Truncated and fusion proteins

Abstract

The present invention relates to productive tissue repair and regeneration, and in particular to polypeptides, compositions comprising said polypeptides, and methods for productive tissue repair and regeneration using said polypeptides or compositions.In one embodiment, the present invention provides a polypeptide comprising, consisting essentially of, or consisting of a C-terminal portion of NAMPT comprising a truncated cytokine finger motif (cif) motif.In another embodiment, the present invention provides a fusion protein comprising, consisting essentially of, or consisting of a polypeptide of full-length NAMPTcif or a truncated variant and a portion that enhances delivery to or retention in tissue.

Description

[Technical field]

[0001] The present invention relates to productive tissue repair and regeneration, and in particular to polypeptides, compositions comprising said polypeptides, and methods for productive tissue repair and regeneration using said polypeptides or compositions. [Background technology]

[0002] Related Applications This application claims priority from Australian Provisional Application No. 2021903439, filed October 27, 2021, the entire contents of which are incorporated herein by reference.

[0003] 2. Background of the Invention Skeletal muscle typically forms approximately 40% of adult body weight. Skeletal muscle is formed during development by myogenesis, where pairs of paraxial mesoderm blocks known as somites give rise to transient myotomes that form muscle stem cells, which expand to form an integrated and complex musculature through the fusion of myoblasts to the myotube surface. In further stages of myogenesis, muscle stem cells (called satellite cells) migrate to occupy the sarcolemmal and basement membrane niches of individual muscle fibers. Amniotes are born with a full set of muscle fibers, and in adults, muscle repair is generally achieved through an increase in the size of existing fibers. Throughout life, muscle tissue homeostasis, growth, regeneration, and repair are driven by mesodermally derived skeletal muscle resident stem cells. At the molecular level, quiescent satellite cells require and express the transcription factor PAX7, and also express PAX3. After injury to skeletal muscle, a portion of satellite cells become activated, differentiate and fuse to form new myofibers or form myoblasts that merge with and repair damaged myofibers. This myogenic program is governed by the myogenic regulators MYF5, MYOD, MYOG and MRF4. Many other factors and cells associated with the muscle niche are thought to participate in the complex cellular processes and eventual production of functional tissue in the context of homeostasis and regeneration. Therefore, until now, it has been difficult to identify the source and nature of the signals that stimulate satellite cell activation and proliferation.

[0004] Satellite cells are the prototype of unipotent tissue-resident stem cells that occupy specific anatomical niches in differentiated tissues. Decades of research have revealed the remarkable ability of this system to effectively orchestrate muscle repair in response to a wide variety of insults. Despite this demonstrated regenerative potential, transplantation of isolated muscle stem cells has yet to have a therapeutic impact, and pro-regenerative procedures to stimulate muscle stem cells are currently completely lacking.

[0005] There is a need for new and / or improved compositions and methods for use in myoblast-based therapies.

[0006] The reference to any prior art herein is not an admission or suggestion that the prior art forms part of common general knowledge in any jurisdiction, or that the prior art could reasonably be expected by a person of ordinary skill in the art to be understood, considered in relation to other prior art, and / or taken in combination with other prior art. Summary of the Invention

[0007] In certain embodiments, the present invention provides truncated or modified NAMPT cytokine finger (cif) polypeptides.

[0008] In this embodiment, the invention provides a NAMPT polypeptide fragment comprising, consisting essentially of or consisting of a C-terminal portion of NAMPT comprising a truncated cif motif. Preferably, the only amino acid sequence of said polypeptide that is derived from or has homology or identity to the NAMPT protein is the truncated cif motif.

[0009] In this aspect, the invention provides a polypeptide comprising, consisting essentially of or consisting of a C-terminal portion of NAMPT comprising a truncated cif motif, preferably the only amino acid sequence of said polypeptide that is derived from or has homology or identity to the NAMPT protein is the truncated cif motif.

[0010] In any embodiment, the polypeptide binds to CCR5 and / or stimulates proliferation of muscle progenitor cells.

[0011] In any embodiment, the only amino acid sequence of the polypeptide that binds to CCR5 and / or stimulates stimulation of muscle progenitor cells is a truncated cif motif.

[0012] In any embodiment, the cif motif comprises or consists of the amino acid sequence depicted in any of SEQ ID NO: 1, 2, or 3. Preferably, the NAMPT polypeptide comprises, consists essentially of, or consists of the amino acid sequence of a truncated SEQ ID NO: 1.

[0013] In any embodiment, the truncation of the cif motif may be an N-terminal and / or C-terminal truncation. In certain embodiments, the N-terminal and / or C-terminal truncation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids. Preferably, the truncation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids at the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence equivalent to SEQ ID NO:1.

[0014] In any embodiment, the truncation is of 1 to 12, 1 to 20, 1 to 28, or 1 to 35 residues at the N-terminus of the cif motif. Preferably, the truncation is of 1 to 12, 1 to 20, 1 to 28, or 1 to 35 residues at the N-terminus of the amino acid sequence depicted in SEQ ID NO:1, or an equivalent thereof.

