims.

[0118] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0119] All of the patents and publications referred to herein are incorporated by reference in their entirety.1005837019 42

[0120] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0121] The general chemical terms used in the formulae herein have their usual meaning.

[0122] The present disclosure arises from the identification of an important neuro- immuno-regenerative axis following acute tissue injury and highlights the intricate interplay between nociceptors, immune cells, and the tissue healing processes. The inventors found that Nav1.8+sensory neurons expressing CGRP, which mostly represent nociceptors, extend into the granulation tissue formed after skin and muscle injuries and profoundly modulate neutrophil and macrophage dynamics during tissue healing. This neuro-immune modulation promotes the transition towards an anti-inflammatory and pro- repair phase, which ultimately facilitates the healing process. Mechanistically, the inventors demonstrated that CGRP signalling in neutrophils and macrophages induces the expression of TSP-1, which in turn limits their accumulation and accelerates cell death in the presence of inflammatory cytokines. In addition, CGRP promotes neutrophil clearance via efferocytosis and boosts macrophage polarisation into an anti-inflammatory and pro-repair phenotype via autocrine and paracrine effects of TSP-1.

[0123] Further, the present disclosure relates to, amongst other things, fusion proteins with several advantages. CGRP is a small peptide, which poses a challenge in achieving sustained effects without immediate burst signalling when delivered locally into tissue as it can rapidly signal to cells, diffuse away from the delivery site, and undergo degradation. The fusion proteins include CGRP engineered to enhance retention and protection at delivery sites by fusing it to a sequence with a high affinity for ECM components. The ECM-binding sequence was fused to CGRP, preferably the N-terminus of CGRP, followed by a plasmin-sensitive sequence to allow the release of CGRP from ECM via proteolytic activity. These modifications did not impair CGRP activity, whilst promoting greater retention of CGRP after delivery into tissues, greater wound closure and muscle regeneration and lesser peripheral nociceptive sensitisation than the corresponding CGRP polypeptide not linked to an ECM binding polypeptide.

[0124] Collectively, the present disclosure, in particular fusion proteins described herein, have application in regenerative medicine such as tissue regeneration and wound1005837019 43 repair, particularly for patients with peripheral neuropathies, including those associated with conditions such as diabetes. Fusion proteins

[0125] In general, preparation of the fusion proteins of the invention can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques involving, e.g., polymerase chain amplification reactions (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, transformation or transfection of a host, culturing of the host. Additionally, the fusion proteins can be isolated and purified using chaotropic agents and well known electrophoretic, centrifugation and chromatographic methods.

[0126] The invention further provides nucleic acid sequences and particularly DNA sequences that encode the present fusion proteins. Preferably, the DNA sequence is carried by a vector suited for extrachromosomal replication such as a phage, virus, plasmid, phagemid, cosmid, YAC, or episome. In particular, a DNA vector that encodes a desired fusion protein can be used to facilitate preparative methods described herein and to obtain significant quantities of the fusion protein. The DNA sequence can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. A variety of host-vector systems may be utilized to express the protein- coding sequence. These include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

[0127] In general, a preferred DNA vector according to the invention comprises a nucleotide sequence linked by phosphodiester bonds comprising, in a 5' to 3' direction a first cloning site for introduction of a first nucleotide sequence encoding a CGRP polypeptide or CGRP receptor agonist polypeptide, operably linked to a nucleotide sequence encoding an ECM binding peptide.1005837019 44

[0128] In most instances, it will be preferred that each of the fusion protein components encoded by the DNA vector be provided in a "cassette" format. By the term "cassette" is meant that each component can be readily substituted for another component by standard recombinant methods.

[0129] The fusion proteins described herein are preferably produced by standard recombinant DNA techniques. The resultant hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein. The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered (i.e., the DNA molecules are ligated to each other in-frame). The resulting DNA molecules encode an in-frame fusion protein.

[0130] A number of strategies can be employed to express the fusion proteins of the invention. For example, the gene fusion construct described above can be incorporated into a suitable vector by known means such as by use of restriction enzymes to make cuts in the vector for insertion of the construct followed by ligation. The vector containing the gene construct is then introduced into a suitable host for expression of the fusion protein. Selection of suitable vectors can be made empirically based on factors relating to the cloning protocol. For example, the vector should be compatible with, and have the proper replicon for the host that is being employed. Further the vector must be able to accommodate the DNA sequence coding for the fusion protein that is to be expressed. Suitable host cells include eukaryotic and prokaryotic cells, preferably those cells that can be easily transformed and exhibit rapid growth in culture medium. Specifically, preferred hosts cells include prokaryotes such as E. coli, Bacillus subtillus, etc. and eukaryotes such as animal cells and yeast strains, e.g., S. cerevisiae. Mammalian cells are generally preferred, particularly J558, NSO, SP2-O or CHO. Other suitable hosts include, e.g., insect cells such as Sf9. Conventional culturing conditions are employed. Stable transformed or transfected cell lines can then be selected. Cells expressing fusion proteins according to the invention can be determined by known procedures.

[0131] Nucleic acid encoding a desired fusion protein can be introduced into a host cell by standard techniques for transfecting cells. The term "transfecting" or "transfection" is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAE-1005837019 45 dextran-mediated transfection, lipofection, electroporation, microinjection, viral transduction and / or integration.

[0132] The present disclosure further provides a production process for isolating a fusion protein of interest. Methods for isolating a polypeptide are known in the art and / or described herein.

[0133] In the process, a host cell (e.g., a yeast, fungus, insect, bacterial or animal cell), into which has been introduced a nucleic acid encoding the fusion protein operatively linked to a regulatory sequence, is grown at production scale in a culture medium. Subsequently, the fusion protein of interest is isolated from harvested host cells or from the culture medium. Standard protein purification techniques can be used to isolate the fusion protein from the medium or from the harvested cells. In particular, the purification techniques can be used to express and purify a desired fusion protein on a large-scale (i.e. in at least milligram quantities) from a variety of implementations including roller bottles, spinner flasks, tissue culture plates, bioreactor, or a fermentor.

[0134] The skilled artisan will be aware that a polypeptide can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or an influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting polypeptide is then purified using methods known in the art, such as, affinity purification.

[0135] An expressed fusion protein can be isolated and purified by known methods. Typically, the culture medium is centrifuged and then the supernatant is purified by affinity or immunoaffinity chromatography, e.g. Protein-A or Protein-G affinity chromatography or an immunoaffinity protocol comprising use of monoclonal antibodies that bind the expressed fusion protein. The fusion proteins of the present disclosure can be separated and purified by appropriate combination of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electrical charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatograph, methods utilizing a difference in hydrophobicity such as reverse-phase high1005837019 46 performance liquid chromatograph and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA.

[0136] It is preferred that the fusion proteins of the present disclosure be substantially pure. That is, the fusion proteins have been isolated from cell substituents that naturally accompany it so that the fusion proteins are present preferably in at least 80% or 90% to 95% homogeneity (w / w). Fusion proteins having at least 98 to 99% homogeneity (w / w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the fusion protein should be substantially free of contaminants for therapeutic applications. Once purified partially or to substantial purity, the soluble fusion proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.

[0137] As used herein "polypeptide" refers to any sequence of two or more amino acids, regardless of length, post-translation modification, or function. Polypeptides can include natural amino acids and non-natural amino acids. Polypeptides can also be modified in any of a variety of standard chemical ways (e.g., an amino acid can be modified with a protecting group; the carboxy-terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be modified with groups to, e.g., enhance lipophilicity; or the polypeptide can be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications can include the attachment of another structure such as a cyclic compound or other molecule to the polypeptide and can also include polypeptides that contain one or more amino acids in an altered configuration (i.e., R or S; or, L or D).

[0138] As used herein, an ECM binding polypeptide binds to one or more or all extracellular matrix proteins selected from the group consisting of: collagen, vitronectin, fibronectin, tenascin C, osteopontin, fibrinogen, heparan sulfate, and heparan sulfate proteoglycans; in preference to other proteins. In one embodiment an ECM binding polypeptide that binds to one or more or all ECM proteins binds with high affinity, preferably with a dissociation constant (KD) of less than about 300 nM, or less than about 200 nM, or less than about 100 nM, or less than about 40 nM, or less than about 25 nM or less than about 15 nM or less than about 10 nM.1005837019 47

[0139] By “derivative” is meant an agent or active that has been derived from the polypeptide or fusion protein by modification of the amino acid sequence, or, for example by conjugation or complexing or expression (eg, as a fusion protein) with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term “derivative” also includes within its scope alterations that have been made to a parent sequence including additions, or deletions that provide for functionally equivalent or functionally enhanced molecules.

[0140] By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0141] The term “subject,” includes patient, and refers to any subject of medical or veterinary interest. Subjects may be a vertebrate subject, such as mammalian subject (e.g, bovines, pigs, dogs, cats, equine, lama, camelids, etc.), non-mammals, reptiles birds, fish. The subject includes a human, for whom prophylaxis or therapy is desired. The subject may be in need of prophylaxis or treatment for a wound care, sarcopenia or other pathology, disease, disorder or condition associated with tissue degeneration or injury, including as described elsewhere herein.

[0142] The term “polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.

[0143] The term sequence “identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present disclosure, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the1005837019 48 reference manual accompanying the software. Amino acid sequence identity may also be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk / Tools / emboss / align / . This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix “Blosum62” is utilized for amino acid sequences and the default matrix.

[0144] The term sequence “similarity” refers to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions as defined in Table 2 above. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0145] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rded., vol.1- 3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001); and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates).

[0146] Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Current Protocols in Immunology, ed. Coligan et al., Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates); Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, Mass., (2007); Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl, (2006); Medical Immunology, 6thed., edited by Gabriel Virella, Informa Healthcare Press, London, England, (2007); and Harlow and Lane ANTIBODIES: A Laboratory Manual, Second edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014).

[0147] Conventional methods of gene transfer and gene therapy may also be adapted for use in the present disclosure. See, e.g., Gene Therapy: Principles and1005837019 49 Applications, ed. T. Blankenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; Viral Vectors for Gene Therapy: Methods and Protocols, ed. Otto-Wilhelm Merten and Mohammed Al-Rubeai, Humana Press, 2011; and Nonviral Vectors for Gene Therapy: Methods and Protocols, ed. Mark A. Findeis, Humana Press, 2010. Amino Acids.2018 Jan; 50(1):39-68. doi: 10.1007 / s00726-017-2516-0. Epub 2017 Nov 28.

[0148] An orthologue as used herein is the equivalent of the protein or peptide used in the fusion protein whose sequence is derived from a non-human animal, preferably a mammal, such as a mouse, rat or pig.

[0149] Functional homologues or variants may be derived by insertion, deletion or substitution of amino acids in, or chemical modification of, the native carboxyl-terminal sequence. Amino acid insertion variants include amino and / or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertion amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletion variants are characterised by the removal of one or more amino acids from the sequence.

[0150] Substitution amino acid variants are those in which at least one amino acid residue in the sequence has been replaced by another of the twenty, primary protein amino acids, or by a non-protein amino acid. In one embodiment substitutions are with conservative amino acids.

[0151] A “conservative amino acid substitution” is one in which the naturally or non- naturally occurring amino acid residue is replaced with a naturally or non-naturally occurring amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., Lys, Arg, His), acidic side chains (e.g., Asp, Glu), uncharged polar side chains (e.g., Gly, Asn, Gln, Ser, Thr, Tyr, Cys), nonpolar side chains (e.g., Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), beta-branched side chains (e.g., Thr, Val, Ile) and aromatic side chains (e.g., Phe, Trp, His). Thus, a predicted nonessential amino acid residue, for example, may be replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or1005837019 50 other properties (e.g.2-thienylalanine for phenylalanine). A full amino acid sub- classification is set out in Table 1 and exemplary substitutions are set out in Table 2. Table 1. Amino acid sub-classification Sub-classes Amino acids Acidic Aspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic: Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine, Histidine Small Glycine, Serine, Alanine, Threonine, Proline Polar / neutral Asparagine, Histidine, Glutamine, Cysteine, Serine, Threonine Polar / large Asparagine, Glutamine Hydrophobic Tyrosine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan Aromatic Tryptophan, Tyrosine, Phenylalanine Residues that influence chain Glycine and Proline orientation Table 2. Exemplary and Preferred Amino Acid Substitutions Original residue Exemplary substitutions Preferred substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu, Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser1005837019 51 Original residue Exemplary substitutions Preferred substitutions Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

[0152] The polypeptides and peptides of the present disclosure comprise amino acids. Reference to “amino acid” includes naturally occurring amino acids or non- naturally occurring amino acids.