[0015] In certain embodiments, the polypeptide comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11, or a sequence 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 of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11. Alternatively, the polypeptide comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11, or a sequence 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 any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11.

[0016] In certain embodiments, "% identity" or "~% identical" to a sequence means that the polypeptides have the same length, e.g., the same number of amino acids, but the amino acids over 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 sequence identity are due to conservative substitutions (e.g., those summarized in Table 3 below).

[0017] In certain embodiments, the polypeptide has a length of about 110, about 109, about 108, about 107, about 106, about 105, about 104, about 103, about 102, about 101, about 100, about 99, about 98, about 97, about 96, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85 , about 84, about 83, about 82, about 81, about 80, about 79, about 78, about 77, about 76, about 75, about 74, about 73, about 72, about 71, about 70, about 69, about 68, about 67, about 66, about 65, about 64, about 63, about 62, about 61, about 60, about 59, about 58, about 57, or about 56 amino acids.

[0018] In certain embodiments, the polypeptide is equal to or less than 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, or 56 amino acids in length.

[0019] In certain embodiments, the amino acid sequence of a polypeptide identical 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 of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11 is about 110, about 109, about 108, about 107, about 106, about 105, about 104, about 106, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 200, about 2010, about 2011, about 2012, about 2013, about 2014, about 2015, about 2016, about 2017, about 2018, about 2019, about 210, about 211, about 212, about 213, about 214, about 215, about 216, about 217, about 218, about 219, about 220, about 221, about 222, about 223, about 224, about 225, about 226, about 227, about 228, about 229, about 230, about 231, about 232, about 233, about 234, about 235, about 236 about 103, about 102, about 101, about 100, about 99, about 98, about 97, about 96, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80, about 79, about 78, about 77, about 76, about 75, about 74, about 73, about 72, about 71, about 70, about 69, about 68, about 67, about 66, about 65, about 64, about 63, about 62, about 61, about 60, about 59, about 58, about 57, or about 56 amino acids.

[0020] In certain embodiments, the amino acid sequence of a polypeptide that is identical 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 any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11 is of length 110, 109, 108, 107, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 06, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, or 56 amino acids.

[0021] In any embodiment, the polypeptide or fusion protein comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 conservative (e.g., those summarized in Table 3 below) or non-conservative amino acid substitutions, deletions, or additions relative to the sequence, and retains the activity of interacting with CCR5 or tissue stem cells.Preferably, the conservative or non-conservative amino acid substitutions, deletions, or additions are not substitutions, deletions, or additions of amino acids at positions 431 to 435 or 472 to 491 (e.g., numbers corresponding to human NAMPT in SEQ ID NO: 19).

[0022] In one embodiment, the polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 8 with an N-terminal truncation. Preferably, the N-terminal truncation is less than 6 amino acids.

[0023] In certain embodiments, the polypeptides described herein comprise a C-terminal alpha helix present in the cif motif.

[0024] In one embodiment, the polypeptide consists of an amino acid sequence identical to or having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 4 or 5. Preferably, the amino acid sequence of the polypeptide is identical to or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 5.

[0025] In certain embodiments, the polypeptide consists of an amino acid sequence identical to or having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 7. Preferably, the amino acid sequence of the polypeptide is identical to or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6 or 7.

[0026] In certain embodiments, the polypeptide consists of an amino acid sequence identical to or having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 8 or 9. Preferably, the amino acid sequence of the polypeptide is identical to or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 9.

[0027] In one embodiment, the polypeptide consists of an amino acid sequence identical to or having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 10 or 11. Preferably, the amino acid sequence of the polypeptide is identical to or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11.

[0028] In one embodiment, the truncated NAMPT cytokine finger (cif) polypeptide has an N-terminal truncation of, or an equivalent N-terminal truncation of, residues 402-413 of SEQ ID NO: 1, and said polypeptide has at least 85% sequence identity to the amino acid sequence of residues 414-491 of SEQ ID NO: 1. Preferably, the amino acid sequence of said polypeptide is equal to, or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to, the amino acid sequence of residues 414-491 of SEQ ID NO: 1.

[0029] In one embodiment, the truncated NAMPT cytokine finger (cif) polypeptide has an N-terminal truncation of, or an equivalent N-terminal truncation of, residues 402-421 of SEQ ID NO: 1, and said polypeptide has at least 85% sequence identity to the amino acid sequence of residues 422-491 of SEQ ID NO: 1. Preferably, the amino acid sequence of said polypeptide is equal to, or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to, the amino acid sequence of residues 422-491 of SEQ ID NO: 1.

[0030] In one embodiment, the truncated NAMPT cytokine finger (cif) polypeptide has an N-terminal truncation of, or an equivalent N-terminal truncation of, residues 402 to 429 of SEQ ID NO: 1, and said polypeptide has at least 85% sequence identity to the amino acid sequence of residues 430 to 491 of SEQ ID NO: 1. Preferably, the amino acid sequence of said polypeptide is equal to, or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, the amino acid sequence of residues 430 to 491 of SEQ ID NO: 1.

[0031] In one embodiment, the truncated NAMPT cytokine finger (cif) polypeptide has an N-terminal truncation of residues 402-435 of SEQ ID NO:1, or an equivalent truncation, and said polypeptide has at least 85% sequence identity to the amino acid sequence of residues 436-491 of SEQ ID NO:1. Preferably, the amino acid sequence of said polypeptide is equal to or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of residues 436-491 of SEQ ID NO:1.

[0032] In some embodiments, the polypeptide may be in the form of a monomer, dimer or multimer. To promote dimer formation, any polypeptide described herein may be modified to allow homodimer formation or heterodimer formation. The modification may be, for example, the addition of an amino acid that forms a covalent bond, such as a cysteine ​​that forms a disulfide bond, either natural or non-natural. Typically, the addition of an amino acid that forms a covalent bond, such as a cysteine, is added at the N-terminus or C-terminus of the polypeptide. In some embodiments, the polypeptide comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11, with an additional cysteine ​​at the N-terminus or C-terminus.

[0033] In another embodiment, there is provided a dimeric polypeptide formed by covalent bonding, preferably disulfide bonding, between monomers of the polypeptides described herein.

[0034] In another embodiment, the polypeptide comprises, consists essentially of, or consists of two or more truncated cif motifs as described herein, preferably separated by a linker.

[0035] The peptide linker may be one or more repeats of Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS), or any of their variations. In some embodiments, the linker may comprise or consist of the sequence GGGGSGGGSGGGGGS (G4S)3. In some embodiments, the peptide linker may comprise the amino acid sequence GGGGS (a linker of length 6 amino acids) or a longer sequence. The linker may be a repeating series of glycine and serine residues (GS) of various lengths, i.e., (GS)n, where n is any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS), or even longer (GS)11, or even longer. It will be understood that n may be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more.

[0036] In another aspect, the invention provides fusion proteins comprising, consisting essentially of, or consisting of a polypeptide of the invention as described herein and a moiety that enhances delivery to or retention in a tissue, such as one or more ECM and / or other tissue-specific binding moieties.

[0037] In another aspect, the present invention provides a fusion protein comprising, consisting essentially of, or consisting of a full-length NAMPT cytokine finger (cif) polypeptide and a moiety that enhances delivery to or retention in a tissue, such as one or more ECM and / or other tissue-specific binding moieties. In one embodiment, the full-length NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of, or consists of the amino acid sequence depicted in SEQ ID NO:1.

[0038] In any embodiment, the ECM binding moiety binds to any one or more of the following ECM molecules: collagen, fibronectin, tenascin-C, osteopontin, fibrinogen, and heparan sulfate proteoglycans.

[0039] In any embodiment, the ECM binding moiety is derived from placental growth factor (PlGF), amphiregulin (Areg), collagenase (col), or von Willebrand factor (vWF). Preferably, the PLGF, Areg, col, or vWF is human.

[0040] In any embodiment, the ECM binding moiety 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, consist essentially of, or consist of RRRPK, RKKK, KRRR, or any other sequence described herein, including SEQ ID NOs: 12 and 13. In another embodiment, the ECM binding moiety comprises at least two contiguous sequences of positively charged amino acid residues.

[0041] In any embodiment, the ECM binding moiety comprises, consists essentially of, or consists of any of SEQ ID NOs:12-16. In another embodiment, the ECM binding portion comprises, consists essentially of, or consists of an amino acid sequence identical 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 of SEQ ID NOs: 12-16, wherein the ECM binding portion binds to one or more ECM proteins with the same affinity, an affinity that does not significantly differ, or an affinity that is at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the affinity of the ECM binding portion of any of SEQ ID NOs: 12-16 from which the ECM binding portion is derived.

[0042] Some embodiments include extracellular matrix (ECM) binding moieties known in the art. Exemplary ECM binding peptides are described in US Publication No. 2014 / 0011978 and US Publication No. 20140010832. Standard methods are used to conjugate drugs or peptides to binding moieties such as ECM binding moieties with or without linkers.

[0043] In certain embodiments, the polypeptides or fusion proteins described herein include one or more signaling enhancing moieties, such as syndecan binding moieties. Typically, syndecan binding sites are included to provide for the persistence or enhancement of CCR5 signaling through syndecan.

[0044] In any embodiment, a polypeptide or fusion protein described herein binds to satellite cells and stimulates satellite cell activation, myoblast proliferation, and / or muscle regeneration.