[0153] Peptide compounds are generally and conventionally modifiable by addition of moieties, flanking peptide residues, and substitutions within understood parameters. Peptides can furthermore comprise routine modified backbones, side chains, peptide bond replacements, and terminal modifications using standard peptide chemistries.

[0154] The amino acids incorporated into the amino acid sequence described herein may be L-amino acids, D-amino acids, L- β -homo amino acids, D- β -homo amino acids or N-methylated amino acids, sugar amino acids, and / or mixtures thereof. Non-natural amino acids may not be recognised by proteases and may therefore alter the half-life. In one embodiment, the D-retro inversion sequence is employed.

[0155] Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid. Examples of unnatural amino acids and derivatives include, but are not limited to, 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, nor leucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and / or D-isomers of amino acids.

[0156] In one embodiment, peptides are modified to enhance their pharmacodynamics properties using art recognised modifications. Peptides may be substituted, such as alanine substituted, or substituted with cross linkable moieties and / or linked. Suitable residues may comprise additional alpha-carbon substitutions selected from hetero- lower alkyl, hetero- methyl, ethyl, propyl and butyl. Peptide bond replacements such as trifluoroethylamines are used to produce more stable and active peptidomimetics.1005837019 52

[0157] In preferred embodiments, fusion peptide is amidated at the C-terminus. Suitable amidation methods are known to those skilled in the art. Constructs / Vectors

[0158] “Expression construct” and “vector” are used interchangeably herein. A construct or vector for expressing a polypeptide or fusion protein described herein in a host cell can comprise one or more DNA regions comprising a promoter operably linked to a nucleotide sequence encoding the peptide. The promoter can be inducible or constitutive. Examples of suitable constitutive promoters include, e.g., an immediate early cytomegalovirus (CMV) promoter, an Elongation Growth Factor - la (EF-la) gene promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. A promoter may be tissue-specific or cell-specific for tropic expression. An inducible or ‘switchable’ promoter may be used for tunable, temporal expression.

[0159] A construct or vector of the disclosure may further comprise one or more control sequences to control, enhance and / or alter the spatial and / or temporal expression of one or more nucleic acid sequences encoding a polypeptide or fusion protein described herein. Control sequences, also referred to as regulatory sequences, include but are not limited to: transcription initiators, transcription terminators, enhancers, silencers, polyA tail sequences, ribosome entry sequences (eg Kozak), and signal sequences (such as nuclear localization signals, signals to direct expression to the cell membrane or specific organelle). Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).1005837019 53

[0160] The expression constructs may be generated by any suitable method including recombinant or synthetic techniques, utilizing a range of vectors known and available in the art such as plasmids, bacteriophage, baculovirus, mammalian virus, artificial chromosomes, among others. The expression constructs can be circular or linear, and should be suitable for replication and integration into eukaryotes. Non viral vectors include, but are not limited to, plasmids, bacmids, cosmids, linear DNA (eg doggyboneDNA (dbDNA)). Viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses (AAVs, including recombinant AAVs (rAAVs) and self-complementary AAVs (scAAVs), herpes viruses and lentiviruses. Preferably, the viral vector is unable to self-replicate. A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a host cell or a target cell of a subject. A number of viral and non-viral vector systems are known in the art.

[0161] In a specific embodiment of the present disclosure, where the fusion protein or TSP-1 polypeptide is provided as a nucleic acid encoding the fusion protein or TSP-1 polypeptide, the nucleic acid may be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular (e.g., by use of a retroviral vector, an AAV vector, by direct injection, by use of microparticle bombardment, by coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide or other intracellular targeting moiety. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression.

[0162] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0163] Nucleic acids are said to have “5’ ends” and “3’ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’1005837019 54 phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements.

[0164] “Codon optimization” may be used and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding a protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge).

[0165] A nucleic acid molecule as described herein may in any form such as DNA or RNA, including in vitro transcribed RNA or synthetic RNA. Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules and modified forms thereof. A nucleic acid molecule may be single stranded or double stranded and linear or closed covalently to form a circle. The RNA may be modified by stabilizing sequences, capping, and polyadenylation. RNA or DNA and may be delivered as plasmids to express the fusion protein or polypeptide. RNA-based approaches are routinely available.

[0166] The term “RNA” relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2’-position of a β-D-1005837019 55 ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0167] An optimised mRNA based composition could comprise a 5’ and 3’ non translated region (5’-UTR, 3’-UTR) that optimises translation efficiency and intracellular stability as known in the art. In one embodiment, removal of uncapped 5 ‘-triphosphates can be achieved by treating RNA with a phosphatase. RNA may have modified ribonucleotides in order to increase its stability and / or decrease cytotoxicity. For example, in one embodiment, in the RNA, 5-methylcytidine is substituted partially or completely, for cytidine. In one embodiment, the term “modification” relates to providing an RNA with a 5’-cap or 5’-cap analog. The term “5’-cap” refers to a cap structure found on the 5’-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5’ to 5’ triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position. The term “conventional 5’- cap” refers to a naturally occurring RNA 5’-cap, preferably to the 7-methylguanosine cap. The term “5’-cap” includes a 5’-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and / or enhance translation of RNA. Providing an RNA with a 5’-cap or 5’-cap analog may be achieved by in vitro transcription of a DNA template in the presence of said 5’-cap or 5’-cap analog, wherein said 5’-cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5’-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0168] A further modification of RNA may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5’- or 3 ‘-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said1005837019 56 RNA, for example, the exchange of the existing 3’-UTR with or the insertion of one or more, preferably two copies of a 3’-UTR derived from a globin gene, such as alpha2- globin, alphal-globin, beta-globin. RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence. In order to increase stability and / or expression of the RNA it may be modified so as to be present in conjunction with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues. In order to increase expression of the RNA it may be modified within the coding region so as to increase the GC-content to increase mRNA stability and to perform a codon optimization and, thus, enhance translation in cells. Modified mRNA may be synthesised enzymatically and packaged into nanoparticles such as lipid nanoparticles and administered, for example intramuscularly. Methods of use

[0169] The fusion proteins, nucleic acids, vectors, cells, and compositions of the invention are useful in wound healing. Most wounds in skin and other organ systems are characterized by a loss of cells and connective tissue matrix from the protective outer layer as well as the underlying layers and tissues. In the case of skin wounds, the epidermis is the outer layer that is lost. The epidermis overlies the dermis as well as deeper structures such as fat, muscle and bone. Closure of large wounds in skin and other organ systems typically requires the production of billions of cells, nutrition through a vascular network and mechanical strength from proteins and glycosaminoglycans present in a nascent extracellular matrix (ECM).

[0170] The term “wound,” for purposes herein, refers broadly to an injury to an organ or organ system. In the case of the skin, the injury may be to the epidermis, the dermis and / or the subcutaneous tissue. Skin wounds may be classified into one of four grades depending on the depth of the wound: i) Grade I: wounds limited to the epithelium; ii) Grade II: wounds extending into the dermis; iii) Grade III: wounds extending into the subcutaneous tissue; and1005837019 57 iv) Grade IV (or full-thickness wounds): wounds wherein bones are exposed (e.g., a bony pressure point such as the greater trochanter or the sacrum). The term “partial thickness wound” refers to wounds that encompass Grades I-III; examples of partial thickness wounds include burn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers. The term “deep wound” includes both Grade III and Grade IV wounds. The methods of the invention are useful for treating all grades of wounds, including chronic and acute wounds.

[0171] The term “chronic wound” may refer to a wound that has not healed, or is not predicted to heal, within 30 days. An example of a chronic wound is an infectious wound or ulcer.

[0172] The term “acute wound” may refer to a wound that has healed, or is predicted to heal, within 30 days. An example of an acute wound are burns, skin grafts, and dehisced surgical wounds.

[0173] The term “promoting wound healing,” for purposes herein, refers to enabling reconstitution of the normal physiologic barrier of an organ or organ system. In the case of skin wounds, promoting wound healing may include the induction of the formation of granulation tissue, and / or the induction of wound contraction, and / or the induction of revascularization, and / or the induction of epithelialization (i.e., the generation of new cells in the epithelium).

[0174] The types of wounds to be treated by the methods of the invention include various kinds of wounds including, but are not limited to: surgical wounds; traumatic wounds; radiation injury wounds; toxic epidermal necrolysis wounds; infectious wounds; neoplastic wounds; full-thickness wounds; partial-thickness wounds; and burn wounds, as well as wounds arising from various types of ulcers, such as skin ulcers, corneal ulcers, arterial obstructive ulcers, continuous pressure-induced decubital and diabetic ulcers, burn ulcers, injury ulcers, radiation ulcers, drug-induced ulcers, post-operative ulcers, inflammatory ulcers, ulcers of the gastrointestinal tract, simple ulcers and other types of angiopathic ulcers, and chronic (intractable) ulcers.

[0175] The methods of various embodiments of the invention may be particularly useful in treating complex wounds or difficult-to-heal wounds. Many factors can adversely affect the wound healing process, including infection, radiated tissue, systemic illness,1005837019 58 medications, patient age, patient health, and the nutritional status of the subject. In addition, any process that impedes peripheral blood circulation, such as arteriosclerosis, prolonged pressure, varicose vein disease, and venous stasis, can adversely affect the delivery of oxygen, nutrients, chemical signals, and appropriate cell types to mediate healing in an injured subject, will impair wound healing. Factors which inhibit wound healing include wound desiccation, medication, such as chemotherapy or steroids, and poor patient health and / or nutrition. Certain partial and full thickness injuries, such as burns, skin grafts, and various types of ulcers, resist repair and produce significant pain and discomfort for the subject.

[0176] The general physical condition of the patient is also important in wound healing. As age increases, the ability to repair injured tissue decreases as the skin becomes thinner and the number of fibroblasts and amount of total skin collagen decrease. Disease states such as alcoholism, anemia, diabetes, malnutrition, shock, and uremia lead to impaired oxygen and nutrient delivery to the wound site, thereby inhibiting the healing process. Also, diseases leading to monocytopenia can significantly impair wound healing.

[0177] Medications used to treat disorders can produce impaired wound healing. Chemotherapy, used to eliminate dividing cells in cancer patients, also suppresses the ability of such a patient to heal wounds, which is also dependent upon new cell growth. Steroids negatively impact all three phases of wound repair, inhibiting the initial inflammatory response, slowing the production of new epithelium and vascular tissue, and weakening the collagen matrix in the scar tissue.

[0178] Bacterial wound infection is a common local cause for prolonged wound healing. Human skin is typically colonized by a number of microorganisms, including Candida albicans, Staphylococcus epidermidis, Staphylococcus aureus, and some Streptococcus strains. Thus, any wound which exposes underlying tissues to the environment becomes infected with at least resident microbial flora. Wounds which are well tended and in highly vascularized tissue resist infection, while those in ischemic tissue are much more susceptible to infection.

[0179] In some embodiments the subject may have a skin wound. In other embodiments the subject may have an ocular condition such as an ocular wound (e.g., dead, damaged or infected ocular cells) in, for example, the corneal epithelium. Thus,1005837019 59 the corneal epithelium may be wounded in a subject having an ocular condition in accordance with the invention.

[0180] Another use for the compositions of the invention is in tissue regeneration (eg skin regeneration, muscle regeneration). Wound healing can be achieved through either tissue repair or tissue regeneration. In comparison to repair, which usually results in a formation of a scar, tissue regeneration provides complete morphological and functional restoration of normal structures. Spontaneous tissue regeneration does not happen in postnatal life; however, it can be at least partially aided by exogenous biological matrices, such as the scaffolds, clinically known as INTEGRA® (Integra LifeSciences, Plainsboro, N.J.), which have been approved by the U.S. Food and Drug Administration for use in massively burned patients and for the treatment of reconstructive defects and chronic wounds. The regenerated skin is mechanically competent, fully vascularized, and sensitive to touch and heat or cold, but is lacking critical skin appendages, e.g., hair follicles and sweat glands. Administration

[0181] In accordance with this disclosure, the fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell disclosed herein can be administered to patients for wound healing or to delay, maintain, or regenerate tissue (eg muscle, skin) in various conditions associated with tissue loss (eg muscle loss; skin wound) or diminished ability to regenerate functionally.

[0182] The fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell may be delivered by injection, by topical or mucosal application, by inhalation or via oral route including modified release modes, over periods of time and in amounts which are effective to stimulate muscle regeneration levels in a subject.

[0183] Administration may be topical or systemic (e.g., parenteral via for example intravenous, intraperitoneal, intradermal, sub cutaneous or intramuscular routes) or targeted.

[0184] In one embodiment, administration of the fusion protein, TSP-1 agonist, TSP-1 polypeptide, or composition thereof is systemic or directly to a wound in need of repair or a tissue in need of regeneration. Sub cutaneous or intramuscular routes may be directly to an affected tissue.1005837019 60

[0185] A polypeptide, fusion protein, nucleic acid, vector or cell described herein can be formulated in the form of ointments, creams, patches, powders, or other formulations suitable for topical formulations. Small molecular weight polypeptide or fusion protein formulations can deliver the agent from skin to deeper muscle tissue. Accordingly, such formulations may comprise one or more agents that enhance penetration of active ingredient through skin. For topical applications, the polypeptide, fusion protein, nucleic acid, or vector can be included in wound dressings and / or skin coating compositions.

[0186] The amount of the agent to be administered may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the nature of the agent and other clinical factors (such as the condition of the subject their weight, age, other conditions, the route of administration and type of composition (cellular, scaffolded, hydrogel baes or oral formulations). The precise dosage to be therapeutically or prophylactically effective and non-detrimental can be determined by those skilled in the art. Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington, the Science and Practice of Pharmacy, 20thEdition, Remington, J., ed. (2000) and later editions.

[0187] Reference to an effective amount includes a therapeutically or physiologically or regeneratively effective amount. A “therapeutically-effective amount” as used herein means that amount of, e.g. fusion protein, TSP-1 agonist, TSP-1 polypeptide, composition, nucleic acid, vector, or cell, which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. For example, an amount of a polypeptide or fusion protein administered to a subject that is sufficient to produce a statistically significant, measurable wound, skin or muscle repair or regeneration. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject’s history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.

[0188] As used herein, the term “administer” refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the active ingredient or composition or medicament at a desired site such that desired effect1005837019 61 is produced. Routes of administration suitable for the instant compositions with vary depending upon its format and include both local and systemic administration. Generally, local administration results in more polypeptide or fusion protein or cell treated with a polypeptide or fusion protein being delivered to a specific location as compared to the entire body of the subject, whereas, systemic administration results in delivery to essentially the entire body of the subject. One method of local administration is by topical or intramuscular injection.

[0189] As described herein regeneration of muscle tissue by the present methods may be associated with minimal fibrosis. Specifically, the methods and agents described herein may reduce and / or inhibit formation of scar-like tissue in the damaged or non-regenerating or atrophying tissue (eg muscle tissue). Accordingly, in some embodiments, formation of scar-like tissue formation in the damaged tissue is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% relative to a control without the present agents. Adipose deposition may be similarly reduced.

[0190] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0191] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some instances ±5%, in some instances ±1%, and in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0192] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed1005837019 62 subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Examples Example 1: Materials and methods

[0193] Animal ethics

[0194] Animal experiments were approved by the Monash Animal Research Platform ethics committee and the Animal Research Committee of the Research Institute for Microbial Diseases of Osaka University (approval numbers 13294, 13335, 17075, 14013, 23006).

[0195] Animals

[0196] Wild-type C57BL / 6J mice were from the Monash Animal Research Platform. Sperm from Nav1.8Cre+ / +mice (B6.129-Scn10atm2(cre)Jnw / H B6, stock ID EM:04582, European Mouse Mutant Archive) were used for in vitro fertilisation to generate Nav1.8Cre+ / -mice on a C57BL / 6J background. Rosa26DTA+ / +(B6.129- Gt(ROSA)26Sortm1(DTA)Mrc / J, strain 010527, Jackson Laboratory) were maintained on a C57BL / 6J background. To delete sensory neurons expressing Nav1.8, Nav1.8Cre+ / -mice were bred with Rosa26DTA+ / +to generate Nav1.8Cre+ / - / Rosa26DTA+ / -mice. Nav1.8Cre- / - / Rosa26DTA+ / -littermates were used as controls. For visualising Nav1.8+ neurons, Rosa26tdTreporter mice (B6.Cg-Gt(ROSA)26Sortm14strain 007914, Jackson Laboratory) were bred with Nav1.8Cre+ / +mice to generate Nav1.8Cre+ / - / Rosa26Tdt+ / -. Leprdb / db(BKS.Cg-Dock7m+ / +Leprdb / J, strain 000642) were obtained from Jackson Laboratory. Mice were bred as heterozygotes to generate Leprdb / dband Leprdb / +littermates. B6.129S2-Ramp1<tm1.2Tsuj>sperm was kindly provided by Prof. Kazutake Tsujikawa (Graduate School and School of Pharmaceutical Sciences, Osaka University) and used for in vitro fertilisation to generate Ramp1fl / +mice. Ramp1- / -mice were generated by crossing Ramp1fl / flmice with CAGcre mice (C57BL / 6-Tg(CAG-cre)13Miya, RIKEN BioResource Research Center, strain 09807). Specific deletion of Ramp1 in myeloid cells (LysMCre+ / - / Ramp1fl / flmouse) was done by crossing Ramp1fl / flwith LysMCre+ / +mice (B6.129P2-Lyzs<tm1(cre)Ifo>, RIKEN BioResource Research Center, strain 02302). LysMCre+ / -littermates were used as controls. To obtain mice constitutively1005837019 63 expressing tdTomato, Rosa26tdTmice were crossed with B6.C-Tg(CMV-cre)1Cgn / J mice from Jackson Laboratory (strain 006054).

[0197] Full-thickness skin wound model

[0198] Male mice (10-12-weeks-old) were used for most experiments except for experiment with CGRP variant delivery in which female mice were used (10-12-weeks- old, non-diabetic; 12–14-weeks-old, diabetic). Full-thickness punch-biopsy wounds (5 mm in diameter) were created while under isoflurane anaesthesia as described previously (Martino, M. M. et al. Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing. Science 343, 885-888 (2014); Tan, J. L. et al. Restoration of the healing microenvironment in diabetic wounds with matrix-binding IL-1 receptor antagonist. Commun Biol 4, 422 (2021)). For analgesia, mice received subcutaneous administration of 0.1 mg / kg buprenorphine. In non-diabetic mice, wounds were covered with a round seal spot plaster (22.5 mm, Livingstone International, Australia) secured with 3M surgical tape. In experiments involving CGRP delivery, a nylon ring (Zenith 5”16" And M8 Nylon Washer, Australia) was attached with superglue (UHU GmbH & Co. KG, Germany) to prevent wound contraction. Wounds received topical treatment with either 10 μl of saline (PBS) or a CGRP variant in PBS. Solutions were applied in two different dosages: 250 ng of CGRP or equimolar eCGRP on D1 post-injury for the low dose, and 500 ng of CGRP or equimolar eCGRP on D1 and D4 post-injury for the high dose. For diabetic mice, four wounds were created and treated with PBS or CGRP variant in PBS (500 ng CGRP or equimolar eCGRP) on D1 and D3.

[0199] Volumetric muscle loss

[0200] Non-diabetic (10-12-weeks-old) and diabetic (12-14-weeks-old) male mice underwent isoflurane anaesthesia. For analgesia, mice received subcutaneous administration of 0.1 mg / kg buprenorphine. A 1 cm unilateral incision was made, exposing the fascia. Muscle injuries were created either with a 3 mm biopsy punch or by excising a 3 mm x 5 mm segment of the quadriceps, including the rectus femoris muscle. In experiments involving CGRP delivery, muscle defects were covered with a fibrin matrix (40 µl total, 8 mg / ml fibrinogen (Enzyme Research Laboratories), 12 U / ml bovine thrombin (Sigma), 5 mM CaCl2, and 17 μg / ml aprotinin (Roche, Sigma)) containing CGRP or eCGRP (250 ng or 1 μg for non-diabetic mice and 1 μg for diabetic mice). The incision site was sutured with absorbable sutures.1005837019 64

[0201] Adoptive transfer of bone marrow cells

[0202] Bone marrow cells (1 × 107) from 6-week-old Ramp1– / –or wildtype C57BL / 6J mice were intravenously injected into lethally irradiated 6-week-old recipient wildtype or Ramp1– / –mice that received 10 ml / l neomycin sulfate for 2 weeks post-irradiation. Skin or muscle defect surgeries were performed 6 weeks after transplantation.

[0203] Histological analysis

[0204] Skin wounds were harvested using an 8 mm biopsy punch, fixed in 10% formalin at room temperature for 24 hours, cut at the edge of the wounds, embedded in paraffin and sectioned at 4 µm until the centre of the wound was passed. Re- epithelialisation was measured through histomorphometric analysis. Slides were stained with haematoxylin and eosin, and the wound's centre was determined by measuring the distance between the panniculus carnosus muscle gap using Aperio ImageScope Viewer (Leica Biosystems). Closure was calculated as the ratio of epidermis closure to the length of the panniculus carnosus gap. Muscle injury sites, including the proximal and distal quadriceps segments, were harvested, fixed in 10% formalin solution for 24 hours, embedded in paraffin, and sectioned at 4 μm thickness for 5 depths, starting from the patella's edge, passing the wound's centre, up to the proximal end of the defect site. Cross-sections were stained with Masson's Trichrome. Muscle regeneration was determined by averaging the percentage of blue-stained fibrotic area (normalised to the total area) and the remaining non-fibrotic muscle area across 5 tissue section depths, using Aperio ImageScope.

[0205] Immunohistochemistry for neuropeptides, TSP-1, and myeloid cells

[0206] Tissues and DRGs (L1-L6 vertebrae) were fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and embedded in O.C.T. compound for 10 μm cryo- sections. Sections were stored at -20°C, thawed, permeabilized, and blocked with 1% BSA, 10% NGS or NDS in PBS for 1 hour. Sudan Black B solution was applied for 10 minutes. For neuropeptide detection, primary antibodies were added in staining buffer (0.5% BSA, 5% NGS or 0.5% BSA, 5% NDS in PBS) overnight at 4°C. The primary antibodies included rabbit anti-CGRP (66.7 μg / ml, Sigma, #C8198), rabbit anti- substance P (1:500, Thermo Fisher Scientific, #20064), rabbit anti-VIP (1:500, Thermo Fisher Scientific, #20077), and goat anti-galanin (1 μg / ml, Abcam, #99452). For TSP-11005837019 65 detection, sections were incubated with AffiniPure Fab Fragment goat anti-mouse IgG (H+L) at 100 μg / ml (Jackson ImmunoResearch Labs, #115-007-003) in PBS for 2 hours at room temperature, followed by mouse anti-thrombospondin-1 (5 μg / ml, Thermo Fisher Scientific, #14-9756-82). For myeloid cell detection, slides were incubated with rat anti-mouse CD11b (5 μg / ml, Thermo Fisher Scientific, #14-0112-82). Sections were washed and incubated with respective secondary antibodies for 1 hour at room temperature. The secondary antibodies included F(ab')2-Goat anti-Rabbit IgG Alexa Fluor 488 (2.6 μg / ml, Thermo Fisher Scientific, #A-11070), donkey anti-goat IgG Alexa Fluor 488 (2.6 μg / ml, Thermo Fisher Scientific, #A-11055), goat anti-mouse IgG Alexa Fluor Plus 488 (2.6 μg / ml, Thermo Fisher Scientific, #A48286TR), and goat anti-rat IgG Alexa Fluor Plus 594 (2.6 μg / ml, Thermo Fisher Scientific, #A48264). Counterstaining with DAPI for 10 minutes and mounting with Fluoroshield followed. Imaging was done using Leica DMi8 fluorescent microscope and Leica SP8 inverted confocal microscope.