[0045] In one embodiment, the fusion protein comprises, consists essentially of, or consists of an amino acid sequence of any one of SEQ ID NOs: 12 to 16 fused, linked, or directly linked to an amino acid sequence of any one of SEQ ID NOs: 1 to 11. Preferably, the fusion protein comprises, consists essentially of, or consists of an amino acid sequence of any one of SEQ ID NOs: 12 to 16 fused, linked, or directly linked to the amino acid sequence of SEQ ID NO: 1. Preferably, the fusion protein comprises, consists essentially of, or consists of, an amino acid sequence of any one of SEQ ID NOs: 12 to 16, and an amino acid sequence of any one of SEQ ID NOs: 1 to 11 fused, linked, or directly linked thereto, in the order from the N-terminus to the C-terminus. In one embodiment, the protein comprises, consists essentially of, or consists of an amino acid sequence encoded by any one of SEQ ID NOs: 29 to 31.

[0046] In any embodiment, the polypeptide or fusion protein described herein stimulates proliferation of muscle precursor cells (e.g., myoblasts) to a level equal to or greater than full-length NAMPT, e.g., comprising, consisting essentially of, or consisting of SEQ ID NO: 19, or to a level equal to or greater than full-length NAMPTcif, e.g., comprising, consisting essentially of, or consisting of SEQ ID NO: 1 or 2. Preferably, proliferation of muscle precursor cells (e.g., myoblasts) is determined by an assay described herein, including in Example 1.

[0047] In any embodiment, the polypeptide or fusion protein described herein stimulates the activation of TLR4 to a level significantly lower than full-length NAMPT. For example, it stimulates the activation of TLR4 to a level equivalent to or 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% lower than full-length NAMPT (e.g., a polypeptide that includes or consists of the amino acid sequence of SEQ ID NO: 19). In any embodiment, the polypeptide or fusion protein stimulates the activation of TLR4 to a level that is the same as or not significantly different from the cytokine finger motif of NAMPT (e.g., a polypeptide that consists of the amino acid sequence of SEQ ID NO: 1). The level of TLR4 activation may be determined using the assays described herein, including Example 1.

[0048] In another aspect, the present invention also provides an isolated nucleic acid encoding a polypeptide or fusion protein comprising, consisting essentially of, or consisting of an amino acid sequence described herein, e.g., any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 17, or 18. In one embodiment, a nucleic acid molecule encoding a polypeptide or fusion protein described herein comprises, consists essentially of, or consists of a polynucleotide sequence set forth in any of SEQ ID NOs: 25-31, or a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0049] The nucleic acid molecule may be RNA or DNA, or RNA:DNA, or chemically modified forms thereof. For example, the nucleic acid may be in the form of a viral or non-viral vector.

[0050] In another aspect, the present invention provides a vector comprising the nucleic acid as described above, optionally linked to a regulatory sequence.

[0051] In another aspect, the invention provides host cells comprising said vectors and methods for producing said polypeptides or fusion proteins, and optionally, methods for recovering said polypeptides or fusion proteins.

[0052] In another aspect, the invention provides a cell expressing a polypeptide or fusion protein described herein.

[0053] In another aspect, the present invention provides a composition for use in stimulating muscle regeneration, providing an activity of interacting or binding with chemokine receptors or an activity of interacting with muscle tissue stem cells.In one embodiment, the present application provides a composition for use in stimulating muscle regeneration without or substantially without fibrosis, providing an activity of interacting or binding with chemokine receptors or an activity of binding or interacting with satellite cells.In one embodiment, the chemokine receptor is a CCR5 chemokine receptor or a tissue stem cell receptor that binds to NAMPT, including a tissue stem cell receptor that binds to NAMPTcif.In one embodiment, the composition comprising cells or other drugs that provide an activity of interacting with CCR5 binds to tissue stem cells, particularly muscle stem cells.

[0054] In some embodiments, the composition comprises a polypeptide, a fusion protein, a nucleic acid, a vector, or a cell described herein, and a pharma- ceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition may further comprise one, two, or all of: (a) tissue stem cells (such as satellite cells) or their progenitor cells or their progeny; (b) macrophages or their progenitor cells or their progeny; and (c) a scaffold or retentive material.

[0055] In certain non-limiting embodiments, promoting muscle stem cell chemokine receptor signaling is particularly useful in treating subjects with muscle damage, including volumetric muscle loss injury or muscle degeneration / atrophy, or muscle or neuromuscular disorders, muscle degeneration or neuromuscular degenerative conditions, myopathy, or a tendency thereto. In certain embodiments, the activity of binding to a chemokine receptor is provided in the form of cells, such as macrophages or stem cells, that express a polypeptide or fusion protein described herein.

[0056] In another aspect, the invention provides a method of stimulating stem cell proliferation, such as satellite cell proliferation, comprising administering to a cell or a subject an effective amount of a polypeptide, fusion protein, composition, or cell described herein, thereby stimulating stem cell proliferation.

[0057] In one embodiment, the chemokine receptor is a CCR5 receptor.

[0058] In one embodiment, the CCR5 receptor is a CCR5 receptor of a tissue stem cell or a progeny cell of a tissue stem cell.In one embodiment, the CCR5 receptor is a CCR5 receptor of a satellite cell or a progeny cell of a satellite cell.