[0207] Evaluation of neuropeptide expression

[0208] Skin and muscle samples from male Nav1.8Cre+ / - / Rosa26Tdt+ / -mice (10-12-week- old) were immunostained as detailed above, imaged on a Leica DMi8 fluorescent microscope, and processed using Fiji (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682 (2012)). Binary images were created with an optimal threshold, and overlapping areas were determined by combining region of interest binary images. Area fraction values, indicating neuropeptide expression in Nav1.8+ nerves, were calculated based on pixel ratios and converted using a built-in scale bar (Price, T. J. & Flores, C. M. Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse. J Pain 8, 263-272 (2007); Adler, J. & Parmryd, I. Colocalization analysis in fluorescence microscopy. Methods Mol Biol 931, 97-109 (2013)).

[0209] Immunofluorescence for Ki-67 and K14

[0210] Paraffin sections underwent 20-minute antigen retrieval in 10 mM sodium citrate buffer (pH 6.0), followed by PBS washes and 5-minute permeabilization (0.2% Triton X-100 in PBS). Blocking with 10% NGS in 1% BSA / PBS occurred for 2 hours, and endogenous IgG was blocked with unconjugated affinity-purified F(ab) fragment1005837019 66 anti-mouse IgG (H+L) (Jackson ImmunoResearch, #AB_2338476) for 1 hour at room temperature. Staining overnight at 4 °C utilized rat anti-mouse Ki-67 (5 μg / ml, Thermo Fisher Scientific, #14-5698-82) and mouse anti-mouse cytokeratin 14 (4 μg / ml, Thermo Fisher Scientific, #MA5-11599) in 1% NGS in PBS with 0.1% BSA. After PBS-T washes, incubation with secondary antibodies occurred: goat anti-mouse Alexa Fluor-647 (2 μg / ml, Thermo Fisher Scientific, #A-21235) and goat anti-rat Alexa Fluor-488 (2.67 µg / ml, Thermo Fisher Scientific, # A48262TR) for 1 hour at room temperature, followed by PBS-T wash. Counterstaining with DAPI (1 μg / ml) for 10 minutes at room temperature preceded mounting with Fluoroshield.

[0211] TUNEL assay

[0212] The In Situ Cell Death Detection Kit, TMR red (Roche, #12156792910) was used, following the manufacturer's instructions on muscle and skin tissue cryosections. To detect CD11b+ cells, sections were incubated overnight at 4°C with rat anti-mouse CD11b (5 μg / ml, M1 / 70, Thermo Fisher Scientific, #14-0112-82) in staining buffer. After PBS-T washes, sections were incubated with a AlexaFluor-488 goat anti-rat antibody (2.67 µg / ml, Thermo Fisher Scientific, # A48262TR), washed with PBS-T, and counterstained with DAPI (1 μg / ml) before mounting with Fluoroshield. Two tissue section levels were evaluated per sample to determine the percentage of TUNEL+ apoptotic cells over total CD11b+ cells, examining three fields per section within the injury site.

[0213] Fibroblast, keratinocytes, myoblasts, and endothelial cell maintenance

[0214] Human umbilical vein endothelial cells (HUVECs; Sigma, 200P-05N) cultured in EGM-2 medium (Lonza, CC-4176) up to 3 passages, and primary mouse fibroblasts from C57BL / 6J mouse tails26 (passages 2-3) were used. MCDB-131 medium (Thermo Fisher Scientific) with 100 mg / ml penicillin / streptomycin and 2 mM glutamine was employed for proliferation assays. C2C12 mouse myoblasts (CellBank Australia) were cultured in a 1:1 ratio of DMEM to F10 medium (2 mM glutamine, 10% FBS, 100 units / ml penicillin / streptomycin). HaCaT keratinocytes (gifted by Professor Richard Boyd, Monash University) were cultured in DMEM without Ca2+ and Mg2+ (2 nM glutamine, 10% Chelex-treated FBS, 0.03 nM calcium chloride, 100 units / ml penicillin / streptomycin) for at least 3 passages. For proliferation assays, FBS was reduced to 2% or kept at 10%, and cells were starved for 24 hours. Detached with1005837019 67 TrypLE, cells were seeded (2000 cells / well for HUVECs, fibroblasts, HaCaTs; 1000 cells / well for C2C12) and treated with CGRP (1 or 20 nM) or 10-20% FBS. Incubation for 48 hours (fibroblasts, C2C12) or 72 hours (HUVECs, keratinocytes) at 37°C with 5% CO2 followed. Proliferation was determined using the CyQUANT Cell Proliferation Assay (Invitrogen), presented as fold change over basal proliferation (medium only).

[0215] Flow cytometry with tissue samples

[0216] Skin wounds were harvested using an 8 mm biopsy punch, and muscle defects were dissected to isolate the quadriceps. Samples were minced with scissors and subjected to two serial digestions with collagenase XI (1 mg / ml) at 37°C (30 minutes for skin, 20 minutes for muscle). After the first digestion, the supernatant was collected and mixed with neutralisation buffer (DMEM / F12 with 10% FBS and 5 mM EDTA). The first collection was kept on ice, and fresh collagenase XI was added to the undigested tissue for the second digestion. Digestion mixtures were passed through a 70 μm cell strainer and stained with LIVE / DEAD Fixable Aqua dye (Thermo Fisher Scientific, 1:400 dilution in PBS) for 20 minutes on ice. Cells were incubated with TruStain FcX anti-CD16 / 32 (10 μg / ml; clone 93, BioLegend) diluted in staining buffer (5% FBS and 2 mM EDTA in PBS) for 20 minutes and subsequently incubated with primary antibodies in staining buffer for a further 30 minutes on ice.

[0217] The following anti-mouse antibodies from BioLegend were used: FITC anti- CD11b (clone M1 / 70, 6.6 μg / ml) or BV711 anti-CD11b (clone M1 / 70, 2 μg / ml); PE anti- F4 / 80 (clone BM8, 4 μg / ml); BV421 anti-Ly6G (clone 1A8, 2 μg / ml); BV711 anti-Ly6C (clone HK1.4, 1 μg / ml) or FITC anti-Ly6C (clone HK1.4, 5 μg / ml); PE-Cyanine7 anti- CD206 (clone C068C2, 2.6 μg / ml); PE-Cyanine7 anti-CD3 (clone 17A2, 4 μg / ml); APC anti-CD4 (clone GK1.5, 2 μg / ml); BV421 anti-CD8 (clone 53-6.7, 2 μg / ml); PE anti-TCR β (clone H57-597, 2 μg / ml); APC / Fire 750 anti-TCR γ / δ (clone GL3, 2 μg / ml); PerCP anti-CD11c (clone N418, 2 μg / ml); APC / Fire 750 anti-MHC Class II (clone M5 / 114.15.2, 2 μg / ml). Cells were washed once with a large volume of staining buffer before analysis with BD LSR Fortessa X-20 and FlowJo software (BD Biosciences).

[0218] Mouse bone marrow neutrophil and monocyte isolation

[0219] Bone marrow cells were flushed from femora and tibiae of C57BL / 6J mice (8- 12-weeks-old) with HBSS without Ca2+ and Mg2+ containing 2% FBS and 1 mM EDTA.1005837019 68 Cell suspension was passed through a 70 μm strainer. Next, EasySep Mouse Neutrophil Enrichment Kit or EasySep Mouse Monocyte Isolation Kit (STEMCELL Technologies) was used to isolate neutrophils or monocytes according to the manufacturer’s instructions. Neutrophils were resuspended in RPMI containing 100 units / ml penicillin / streptomycin and 10% FBS for cell migration assay and cell death assay or 2% FBS for efferocytosis. Monocytes were cultured in DMEM / F12 (Thermo Fisher Scientific) containing 10% FBS, 2-10 ng / ml M-CSF (PeproTech Inc.) and 100 units / ml penicillin / streptomycin for subsequent experiments.

[0220] Neutrophil cell death

[0221] Bone marrow-isolated neutrophils were cultured in RPMI 1640 medium (10% FBS). Cells were incubated with CGRP (1-20 nM, Tocris Bioscience, #83651-90-5) for 10 minutes, followed by treatment with IL-1 (5 ng / ml) and TNF-α (50 ng / ml) for 12 hours at 37°C with 5% CO2 to induce cell death. After 12 hours, cells were washed with PBS and incubated with LIVE / DEAD Fixable Aqua dye (Thermo Fisher Scientific, 1:400 dilution) in PBS on ice for 20 minutes. Cell death was assessed using BD LSR Fortessa X-20 and FlowJo software (BD Biosciences).

[0222] Macrophage death and polarisation marker expression

[0223] Bone marrow cells from 8-12-week-old C57BL / 6J mice were flushed, filtered, and cultured in conditioned medium (DMEM / F12 with 10% heat-inactivated FBS, 100 units / ml penicillin / streptomycin, and 20% L929 fibroblasts-conditioned medium) at 37°C with 5% CO2. After 7-9 days, differentiated macrophages were harvested and seeded in 12-well or 6-well plates. The next day, cells were treated with CGRP (1 or 20 nM, Tocris Bioscience, #83651-90-5) before exposure to mouse IL-1 (5 ng / ml) and TNF-α (50 ng / ml), IL-4 (2 ng / ml) and IL-13 (2 ng / ml), or IL-10 (2 ng / ml) (PeproTech Inc) for 24 or 72 hours. Macrophages were detached with TrypLE (Gibco) containing 3 mM EDTA, stained with LIVE / DEAD Aqua dye for 20 minutes on ice, and incubated with blocking solution (10 μg / ml TruStain FcX anti-CD16 / 32 (clone 93, BioLegend)) before staining with antibodies for 30 minutes on ice.

[0224] Antibodies from BioLegend included PE anti-CD11b (clone M1 / 70, 1 μg / ml), BV711 anti-F4 / 80 (clone BM8, 2 μg / ml), APC anti-CD80 (clone 16-10A1, 0.5 μg / ml), and PE-Cyanine7 anti-CD206 (clone C068C2, 1 μg / ml). For intracellular staining, cells1005837019 69 were fixed and permeabilized using FluoroFix Buffer and Intracellular Staining Permeabilization Wash Buffer (Perm buffer, BioLegend). APC anti-mouse arginase 1 (ThermoFisher, Clone AlexF5, 1 μg / ml) was added to the Perm buffer and incubated with the cells for 30 minutes on ice. After washing with Perm buffer and staining buffer, cells were analysed using BD LSR Fortessa X-20 and FlowJo software (BD Biosciences).

[0225] Neutrophil and macrophage migration assays

[0226] Assays were conducted using 6.5-mm-diameter culture plate inserts (Corning) with 5 μm and 3 μm pore sizes for macrophages and neutrophils, respectively. Macrophages (1 × 105) or neutrophils (3 × 105) in migration media (DMEM / F12 with 0.25% BSA) were added to the inserts. The lower chambers contained migration buffer alone or chemoattractant (mouse CCL-210 ng / ml for macrophages or mouse CXCL1 / KC 150 ng / ml for neutrophils, PeproTech Inc.) with or without CGRP. Cells were allowed to migrate through the insert membrane for 3-4 hours at 37°C with 5% CO2. For macrophages, the inserts were then fixed with 4% paraformaldehyde, and cells on the upper side were removed. DAPI (1 μg / ml) was used to stain cells on the bottom side, and they were counted using a fluorescent microscope. For neutrophils, cells that migrated into the lower chamber were collected and counted using a haemocytometer. The data are presented as the fold change, calculated by dividing the number of cells that migrated in response to treatments by the number of cells that migrated spontaneously (migration media only).

[0227] Efferocytosis assay

[0228] An efferocytosis assay kit (Cayman, #601770) was used following the manufacturer's instructions. Neutrophils were labelled with CFSE and cultured in RPMI with 2% serum for 12 hours to induce cell death. Bone marrow-derived macrophages cultured for 7 days in vitro were seeded at a density of 4 x 105 cells / well in a 6-well plate with DMEM / F12 containing 10% FBS and 100 units / ml penicillin / streptomycin. Prior to the assay, macrophages were pre-treated with CGRP (1 or 20 nM) for 24 hours. Macrophages were harvested, labelled with CytoTell Blue, and then incubated with CFSE-labelled dead / dying neutrophils at different ratios (1:1, 1:2, and 1:4) at 37°C for 15 minutes. The reaction was stopped by washing cells with ice-cold PBS containing 5% FBS and 1 mM EDTA. Cells were analysed with BD LSR Fortessa X-20 and FlowJo1005837019 70 software (BD Biosciences). Macrophages were identified by CytoTell Blue-positive staining, and the efferocytosis index was calculated as the percentage of CFSE-positive cells in CytoTell Blue-labelled macrophages.

[0229] Adoptive transfer of TdTomato+ cells for migration and efferocytosis assay

[0230] TdTomato+ bone marrow cells from CMV-cre / Rosa26TdTomatomale mice (8-12- week-old) were adoptively transferred into Nav1.8Cre / Rosa26DTAand Rosa26DTAmice either directly after red blood cell lysis (migration assay) or following neutrophil isolation (efferocytosis assay). In the migration assay, 1 x 107cells were intravenously delivered D2 after skin or muscle injury. On D3, harvested tissues were analysed via flow cytometry to detect TdTomato+ cells. For the efferocytosis assay, neutrophils were cultured in low serum (2 %) for 24 hours to induce cell death, and 2 x 106apoptotic neutrophils were intradermally injected at the skin wound border on D3 post-injury. After 12 hours, harvested tissues were assessed via flow cytometry to quantify efferocytosis as the number of monocytes / macrophages that had taken up TdTomato+ apoptotic neutrophils. Results were presented as fold change relative to Rosa26DTAcontrol mice.

[0231] RT-PCR, qPCR, and RNA sequencing

[0232] Isolated neutrophils were treated with CGRP (1 nM) in RPMI with 10% FBS and 100 units / ml penicillin / streptomycin for 4 hours at 37°C with 5% CO2. Monocytes, cultured in DMEM / F12 with 10% FBS, 100 units / ml penicillin / streptomycin, and M-CSF (10 ng / ml) for 3 days, had their medium replaced with CGRP (1 nM) for 4 hours at 37°C with 5% CO2. After harvesting, RNA extraction used the RNeasy Plus Micro Kit (Qiagen).

[0233] For RT-PCR and qPCR, reverse transcription used ReverTra Ace (Toyobo Co., Ltd.). RT-PCR primers were: Human_Calcrl 5’-CATGCACATCCTTATGCAC-3’ (SEQ ID NO: 91) and 5’-CCATCACTGATTGTTGACAC-3’ (SEQ ID NO: 92); Human_Ramp1 5’-GCCAGGAGGCTAACTACG-3’ (SEQ ID NO: 93) and 5’- GAAGAACCTGTCCACCTCTG-3’(SEQ ID NO: 94); Mouse_Calcrl 5’- GGTACCACTACTTGGCATTG-3’ (SEQ ID NO: 95) and 5’- GTCACTGATTGTTGACACTG-3’(SEQ ID NO: 96); Mouse_Ramp15’- GACGCTATGGTGTGACT-3’ (SEQ ID NO: 97) and 5’-GAGTGCAGTCATGAGCAG-1005837019 71 3’(SEQ ID NO: 98); Human / mouse_Gapdh primers from Integrated DNA Technologies (#51-01-07-12 and #51-01-07-13).

[0234] PCR products were analyzed by gel electrophoresis. qPCR, using an ABI PRISM 7500 and TaqMan Assay primers from Thermo Fisher Scientific (Thbs1, Mm00449032_g1; Gapdh, Mm99999915_g1), was performed. For RNA sequencing, RNA quantity / quality assessment, library preparation and sequencing were performed at the Medical Genomics Facility, Monash Health Translation Precinct (MHTP). RNA quantity was assessed using Qubit. RNA samples (20 ng) with RIN value ≥ 7 were used for library preparation.

[0235] First strand synthesis was performed using a dT primer which adds the Illumina P7 (5ʹ-CAAGCAGAAGACGGCATACGAGAT-3ʹ (SEQ ID NO: 99)), 8 bp i7 index for each sample and a 10 bp unique molecular identifier (UMI). The modified reverse transcriptase reaction also adds a template switching sequence at the 5’ end of the RNA during the generation of indexed cDNA. These first stand indexed cDNA were pooled and amplified using primers to P7 and the template switch sequence. Illumina P5 was added by tagmentation by Nextera transposase during amplification. Standard Illumina R1 primer was used (main cDNA read), followed by standard i7 primer for index / UMI. R2 primer was present but not used as it will read into poly-A tail. Sequencing was performed on the NextSeq2000 (Illumina), using NextSeq 1000 / 2000 P2 Reagents (100 cycles) v3 (Illumina) in accordance with the Illumina Protocol 1000000109376 v3 Nov2020.

[0236] Demultiplexing and mapping

[0237] Fastq files were processed using the nfCore / RNAseq (v3.2) pipeline using the umi function (Patel, H., Ewels, P. & Peltzer, A. nf-core / rnaseq: nf-core / rnaseq v3.2 - Copper Flamingo. (2021)). Reads were aligned to the Mus musculus GRCm38 reference using STAR aligner (Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013)).

[0238] Reads were quantified using featureCounts producing the raw genes count matrix and various quality control metrics which were summarised in a multiQC report (Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930 (2014);1005837019 72 Ewels, P., Magnusson, M., Lundin, S. & Kaller, M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047-3048 (2016)).

[0239] Raw counts were analysed with Degust (Powell, D. R., Perry, A. & Milton, M. drpowell / degust 4.1.1 (4.1.1). Degust: Interactive RNA-Seq Analysis. Zenodo (2019)), a web tool which performs normalisation using trimmed mean of M values (TMM) (Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11, R25 (2010)). Differential gene expression analysis was performed using limma / voom (Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol 15, R29 (2014)) in Degust and genes with a False-Discovery Rate (FDR)-adj. p value < 0.05 were considered significantly up- or down-regulated. Volcano plots were made using the web tool, VolcaNoseR (Goedhart, J. & Luijsterburg, M. S. VolcaNoseR is a web app for creating, exploring, labeling and sharing volcano plots. Scientific reports 10, 20560 (2020)). Gene ontology enrichment analysis for biological processes was performed with the web tool, ShinyGO 0.77, by providing all up- or down-regulated DEGs separately as the input for each experimental group (Ge, S. X., Jung, D. & Yao, R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics 36, 2628-2629 (2020)).

[0240] CGRP variants

[0241] CGRP and eCGRP were synthesised by ProteoGenix (France). eCGRP was designed to contain PlGF123-141 at the N-terminus followed by a plasmin-sensitive sequence from vitronectin (KGYR) (Chain, D., Kreizman, T., Shapira, H. & Shaltiel, S. Plasmin cleavage of vitronectin. Identification of the site and consequent attenuation in binding plasminogen activator inhibitor-1. FEBS letters 285, 251-256 (1991)). For both variants, a disulphide bond was formed between the two cysteine residues and the C- terminus phenylalanine was amidated. Peptide purity, determined by high performance liquid chromatography, was 89.63% for CGRP and 87.33% for eCGRP.

[0242] Cleavage of eCGRP by plasmin

[0243] CGRP (4 μg) and equimolar eCGRP in 20 μl of PBS (pH 7.2) were incubated with plasmin (0.0005 U / μg, Sigma) at 37°C for 60 minutes. Aprotinin (25 μg / ml, Sigma)1005837019 73 was added for 5 minutes at 37°C to stop plasmin activity. Samples were analysed by SDS-PAGE.

[0244] Retention of CGRP and eCGRP into skin and muscle

[0245] CGRP (1 μg) or an equal molar amount of eCGRP was intradermally administered to the shaved dorsal skin of male 10-12-weeks-old Nav1.8Cre+ / - / Rosa26Tdt+ / -mice, with injection sites marked using a marker. For muscle, CGRP variants were injected into the quadriceps. After 24 hours, collected injection sites underwent cryosectioning and immunostaining. Fiji was used for image analysis, excluding the co-localisation area of CGRP with tdTomato fluorescence, indicating endogenous CGRP expression.

[0246] cAMP quantification

[0247] Freshly isolated neutrophils or bone marrow-derived macrophages (1 million cells) were treated with CGRP (1 nM) in RPMI with 10% FBS for 30 minutes at 37°C with 5% CO2. cAMP levels were quantified using a cAMP ELISA kit from Cayman Chemical (#581001) according to the manufacturers’ instructions.

[0248] Spontaneous pain behaviour assessment

[0249] Eight mice per group (four males, four females, C57BL / 6J, 10-12 weeks old) were acclimated for 1 hour in empty cages. The right hind paw received an intraplantar injection 1 μg of wild-type mouse αCGRP (SEQ ID NO:77), equimolar amount of eCGRP, 0.05% capsaicin (Sigma, M2028), or 20 μl saline. Mice were immediately place in the cage, and their behaviour was recorded. The number of episodes and the time spent licking, shaking, flinching, and lifting the paw were recorded for first 5 minutes and during 5 minutes after 1, 6, 24 and 48 hours.

[0250] Hot plate test

[0251] Eight mice per group (four males, four females, C57BL / 6J, 10-12 weeks old) received an intraplantar injection of 1 μg of wild-type mouse αCGRP (SEQ ID NO:77), equimolar amount of eCGRP, or 20 μl saline in the right hind paw. After 30 minutes, mice were individually placed on a metal hot plate set to 52°C. The latency, from mouse placement on the surface to the first behavioural sign of nociception (e.g., lifting, shaking, licking the hind paw, or jumping), was measured. Mice were immediately1005837019 74 removed from the hot plate after responding or after a 30 second cut-off. The test was repeated after 1, 6, 24 and 48 hours.

[0252] ELISAs for cytokines and MMPs

[0253] Homogenised skin wound and muscle tissues were incubated for 30 minutes on ice in T-PER Tissue Protein Extraction Reagent (10 ml / g of tissue, Thermo Fisher Scientific) containing 1 tablet of protease inhibitor for 7 ml (Roche). Samples were then centrifuged at 10,000 x g for 5 minutes and supernatants were stored at -80°C. Total protein concentration was measured with a Bradford assay (Millipore). Cytokines and MMPs were detected by ELISA from R&D Systems; Mouse IL-1 beta / IL-1F2 DuoSet ELISA; Mouse CCL2 / JE / MCP-1 DuoSet ELISA, Mouse CXCL2 / MIP-2 DuoSet ELISA; Total MMP-2 Quantikine ELISA Kit; Mouse Total MMP-9 DuoSet ELISA.