[0059] In certain embodiments, in vitro, in vivo, and ex vivo uses are contemplated.

[0060] In another aspect, the invention provides a method of stimulating regeneration of muscle tissue in a subject, the method comprising administering an effective amount of a polypeptide, fusion protein, composition, or cell described herein to a muscle of the subject, thereby stimulating regeneration of muscle tissue.

[0061] In any embodiment, the polypeptide or fusion protein is a CCR5 agonist.That is, it stimulates receptor signaling or downstream events such as the activation and proliferation of satellite cells.In some embodiments, the polypeptide or fusion protein specifically activates tissue stem cells.In some embodiments, the polypeptide or fusion protein specifically activates satellite cells.

[0062] As described herein, in certain embodiments, the tissue regeneration stimulated by the methods is associated with minimal fibrosis, thus in another embodiment, the present application provides polypeptides, fusion proteins, compositions, cells, and methods for reducing the development of fibrosis in a patient or biological tissue subject for regenerative treatment.

[0063] In some embodiments, the present application provides methods suitable for regenerating muscle tissue in vitro, in vivo, or ex vivo. Thus, the polypeptides, fusion proteins, compositions, or cells described herein are intended for use in stem cell-based therapy and tissue engineering. In another embodiment, the polypeptides, fusion proteins, compositions, or cells described herein are for use in the production of artificial meat in vitro.

[0064] In another aspect, the present invention provides a method of stimulating regeneration of muscle tissue, the method comprising administering to muscle an effective amount of a polypeptide, fusion protein, composition, or cell described herein, wherein the polypeptide, fusion protein, composition, or cell described herein binds to satellite cells and stimulates satellite cell activation, myoblast proliferation, and muscle regeneration, and is not accompanied by substantial fibrosis (scar formation).

[0065] In another aspect, the present invention provides a method of stimulating regeneration of muscle tissue in a subject in which inflammation is undesirable, said method comprising administering to muscle an effective amount of a polypeptide, fusion protein, composition, or cell described herein, wherein the polypeptide, fusion protein, composition, or cell described herein thereby stimulates regeneration of muscle tissue in the subject.

[0066] In one embodiment, the unwanted inflammation is inflammation mediated by activation of a TLR, preferably activation of TLR4.

[0067] In certain embodiments, the subject may have been diagnosed with an inflammatory myopathy. Exemplary inflammatory myopathies include polymyositis, dermatomyositis, inclusion body myositis, and necrotizing autoimmune myopathy.

[0068] In this aspect, the invention provides a method of treating an inflammatory myopathy in a subject, the method comprising administering an effective amount of a polypeptide, fusion protein, composition, or cell described herein to a muscle of the subject, thereby treating the inflammatory myopathy. Preferably, the inflammatory myopathy is polymyositis, dermatomyositis, inclusion body myositis, or necrotizing autoimmune myopathy.

[0069] Reference to NAMPT and CCR5 includes their homologs and orthologs, including those from any animal, including mammals, non-mammalian vertebrates, fish, and birds.

[0070] In certain embodiments, the present application provides a method of stimulating regeneration of muscle tissue, the method comprising administering to a muscle a composition comprising an effective amount of a cell comprising or encoding a polypeptide or fusion protein described herein, and optionally a component that enhances delivery to or retention within the muscle, wherein the polypeptide or fusion protein described herein binds to satellite cells and stimulates myoblast proliferation and muscle regeneration.

[0071] In some embodiments, the cells are macrophages. In some embodiments, the macrophages are isolated from tissue. In some embodiments, the macrophages are derived from stem cells, such as bone marrow progenitor cells or iPSCs. In some embodiments, macrophages or macrophage progenitor cells (monocytes) are isolated from a supply tissue, such as, but not limited to, blood, lymph, bone marrow, and then undergo in vitro cell or tissue culture to induce a phenotype directed to a desired tissue niche. In some embodiments, the cell composition is cryopreserved and / or contains a delivery agent.

[0072] As known in the art, macrophages may be generated in vitro from stem cells by various means. Macrophages generated from stem cells such as BMSCs in the presence of IFNγ or LPS are generally considered to be "inflammatory" macrophages, referred to as "M1 macrophages." Macrophages generated in the presence of IL-4 or IL-10 have what is referred to as "anti-inflammatory" activity and are referred to as "M2" macrophages.

[0073] In certain embodiments, the subject's macrophages express M2 macrophage markers.

[0074] In one embodiment, the macrophage cells express one, or two, or three, or four, or five of mmp9, arg2, mmp13a, L-plastin, and cd163.

[0075] In another embodiment, the macrophage subset expresses prox1a and pou2f3.

[0076] In some embodiments, the composition further comprises stem cells and / or macrophage cells.

[0077] In one embodiment, the stem cells are satellite cells. In another embodiment, the stem cells are unipotent or multipotent stem cells.