[0254] siRNA-mediated knockdown

[0255] Macrophages (4 x 105cells / well in a 6-well plate) were transfected with 10 nM scrambled siRNA (Silencer Select Negative Control No.1 siRNA, ThermoFisher, #4390843) or Silencer Select Pre-Designed siRNA against mouse TSP-1 (ThermoFisher, #s124596) using Reduced-Serum Medium (Opti-MEM, Gibco) and Lipofectamine RNAiMAX (Invitrogen, #51985034) for 6 hours. The medium was then replaced with fresh culture medium (DMEM / F12 with 10% FBS). After 24 hours, cells were harvested for the migration assay.

[0256] For the efferocytosis assay, cells were cultured with 1 nM CGRP immediately after transfection. After 24 hours, cells were harvested and co-cultured with dead / dying neutrophils. The evaluation of cell death and polarisation utilised the same methods as those for assessing macrophage death and polarisation marker expression.

[0257] Statistical analysis

[0258] Statistical analyses were performed using GraphPad Prism 9 statistical software (GraphPad, USA). Significant differences were calculated with Student’s t-test, one-sample t-test, and by analysis of variance (ANOVA) when performing multiple comparisons between groups. P < 0.05 was considered as a statistically significant difference.

[0259] Data availability1005837019 75

[0260] RNA sequencing data generated for this study are deposited in NCBI’s Gene Expression Omnibus database (GSE255049). Example 2: Tissue healing without sensory neurons

[0261] To determine the importance of sensory neurons during tissue healing after traumatic injury, the inventors used the Nav1.8Cre / Rosa26DTAmouse. In this mouse, Nav1.8+ dorsal root ganglion neurons, mainly representing nociceptors involved in mechanical, cold, and inflammatory pain, are ablated by the expression of diphtheria toxin fragment A (DTA) Chiu, I. M. et al. Nature 501, 52-57 (2013); Maruyama, K. et al. Cell Rep 19, 2730-2742 (2017); Pinho-Ribeiro, F. A. et al. Cell 173, 1083-1097 e1022 (2018); Yang, D. et al. Cell 185, 4190-4205 e4125 (2022); Pinho-Ribeiro, F. A. et al. Nature 615, 472-481 (2023); Abrahamsen, B. et al. Science 321, 702-705 (2008)). Rosa26DTAlittermates with intact Nav1.8-expressing sensory neurons were used as controls.

[0262] As acute injury models, the inventors chose full-thickness wounds in the dorsal skin (Martino et al.2014; Tan et al.2021) and volumetric muscle loss injuries in quadriceps (Ratnayake, D. et al. Nature 591, 281-287 (2021)). The absence of Nav1.8- expressing sensory neurons resulted in a significant delay in skin wound closure, evident through decreased epithelial migration, a reduced number of proliferative keratinocytes, and wounds remaining largely open after six days (Fig.1a, b, Fig.5a-g). Similarly, muscle regeneration was impaired in Nav1.8Cre / Rosa26DTAmice, characterised by a higher level of fibrotic tissue and less muscle tissue formation (Fig. 1c, d; Fig.5h).

[0263] Nociceptor terminals release neuropeptides in response to danger signals that include inflammatory cytokines commonly present in tissues after acute injury )Donnelly, C. R., Chen, O. & Ji, R. R. Trends Neurosci 43, 822-838 (2020); Udit, S., Blake, K. & Chiu, I. M. Nat Rev Neurosci 23, 157-171 (2022)).. Thus, the inventors investigated the distribution of Nav1.8+ sensory neurons during skin and muscle healing following acute injury.

[0264] To visualise Nav1.8+ sensory neuron distribution and the neuropeptides they express, Nav1.8Cre / Rosa26tdTmice were used, where tdTomato fluorescent protein expression is restricted to Nav1.8+ sensory neurons. Tissue sections of1005837019 76 Nav1.8Cre / Rosa26tdTuninjured skin and muscle showed Nav1.8+ sensory neuron distribution across the epidermis and dermis in skin and nearby connective tissues in muscle (Fig.1e). Following skin and muscle injury, the inventors observed sensory neuron endings growing in clusters into the granulation tissue, establishing innervation within the injured area during the healing process (Fig.1e, Fig.6a). Nav1.8+ neurons exhibited expression of calcitonin gene-related peptide (CGRP) and substance P (SP), and no detectable expression of vasoactive intestinal peptide (VIP) or galanin (GAL) (Fig.1f, Fig.6b-d). Similarly, in the dorsal root ganglia (DRGs) following both skin and muscle injuries, these neurons showed expression of CGRP and SP in cell bodies (Fig. 6e, f). Additionally, the absence of CGRP signal in Nav1.8Cre / Rosa26DTAmice pre- and post-tissue injury underscores Nav1.8+ nociceptors as primary sources of CGRP during skin and muscle healing and suggests that nociceptor-derived CGRP has a pivotal role in these processes (Fig.6g, h). Example 3: CGRP effect on injury immune cells

[0265] To investigate if CGRP mediates neuro-immune interactions that drive tissue healing, mice were generated in which immune cells or non-immune cells were unable to respond to CGRP. Bone marrow cells deficient for receptor activity-modifying protein 1 (Ramp1) (Tsujikawa, K. et al. Proc Natl Acad Sci U S A 104, 16702-16707 (2007)), a co-receptor essential for CGRP signalling, were transplanted into γ-irradiated wildtype or Ramp1- / -mice. Wildtype mice receiving wildtype bone marrow cells or Ramp1- / -mice receiving Ramp1- / -bone marrow cells were controls.

[0266] Skin and muscle healing were severely impaired in wildtype mice reconstituted with Ramp1- / -cells compared to those with wildtype cells. Similarly, transfer of Ramp1- / -cells into Ramp1- / -mice impaired healing, while wildtype cells rescued skin repair and muscle regeneration (Fig.7a-d).

[0267] Considering that myeloid cells such as neutrophils and monocytes / macrophages constitute the majority of immune cells in injured tissues undergoing repair or regeneration and can represent up to 50% of the total wound cells, the inventors hypothesized that CGRP promotes tissue healing by modulating myeloid cells. To investigate this, mice were produced that lacked CGRP signalling in myeloid cells by crossing LysMCremice with Ramp1 floxed (Ramp1fl / fl) mice (Clausen, B. E., Burkhardt, C., Reith, W., Renkawitz, R. & Forster, I. Transgenic Res 8, 265-277 (1999)). Strikingly,1005837019 77 LysMCre+ / - / Ramp1fl / flmice displayed a significant reduction in skin wound closure and muscle regeneration, compared to LysMCre+ / -control mice (Fig.1g-j). The extent of healing impairment was indeed very similar to that observed in Nav1.8Cre / Rosa26tdTmice. Together, these results suggested that CGRP from Nav1.8+ sensory neurons promotes tissue healing via myeloid cells.

[0268] In addition, CGRP treatment in vitro had no significant effect on the proliferation of key cell types involved in skin and muscle healing, including fibroblasts, keratinocytes, myoblasts, and endothelial cells (Fig.7e). Further supporting this observation, the inventors found that keratinocytes and myoblasts have low expression levels of either Ramp1 or calcitonin receptor-like receptor (Calcrl), which together form the CGRP receptor complex (Fig.7f). Lastly, Nav1.8+ nociceptors in granulation tissue were found to be surrounded by CD11b+ myeloid cells, which mainly consist of neutrophils and monocytes / macrophages in the context of tissue healing after acute injury (Fig.7g).

[0269] To gain insights into the effect of Nav1.8+ sensory neurons on immune cells during tissue healing, immune cell dynamics were analysed in injured tissues by flow cytometry. Neutrophils, monocytes / macrophages, dendritic cells, and T cells were assessed, because they constitute the predominant immune populations during tissue healing (Julier, Z., Park, A. J., Briquez, P. S. & Martino, M. M. Acta Biomater 53, 13-28 (2017); Larouche, J., Sheoran, S., Maruyama, K. & Martino, M. MAdv Wound Care (New Rochelle) 7, 209-231 (2018); Tan, J. L. et al. Commun Biol 4, 422 (2021); Peiseler, M. & Kubes, P.. J Clin Invest 129, 2629-2639 (2019); Ratnayake, D. et al. Nature 591, 281-287 (2021))(Fig.8a, b).

[0270] Compared to control mice, Nav1.8Cre / Rosa26DTAmice exhibited an increased number of neutrophils and pro-inflammatory Ly6Chighmonocytes / macrophages in skin and muscle at D3 post-injury (Fig.2a). Likewise, macrophages in Nav1.8Cre / Rosa26DTAmice showed a delayed polarisation towards an anti-inflammatory / pro-repair (M2-like) phenotype in both tissues, characterised by a lower expression of CD206, a well- established M2-like macrophage marker, at later stages of the healing process (Fig.2a). Although the total number of monocytes / macrophages observed 3D post-injury in the skin was lower in Nav1.8Cre / Rosa26DTAmice compared to controls (Fig.2a), this phenomenon was not evident in muscle, a difference that may be attributed to either the1005837019 78 lower density of Nav1.8+ nociceptors in injured muscle or variations in the dynamic accumulation of monocytes / macrophages between the two tissues.

[0271] In both skin and muscle tissues, no major differences were observed in dendritic cells except for a lower number in the skin of Nav1.8Cre / Rosa26DTAat a late time point (D10 post-injury).

[0272] Some variations were noted in the counts of CD4, gamma-delta (γδ), and cytotoxic (CD8) T cells 3D post-injury (Fig.8c). Nevertheless, while these cell populations are recognised for their role in modulating tissue healing, their proportions were considerably lower in both Rosa26DTAand Nav1.8Cre / Rosa26DTA. They constituted 50-20 times fewer cells compared to neutrophils and monocytes / macrophages in skin and muscle, respectively (Fig.8d). Thus, while CGRP signalling on T cells may affect tissue healing to some extent, the profound impairment of tissue healing upon conditional Ramp1 knockout in myeloid cells suggests nociceptor-derived CGRP primarily promotes tissue healing after acute injury by influencing myeloid cells (Fig.1g- j).

[0273] Overall, the data demonstrated that nociceptor absence resulted in an increase in neutrophils and inflammatory monocytes / macrophages. This in turn delayed the transition towards an anti-inflammatory and pro-repair phase, leading to impaired tissue healing.

[0274] Example 4: Effect of CGRP on neutrophils and monocytes / macrophages in vitro

[0275] The increased number of neutrophils and inflammatory monocytes / macrophages observed in the absence of Nav1.8+ sensory neurons, along with the delayed transition of macrophages towards an anti-inflammatory / pro-repair phenotype, suggested that CGRP may regulate these cells through multiple mechanisms. Thus, the inventors investigated the effect of CGRP on neutrophils and macrophages in vitro.

[0276] First, it was verified that these cells strongly express CGRP receptor subunits (Fig.9a, b). Then, migration assays were conducted and it was observed that CGRP severely inhibited neutrophil and macrophage migration towards common chemokines found in wounds (CXCL1 for neutrophils and CCL2 for macrophages) (Fig.2b). Cell1005837019 79 migration was also inhibited to some extent in the absence of chemokines. However, CGRP did not inhibit cell migration in Ramp1- / -neutrophils and macrophages, confirming that CGRP signals via RAMP1 / CALCRL to mediate its effects on these cells (Fig.9e).

[0277] Next, the inventors investigated CGRP effect on neutrophil and macrophage viability. CGRP triggered increased neutrophil and macrophage death in the presence of the inflammatory cytokines IL-1 and TNF-α, typically found in acute injuries. Interestingly, CGRP did not promote macrophage death when cells were cultured without inflammatory cytokines or with cytokines that induce an M2-like polarisation, suggesting that CGRP elicits its effect on macrophage death during the inflammatory phase of tissue healing (Fig.2c, Fig.9c).