[0078] In certain embodiments of the methods, the active ingredient or principal component is or is only a polypeptide or fusion protein described herein, or a pharma- ceutically acceptable salt, hydrate, homolog, ortholog, tautomer, stereoisomer, prodrug thereof.

[0079] Prodrugs refer to drugs that can be converted to the polypeptides or fusion proteins described herein through some chemical or physical process (e.g., enzymatic process and metabolic hydrolysis). That is, the term "prodrug" also refers to a pharma- ceutically acceptable precursor of a biologically active compound. Prodrugs may be inactive when administered to a subject, but are converted to active compounds in vivo. Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in the body. The term "prodrug" is also intended to include any covalently bonded carrier that releases the active compound in vivo when such prodrugs are administered to a subject. Prodrugs of active compounds may be prepared by modifying functional groups present in the active compound such that the modifications are cleaved to the parent active compound, either by routine manipulation or in vivo. Prodrugs include compounds in which a hydroxy, amino, or mercapto group is bonded to any group that is cleaved to form a free hydroxy, free amino, or free mercapto group, respectively, when a prodrug of the active compound is administered to a subject.

[0080] In certain embodiments, the polypeptides or fusion proteins described herein further comprise one or more moieties, such as a linker, a stability enhancing, a signaling enhancing, a delivery enhancing, or a labeling moiety.

[0081] In another aspect, the present invention provides compositions comprising a polypeptide, a fusion protein, a nucleic acid, a vector, or a cell as defined herein. Pharmaceutically and physiologically active compounds are provided. Cellular compositions are expressly provided.

[0082] In some embodiments, the cells are macrophages. In some embodiments, the macrophages are isolated from tissue. In some embodiments, the macrophages are derived from stem cells, such as bone marrow progenitor cells or iPSCs. In some embodiments, macrophages or macrophage progenitor cells (monocytes) are isolated from tissue sources, such as, but not limited to, blood, lymph, bone marrow, and then undergo in vitro cell or tissue culture to induce phenotypes directed to desired tissue niches. In some embodiments, the cell composition is cryopreserved and / or contains a delivery agent.

[0083] As known in the art, macrophages may be generated in vitro from stem cells by various means. Macrophages generated from stem cells such as BMSCs in the presence of IFNg or LPS are generally considered to be "inflammatory" macrophages, referred to as "M1 macrophages". Macrophages generated in the presence of IL-4 or IL-10 have what is referred to as "anti-inflammatory" activity and are referred to as "M2" macrophages.

[0084] In certain embodiments, the subject's macrophages express M2 macrophage markers.

[0085] In one embodiment, the macrophage cells express one, or two, or three, or four, or five of mmp9, arg2, mmp13a, L-plastin, and cd163.

[0086] In another embodiment, the macrophage subset expresses prox1a and pou2f3.

[0087] In some embodiments, the composition includes or is administered with a support such as a hydrogel, glue, foam, or a retaining material, scaffold. Delicate structures are generally suitable to allow for regeneration of more delicate tissues. By way of example, very fast-absorbed materials may be used, such as certain fibrin, collagen, hydrogel, and alginate preparations. Alternatively, slowly absorbed synthetics may be used, such as poly-4-hydroxybutarate. Very smooth products derived from mammalian sources, such as silk fibers, or muscle extracellular matrix, are also contemplated. Non-absorbable synthetics, such as polypropylene and polyethylene, provide support and reliability. In some embodiments, the composition includes a fibrin hydrogel. In another embodiment, a RAFT-acrylamide-based support surface is provided to enhance tissue regeneration and bioavailability of polypeptides, fusion proteins, nucleic acids, vectors, or cells at the target site.

[0088] In one embodiment, a composition is provided comprising a polypeptide, fusion protein, nucleic acid, vector, or cell described herein and any one or two or three or four of the following: (i) a satellite cell or a progenitor cell or a progeny cell thereof, (ii) a macrophage or a progenitor cell or a progeny cell thereof, (iii) a scaffold (semi-solid or solid support) or retention material, (iv) a moiety that enhances delivery to a tissue or an intracellular retention moiety.

[0089] In certain embodiments, the scaffold or retention material is a hydrogel, such as a fibrin or acrylamide hydrogel. In certain embodiments, the tissue delivery enhancing moiety or intracellular retention moiety is an ECM binding moiety.

[0090] As used herein, the term "comprise" and variations of this term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, components, integers, or steps unless the context otherwise requires.

[0091] The meaning of "consisting of" is inclusive and limited to what is listed before the word "consisting of." That is, the word "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present.

[0092] What is meant by "consisting essentially of" is inclusive of any elements recited between the phrases, and is limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. That is, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 additional amino acid residues at the N- or C-terminus of the polypeptide sequence) are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0093] Further aspects of the invention and further embodiments of said aspects set out in the preceding paragraphs will become apparent from the following description, given by way of example, and by reference to the accompanying drawings, in which: [Brief description of the drawings]

[0094] [Figure 1-1]Figure 1. Muscle volumetric injury following NAMPT supplementation in mice. AD, Local delivery of NAMPT promotes muscle regeneration in an adult mouse muscle injury model (schematic, I). (B) Volumetric muscle defects were created and directly treated with NAMPT delivered via fibrin hydrogel. Representative histological sections through the center of the defect, stained with Masson's Trichrome, of mouse rectus femoris (RF) muscle (10 days after treatment) demonstrate that NAMPT delivery significantly increased the area of ​​regenerated muscle (dark red, quantification, C), while simultaneously showing a significant reduction in fibrotic tissue (purple / blue, white dashed lines demarcate fibrotic and healthy myofibers, while the fascia surrounding the muscle is stained blue (quantification, D)). CD, mean ± SEM. One-way ANOVA with Dunnett's post hoc test for multiple comparisons (n ​​= 5 mice per group). Upon EI, exogenous NAMPT supplementation, satellite cells demonstrated enhanced proliferation. Mouse muscle injuries were treated with fibrin-delivered NAMPT (0.5 μg) or fibrin-only control. (EF) The total number of satellite cells (PAX7+) (E) and the number of proliferating satellite cells (PAX7+ / Ki67+) (F) were quantified by flow cytometry in tissues collected 4 days after treatment. Graphs show the fraction of satellite cells per 10,000 cells in collected tissues (fibrin group n=6, NAMPT-treated group n=5). (G) Representative regenerating muscle cryosections stained for PAX7 (satellite cells, yellow), wheat germ agglutinin (WGA, magenta), and nuclei (DAPI, blue) for tissues collected 6 days after treatment. (HI) Centrally nucleated muscle fibers were quantified 6 days after treatment (n=6 mice per group) (H). Representative histology tissue sections with hematoxylin and eosin (I). EF, H, mean ± SEM, two-tailed Student's t-test. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0095] [Figure 2-1] Figure 2. NAMPT binds to the CCR5 receptor present on muscle stem cells and induces proliferation. (A) Exogenous NAMPT supplementation enhances myoblast proliferation. In vitro assay evaluating the effect of exogenously introduced factors on the proliferation of C2C12 myoblasts. Proliferation was determined by EdU incorporation. NAMPT administration (two commercially available sources of NAMPT were tested, hrNAMPT(1) and hrNAMPT(2)) resulted in a dose-dependent increase in myoblast proliferation. This effect is specifically mediated through the CCR5 receptor. Co-administration of NAMPT with the CCR2 / CCR5 dual inhibitor cenicriviroc (CVC) and the CCR5 specific inhibitor maraviroc (MVC) abolishes the proliferation-promoting response of NAMPT, whereas co-administration with the CCT2 inhibitor PF-4136309 (PF) does not prevent the stimulatory effect of NAMPT on myoblast proliferation. Consistent with this finding, the endogenous ligands for CCR5, mrCCL8 and mrCCL4, functioned to enhance proliferation of C2C12, whereas the CCR2-specific ligand, mrCCL2, did not increase proliferation rates above those of controls. The proliferation-promoting function of NAMPT is separate from its intracellular role in energy metabolism, as co-administration of NAMPT with the NAMPT enzyme inhibitor GMX1778 did not affect the effect on myoblast proliferation. Mean + SD. Two-way ANOVA with Tukey's multiple comparison test. (B-C) C-terminal fragments of NAMPT regulate cytokine activity. (B) NAMPT contains a "cytokine finger" (cif) that is conserved in other cytokines. (C) NAMPTcif inhibits the binding of NAMPT to CCR5. Mean ± SEM. [Figure 2-2] Same as above.