[0278] Subsequently, it was explored if CGRP influences neutrophil clearance by regulating macrophage efferocytosis. Stimulation with CGRP resulted in a drastic increase of macrophage efferocytosis, both in the absence and presence of inflammatory cytokines (IL-1 and TNF-α) (Fig.2d). Additionally, CGRP had no direct impact on macrophage Ly6C expression in vitro despite our earlier observation of an increased number of pro-inflammatory monocytes / macrophages (Ly6Chigh) in injured tissues of mice lacking nociceptors (Fig.9d). This suggested that the high number of Ly6Chighmonocytes / macrophages was likely a consequence of delayed neutrophil clearance and / or macrophage polarisation.

[0279] Lastly, it was tested if CGRP accelerates macrophage polarisation towards an M2-like phenotype. In the presence of typical anti-inflammatory cytokines (IL-4 / IL-13 and IL-10), CGRP treatment increased levels of the M2-like markers CD206 and arginase-1, respectively, indicating that CGRP accelerates polarisation into an anti- inflammatory / pro-repair phenotype (Fig.2e). Example 5: Effects of CGRP on neutrophil and monocyte / macrophages in vivo

[0280] To validate inhibitory migration effects of CGRP in vivo, an adoptive transfer model was used in which Tdtomato+ bone marrow cells were systemically administered into Rosa26DTAcontrol and Nav1.8Cre / Rosa26DTAmice following skin and muscle injuries. One day after the transfer, relative accumulation of Tdtomato+ neutrophils and monocytes / macrophages in the injured tissues was assessed by flow cytometry.1005837019 80 Compared to Rosa26DTA, Nav1.8Cre / Rosa26DTAmice showed a significant increase in neutrophils and monocytes / macrophages into injured skin and muscle, suggesting that migration of these cell types into injured tissues is considerably increased (Fig.2f).

[0281] For efferocytosis validation in vivo, dead / dying Tdtomato+ neutrophils were intradermally injected at the border of skin wounds. The relative number of monocytes / macrophages positive for Tdtomato was assessed by flow cytometry and was found be significantly lower in Nav1.8Cre / Rosa26DTAmice, demonstrating an impairment of efferocytosis (Fig.2f).

[0282] Finally, to assess neutrophil and macrophage cell death in vivo, a TUNEL assay was performed on injured tissue sections D3 post-injury. A lower number of CD11b+ cells were TUNEL positive in Nav1.8Cre / Rosa26DTAmice (Fig.2g).

[0283] Together, these data support a model where nociceptor-derived CGRP promotes tissue healing by tightly regulating neutrophil and monocyte / macrophage dynamics, functions, and phenotypes in injured tissues, resulting in a faster transition from a pro-inflammatory to a pro-healing phase. CGRP inhibits neutrophil and monocyte / macrophage migration into injured tissues and may further enhance neutrophil and inflammatory macrophage death in the presence of inflammatory cytokines. Meanwhile, CGRP increases neutrophil clearance by stimulating macrophage efferocytosis, which, together with a direct effect of CGRP on macrophage polarisation, supports macrophages switching towards an anti-inflammatory / pro-repair phenotype. This model aligns well with the function of neutrophils and macrophages during tissue healing. For instance, excessive mobilisation of neutrophils and inflammatory monocytes / macrophages is generally associated with impaired wound healing (Julier, Z., Park, A. J., Briquez, P. S. & Martino, M. M. Acta Biomater 53, 13-28 (2017); Larouche, J., Sheoran, S., Maruyama, K. & Martino, M. M. Adv Wound Care (New Rochelle) 7, 209-231 (2018); Tan, J. L. et al. Commun Biol 4, 422 (2021); Peiseler, M. & Kubes, P. J Clin Invest 129, 2629-2639 (2019); Krzyszczyk, P., Schloss, R., Palmer, A. & Berthiaume, F. Front Physiol 9, 419 (2018)).. Enhanced clearance rate of pro-inflammatory cells within injured tissues is also well-known to prevent excessive inflammation and facilitate the transition towards the pro-repair phase. Indeed, impaired tissue healing has been linked to delayed inflammatory cell death and a decrease in macrophage efferocytosis capability (Chazaud, B. Trends Immunol 41, 481-492 (2020);1005837019 81 Maschalidi, S. et al. Nature 606, 776-784 (2022); Rodrigues, M., Kosaric, N., Bonham, C. A. & Gurtner, G. C. Physiol Rev 99, 665-706 (2019). Example 6: Mediation of CGRP effect by TSP-1

[0284] To further understand the molecular mechanisms by which CGRP modulates neutrophils and macrophages, the inventors analysed the transcriptome of bone marrow-derived neutrophils and macrophages after CGRP stimulation by RNA- sequencing.

[0285] For both cell types, gene ontology (GO) analysis on differentially expressed genes (DEGs) between CGRP-treated and control groups identified biological processes that were congruent with the in vitro effects of CGRP. In addition, the biological processes reflected the differences in neutrophil and macrophage dynamics observed in tissue injuries of control and nociceptor-depleted mice. For both neutrophils and macrophages, pathways enriched in the upregulated DEGs included cell death, while those in the downregulated DEGs included cell migration (e.g. Ccr2, Cx3cr1, Itgav, Itgb3) (Fig.3a, Fig.10a).

[0286] Furthermore, pathways enriched in the upregulated DEGs were associated with tissue remodelling and differentiation in macrophages, featuring genes typically linked to an anti-inflammatory and pro-repair phenotype such as Arg1, Cebpb, Stat3, Sgk1, Tgfb3, Tgm2, and Vegfa35.

[0287] Finally, in response to CGRP, macrophages upregulated genes that are strongly associated with efferocytosis such as Cd14, Clu, S1pr1, Rarg, and Tgm2 (Kourtzelis, I., Hajishengallis, G. & Chavakis, T. Front Immunol 11, 553 (2020); Cunin, P. et al. Cell Death Dis 7, e2215 (2016))(Fig.3a, Fig.10a).

[0288] Importantly, the inventors found that thrombospondin-1 (Thbs1, TSP-1), a multifunctional extracellular matrix (ECM) protein regulating numerous biological processes including tissue healing (Lopez-Dee, Z., Pidcock, K. & Gutierrez, L. S. Mediators Inflamm 2011, 296069 (2011)), was the most upregulated gene in both neutrophils and macrophages (Fig.3b). Further confirming that TSP-1 is expressed in response to CGRP via RAMP1 / CALCRL, Ramp1- / -neutrophils and macrophages did not upregulate Thbs1 after CGRP stimulation (Fig.10b). Moreover, TSP-1 levels after skin and muscle injuries were lower in Nav1.8Cre / Rosa26DTAmice (Fig.10c).1005837019 82

[0289] Interestingly, it has been reported that macrophages are the primary source of TSP-1 in wounds, and the absence of TSP-1 leads to impaired wound healing and prolonged inflammation (Soto-Pantoja, D. R. et al. Matrix Biol 37, 25-34 (2014); Agah, A., Kyriakides, T. R., Lawler, J. & Bornstein, P. Am J Pathol 161, 831-839 (2002)). Furthermore, increasing evidence suggests that TSP-1 is an important immunoregulator (Kaur, S. & Roberts, D. D. Semin Cell Dev Biol (2023)). Thus, the inventors tested whether TSP-1 affects neutrophil and macrophage migration and death, as well as efferocytosis and macrophage polarisation. Although some reports suggested that TSP- 1 promotes neutrophil and macrophage migration, the inventors clearly observed an inhibition of migration when cells were treated with TSP-1, similar to what was observed with CGRP (Fig.3c). Moreover, TSP-1 accelerated death of neutrophils and macrophages in a dose-dependent manner in the presence of inflammatory cytokines (Fig.3d). Efferocytosis of neutrophils by macrophages was also greatly enhanced following macrophage treatment with TSP-1 (Fig.3e). Lastly, TSP-1 enhanced expression of CD206 and arginase-1 in the presence of anti-inflammatory cytokines (IL- 4 / IL-13 or IL-10), demonstrating that TSP-1 accelerates macrophage polarisation towards an anti-inflammatory / pro-repair phenotype (Fig.3f).

[0290] To further confirm that CGRP exerted its effects primarily via an autocrine / paracrine action of TSP-1, the inventors tested the effect of CGRP upon siRNA-mediated knockdown of Thbs1 (Fig.10d). Only macrophages were used, as transfection of neutrophils is very challenging. Thbs1 inhibition abolished the effects of CGRP on cell migration, death, efferocytosis and polarisation, while scramble siRNA showed no effect on all these cellular processes (Fig.10e-h).

[0291] To validate these effects in vivo, TSP-1 was administered in Nav1.8Cre / Rosa26DTAmice following skin and muscle injuries. TSP-1 delivery resulted in reduced numbers of neutrophils, monocytes / macrophages, and inflammatory monocytes / macrophages (Ly6Chigh) at D3 post-injury. Additionally, TSP-1 led to an increase in CD206 expression at D14 post-injury (Fig.10i).

[0292] Further, skin wounds and muscle defects in Leprdb / dbmice were treated with saline or recombinant, purified TSP-1 (20µg; SEQ ID NO:72) (Figure 16). Treatment with TSP-1 improved skin wound closure (Figure 16a-b). Additionally, treatment with TSP-1 reduced fibrosis scarring at a muscle injury site (Figure 16c) and improved1005837019 83 muscle regeneration (as quantified by muscle area, Figure 156, and assessed by muscle histology, Figured 16d).

[0293] Collectively, these results clearly support the role of TSP-1 in mediating the immunomodulatory effect of CGRP on neutrophils and macrophages. Example 7: eCGRP promotes diabetic tissue healing

[0294] Since the inventors found that nociceptor-derived CGRP regulates neutrophils and macrophages to facilitate tissue healing, it was investigated whether local delivery of CGRP could restore the impaired healing observed in mice ablated of nociceptors. CGRP is a small peptide, which poses a challenge in achieving sustained effects without immediate burst signalling when delivered locally into tissue as it can rapidly signal to cells, diffuse away from the delivery site, and undergo degradation. Moreover, for clinical application, a high concentration of CGRP circulating in the body is undesirable due to possible off-target effects (Legrand, J. M. D. & Martino, M. M. Cold Spring Harb Perspect Biol 14 (2022)).

[0295] Thus, the inventors engineered CGRP to enhance retention and protection at delivery sites by fusing it to a sequence with a very high affinity for ECM components (Martino et al.2014; Tan et al.2021; Julier, Z. et al Sci Adv 6, eaba7602 (2020)). The ECM-binding sequence was fused to the N-terminus of CGRP followed by a plasmin- sensitive sequence to allow the release of CGRP from ECM via proteolytic activity (Fig. 11a, b). These modifications did not impair CGRP activity, as demonstrated by the preserved capacity of engineered CGRP (eCGRP) to inhibit neutrophil and macrophage migration and induce cAMP in these cells via RAMP1 / CALCRL (Fig.11c,d). The ECM- binding allowed better retention of CGRP after delivery into tissues (Fig.10e,f).

[0296] The inventors then tested if CGRP variants (wildtype CGRP and eCGRP) could promote closure of splinted skin wounds and regeneration of quadriceps after volumetric muscle loss in Nav1.8Cre+ / − / Rosa26DTAmice. For skin, CGRP variants were delivered topically, while for muscle, they were delivered via a fibrin hydrogel. Both CGRP variants enhanced the extent of wound closure and muscle regeneration upon delivery of 1 μg, compared to saline control. Moreover, at a lower dose (250 ng) eCGRP promoted greater wound closure and muscle regeneration, compared to wildtype CGRP (Fig.11g-j).1005837019 84

[0297] Notably, delivering a relatively high dose of CGRP (10 μg) into mice has been shown to contribute to peripheral nociceptive sensitisation (Shi, X. et al. Anesth Analg 113, 175-183 (2011)). Thus, the inventors tested whether CGRP variant injection induced pain (Fig.11k, l). Administration of wildtype CGRP or eCGRP into mouse hind paws did not elicit significant pain behaviours over the 48-hour experiment. However, a transient and slight increase in thermal sensitivity was observed only for wildtype CGRP. This difference is likely due to eCGRP's binding to the ECM, preventing an immediate burst signalling after delivery (Martino et al.2014; Tan et al.2021; Julier et al.2020).

[0298] The inventors next looked for a model with more clinical relevance, compared to the Nav1.8Cre+ / − / Rosa26DTAmice. Indeed, more than half of diabetic patients develop peripheral neuropathy, characterised by the presence of dysfunctional peripheral nerves and a decrease in intraepidermal nerve fibres (Levy, D. M. et al. Diabetologia 35, 889- 897 (1992); .Pittenger, G. L. et al. Diabetes Care 27, 1974-1979 (2004)).. Consequently, the decrease in neuropeptide levels, including CGRP, may disrupt neuro-immune interactions crucial for tissue healing. Indeed, diabetic patients commonly experience chronic non-healing wounds, which represent the most prevalent and severe complication of the condition, alongside the development of muscle atrophy (Pradhan, L., Nabzdyk, C., Andersen, N. D., LoGerfo, F. W. & Veves, Expert Rev Mol Med 11, e2 (2009); Volmer-Thole, M. & Lobmann, R. Int J Mol Sci 17 (2016); Parasoglou, P., Rao, S. & Slade, J. M. Clin Ther 39, 1085-1103 (2017)).

[0299] The diabetic Leprdb / dbmouse is a model for type 2 diabetes commonly used to study impaired tissue healing, since it mimics some aspects of human chronic wounds, including immune dysregulation and peripheral neuropathies (Martino et al.2014; tan et al.2021; Sullivan, K. A. et al. Neurobiol Dis 28, 276-285 (2007)). Additionally, it has been shown that Leprdb / dbhave impaired muscle regeneration (Nguyen, M. H., Cheng, M. & Koh, T. J. ScientificWorldJournal 11, 1525-1535 (2011)). Thus, the inventors investigated the distribution of CGRP in Leprdb / dbmice skin and muscle to confirm that these mice exhibited peripheral neuropathy.

[0300] A significant reduction in neuron-like structures expressing CGRP was observed in both skin and muscle of Leprdb / dbmice, mirroring the pattern seen in Nav1.8Cre+ / − / Rosa26DTAmice (Fig.4a, Fig.12a). This observation supported the use of diabetic mice to assess the regenerative potential of local CGRP delivery. Remarkably,1005837019 85 wound closure and muscle regeneration was greatly improved when injuries were treated with eCGRP, compared to saline control and wildtype CGRP (Fig.4b-e).

[0301] The inventors also examined TSP-1 expression in granulation tissue post- eCGRP delivery, given CGRP's induction of TSP-1 expression in neutrophils and macrophages. Immunostaining revealed a significant increase in TSP-1 deposition, colocalising to some extent with myeloid cells (CD11b+), in skin and muscle granulation tissues (Fig.12b). Then, since neutrophil and macrophage dynamics were disrupted in Nav1.8Cre+ / − / Rosa26DTAmouse injuries, the inventors then investigated whether eCGRP delivery modulated those cells in injured tissues (Fig.13). eCGRP delivery reduced the number of neutrophils and monocytes / macrophages in both skin and muscle injures, at early time-points post-injury. Moreover, eCGRP delivery led to reduced level of the pro- inflammatory marker Ly6C and increased the level of the anti-inflammatory marker CD206, in skin wounds (Fig.4f).

[0302] To examine the effects of CGRP further, the immunoregulation of neutrophils and macrophages by wild-type mouse αCGRP (SEQ ID NO:77) was compared to the CGRP variants eCGRP (SEQ ID NO: 71) and ^2PI1-8 -CGRP (SEQ ID NO:91). The α2PI1-8 sequence (NQEQVSPL; SEQ ID NO: 89), is a substrate sequence known is the art for a transglutaminase (Factor XIII), that is derived from α2 plasmin inhibitor. The α2PI1-8 sequence allows the covalent linkage of the CGRP polypeptide to ECM polypeptides, such as fibrin, via a lysine (K) acceptor residue. Bioactivity of CGRP variants was assessed using neutrophil and macrophage migration and polarisation assays. Each of the three CGRP molecules induced favourable effects indicative of shifting from a pro-inflammatory phase to a pro-repair phase (Figure 15): reduced neutrophil migration (Figure 15b), reduced macrophage migration (Figure 15b), and increased CD206 expression (Figure 15c) indicative of M2-like macrophages).

[0303] High numbers of neutrophils and inflammatory macrophages in diabetic injured tissues are known to delay healing (Larouche at el.2018; Pieseler and Kubes, 2019; Krzyszcyk et al.2018). An abnormally high number of neutrophils in chronic wounds leads to an over-production of pro-inflammatory cytokines, reactive oxygen species, proteases (Wilgus, T. A., Roy, S. & McDaniel, J. C. Neutrophils and Wound Repair: Adv Wound Care (New Rochelle) 2, 379-388 (2013)), and to increased NETosis (Wong, S. L. et al. Nat Med 21, 815-819 (2015)). Similarly, failure to convert macrophages to an1005837019 86 anti-inflammatory phenotype leads to high levels of pro-inflammatory cytokines and proteases (Krzyszcyk et al.2018).

[0304] Therefore, the inventors measured concentrations of inflammatory cytokines / chemokines and proteases in response to saline and eCGRP treatment. eCGRP resulted in a significant reduction of IL-1, CCL2, CXCL2, MMP-2, and MMP-9 (Fig.13b), which are factors known to impair tissue healing when elevated (Martino et al.2014; Tan et al.2021; Wetzler, C., Kampfer, H., Stallmeyer, B., Pfeilschifter, J. & Frank, S. J Invest Dermatol 115, 245-253 (2000)). Thus, the results overall support that the local delivery of eCGRP accelerates the transition of diabetic injured tissues towards an anti-inflammatory and pro-repair phase.

[0305] Overall, the inventors identified that CGRP promotes neutrophil clearance via efferocytosis and boosts macrophage polarisation into an anti-inflammatory and pro- repair phenotype via autocrine and paracrine effects of TSP-1 (Figure 14). As described herein, this neuro-immune axis can be leveraged to therapeutically address wound healing, particularly of chronic wounds, and other inflammatory conditions, where excessive inflammation impairs tissue healing.

Claims

1005837019 87 CLAIMS 1. A fusion protein comprising: (i) a calcitonin gene-related peptide (CGRP) receptor agonist polypeptide; and (ii) an extracellular matrix (ECM) binding polypeptide.

2. A fusion protein comprising: (i) a calcitonin gene-related peptide (CGRP) polypeptide; and (ii) an extracellular matrix (ECM) binding polypeptide.

3. The fusion protein of claim 1, wherein the CGRP receptor agonist polypeptide is joined to the ECM binding polypeptide via a linker sequence.

4. The fusion protein of claim 2, wherein the CGRP polypeptide is joined to the ECM binding polypeptide via a linker sequence.

5. The fusion protein of claim 3 or claim 4, the CGRP receptor agonist polypeptide or the CGRP polypeptide is joined via a linker sequence to the C-terminus of the ECM binding polypeptide.

6. The fusion protein of any one of claims 3 to 5, wherein the linker sequence is cleavable.

7. The fusion protein of claim 6, wherein the linker sequence comprises or consists of a proteolytic cleavage site.

8. The fusion protein of claim 7, wherein the cleavage site comprises or consists of the amino acid sequence of SEQ ID NO:

70.

9. The fusion protein of any one of claims 1 to 8, wherein the CGRP receptor agonist polypeptide or the CGRP polypeptide is human CGRP or an orthologue thereof.

10. The fusion protein of claim 9, wherein the the CGRP receptor agonist polypeptide or the CGRP polypeptide comprises, consists essentially of, or consists of the1005837019 88 amino acid sequence of any one of SEQ IDs NO: 77-79 or 81-82, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of any one of SEQ IDs NO: 77-79 or 81-82.

11. The fusion protein of claim 9, wherein the CGRP receptor agonist polypeptide or the CGRP polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 80, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

80.

12. The fusion protein of claim 11, wherein the the CGRP receptor agonist polypeptide or the CGRP polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

1.

13. The fusion protein of any one of claims 1 to 12, wherein the ECM binding polypeptide binds or is capable of binding one or more of: collagen, vitronectin, fibronectin, tenascin C, osteopontin, fibrinogen, heparan sulfate and heparan sulfate proteoglycans.

14. The fusion protein of any one of claims 1 to 13, wherein the ECM binding polypeptide is derived from any one of placenta growth factor (PlGF), amphiregulin (AREG), neurturin (NRTN), collagenase (col), alpha2 antiplasmin inhibitor, or von Willebrand factor (vWF).1005837019 89 15. The fusion protein of any one of claims 1 to 14, wherein the ECM binding polypeptide comprises, consists essentially of, or consists of an amino acid sequence of any one of SEQ ID NOs: 4 to 69; or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of any one of SEQ ID NOs: 4 to 69.

16. The fusion protein of any one of claims 1 to 15, wherein the fusion protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO:89, SEQ ID NO: 1 and 87, SEQ ID NO: 1 and 89, or an amino acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 71, SEQ ID NO:89, SEQ ID NO: 1 and 87, or SEQ ID NO: 1 and 89.

17. The fusion protein of any one of claims 1 to 15, wherein the fusion polypeptide comprises, consists essentially of, or consists of the amino acid sequence that is encoded by a nucleic acid sequence that comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 73, SEQ ID NO: 90, or a nucleic acid sequence that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 73 or SEQ ID NO:

90.

18. The fusion protein of any one of claims 1 to 16, wherein the fusion protein is amidated at the C-terminus.

19. An isolated nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 18.

20. A vector comprising the nucleic acid sequence of claim 19.1005837019 90 21. A vector comprising a nucleic acid comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO:83; or a nucleic acid encoding a TSP-1 protein comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:

72.

22. A host cell comprising the isolated nucleic acid sequence of claim 19, or the vector of claim 20 or 21.

23. A method of producing the fusion protein according to any one of claims 1 to 18, comprising introducing into a host cell the isolated nucleic acid sequence of claim 19, or the vector of claim 20.

24. A cell expressing the fusion protein according to any one of claims 1 to 18, or produced by the method of claim 23.

25. A composition comprising the fusion protein according to any one of claims 1 to 18, the nucleic acid of claim 19, the vector of claim 20 or claim 21, the cell of claim 22 or 24.

26. A method for promoting conversion from an inflammatory phase to a pro-repair phase in a subject in need thereof, the method comprising administering the fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24.

27. A fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24 for use in promoting conversion from an inflammatory phase to a pro-repair phase in a subject in need thereof.

28. Use of the fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24 in the manufacture of a medicament for promoting conversion from an inflammatory phase to a pro-repair phase.1005837019 91 29. A method for promoting tissue regeneration in a subject in need thereof, the method comprising administering the fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24.

30. A fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24 for use in promoting tissue regeneration in a subject in need thereof.

31. Use of the fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24 in the manufacture of a medicament for promoting tissue regeneration.

32. A method for promoting wound repair in a subject in need thereof, the method comprising administering the fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24.

33. A fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24 for use in promoting wound repair in a subject in need thereof.

34. Use of the fusion protein according to any one of claims 1 to 18, a TSP-1 agonist, a TSP-1 polypeptide, the composition according to claim 25, the nucleic acid according to claim 19, the vector according claim 20 or claim 21, or the cell of claim 22 or 24 in the manufacture of a medicament for promoting wound repair.

35. The method according to any one of claims 26, 29, or 32, wherein the subject has an injury and administration to the subject is no more than 6 days post-injury.

36. A fusion protein comprising:1005837019 92 (a) a first polypeptide; and (b) a second polypeptide, wherein the first and second polypeptide are joined by a linker that comprises, consists essentially of or consists of the amino acid sequence KGYR (SEQ ID NO: 70).

Images