[0096] [Figure 3-1]Figure 3. N-terminal truncation variants of human NAMPT cytokine finger. Predicted structures of human NAMPT cytokine finger (hNAMPTcif) variants designed by truncating the N-terminal region containing positively charged amino acids. (A) Human NAMPTcif (residues 402-491 of full-length NAMPT, e.g., SEQ ID NO: 19); (B) hNAMPTcif-T1 (residues 414-491 of full-length NAMPT) removes the N-terminal beta strand, loop, and subsequent beta strand. (C) hNAMPTcif-T2 (residues 422-491 of full-length NAMPT) starts with T1 and removes the N-terminal loop and a short helix containing one lysine. (D) hNAMPTcif-T3 (residues 430-491 of full-length NAMPT) starts with T2 and removes the N-terminal loop containing three lysines and two arginines. (E) hNAMPTcif-T4 (residues 436–491 of full-length NAMPT) begins at T3 and removes the N-terminal beta strand and loop containing one histidine. (F) Human NAMPT cytokine finger and its truncated variants stimulate proliferation of muscle progenitor cells. C2C12 mouse myoblasts were treated with 10 nM full-length NAMPT (FL-NAMPT), human NAMPT cytokine finger (hNAMPTcif), or N-terminal truncated variants of hNAMPTcif (hNAMPTcif-T1, hNAMPTcif-T2, hNAMPTcif-T3, where increasing numbers represent shorter hNAMPTcif fragments) for 48 h. Quantification of cell proliferation was performed using a CyQuant Proliferation Assay kit. Data are presented as % increase vs. PBS-treated negative control. 10% fetal bovine serum (FBS) was used as a positive control. n=3-6 technical replicates per condition, two independent experiments. Asterisks indicate significance of one-sample t-test, where *: p<0.05 and **: p<0.01. For Mann-Whitney test against FL-hNAMPT, # indicates p<0.05 and ## indicates p<0.01. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0097] [Figure 4] Figure 4. NAMPTcif fused to the ECM-binding domain retains proliferation-promoting activity. C2C12 mouse myoblasts were treated with 2 nM, 10 nM, or 20 nM of NAMPTcif (NAMPTcif) or NAMPTcif fused to the N-terminal heparin-binding sequence from placental growth factor 2 (PlGF-NAMPTcif) for 48 h. Quantification of cell proliferation was performed using the CyQuant Proliferation Assa kit. Data are presented as % increase vs. PBS-treated negative control. Mean ± SEM. n=4 independent experiments. Unpaired two-tailed t-test. ns: not statistically significant.

[0098] [Diagram 5] Figure 5. NAMPTcif does not induce TLR4 activity compared to full-length NAMPT. HEK-Blue TLR reporter cells were treated with 19 nM NAMPTcif or NAMPTcif fused to the N-terminal heparin-binding sequence from placental growth factor (PlGF-NAMPTcif) for 24 h. To abrogate signaling induced by trace amounts of endotoxin, 5 μg / ml polymyxin B was added to each treatment well. Quantification of TLR4 was determined against a standard curve generated by serial dilutions of bacterial lipopolysaccharide (LPS) and reported as equivalent LPS concentrations. Mean ± SEM. n=2 independent experiments with triplicate wells per condition. One-way ANOVA with Dunnett's multiple comparison test. *: p<0.05.

[0099] [Figure 6]Figure 6. Human NAMPT "cytokine finger" N-terminal truncation variants. Predicted structures of human NAMPT "cytokine finger" variants designed by N- and C-terminal truncation of regions containing structural elements and / or positively charged amino acids. The structures are represented by amino acid numbers corresponding to the full-length human NAMPT of, for example, SEQ ID NO: 19.

[0100] [Figure 7] Figure 7. Human NAMPT variants stimulate proliferation of muscle progenitor cells. C2C12 mouse myoblasts were treated with 20 nM full-length NAMPT (NAMPT) or NAMPT variants (NAMPTxxx-xxx, where xxx is the amino acid number) for 48 h. Quantification of cell proliferation was performed using CyQuant Proliferation Assay kit. Data are presented as % increase vs. PBS-treated negative control. Boxes and whiskers represent median and minimum and maximum values. n=4-5 independent experiments per protein. Statistical significance is shown as exact p-values. Significant increases in proliferation per condition were analyzed by one-sample t-test. Significance between conditions was analyzed by one-way ANOVA with Tukey's post-hoc test.

[0101] [Figure 8] Figure 8. Human NAMPT variants stimulate proliferation of human satellite cells. Human primary satellite cells were treated with 20 nM full-length NAMPT (NAMPT) or NAMPT variants (NAMPTxxx-xxx, where xxx is the amino acid number) for 48 h. The medium contained growth factors IGF-1 and FGF-2. Quantification of cell proliferation was performed using CyQuant Proliferation Assay kit. Data are presented as % increase vs. PBS-treated negative control. Treatment with 20% FBS was used as positive control. Boxes and whiskers represent median and minimum and maximum values. n=6-10 independent experiments per protein. Statistical significance is presented as p-value and determined by one-sample t-test.

[0102] [Figure 9] Figure 9. Human NAMPT variants stimulate the proliferation of human endothelial cells. Human primary endothelial cells derived from umbilical vein were treated with 20 nM full-length NAMPT (NAMPT) or NAMPT variants (NAMPTxxx-xxx, where xxx is the amino acid number) for 48 h. Quantification of cell proliferation was performed using CyQuant Proliferation Assay kit. Data are presented as % increase vs. PBS-treated negative control. Treatment with 10% FBS was used as positive control. Boxes and whiskers represent median and minimum and maximum values. n=6 independent experiments per protein. Statistical significance is shown as p-value and determined by one-sample t-test. [Figure 10] The minimal version of NAMPT protein enhances proliferation in response to muscle injury in zebrafish larvae. Treatment with NAMPT402-491 and NAMPT422-491 after needlestick muscle injury induced a significant increase in cell proliferation within the injury area. The smallest version of NAMPT (NAMPT422-491) stimulated cell proliferation at significantly higher levels compared to human recombinant NAMPT (hrNAMPT), especially in the wound. Representative images are shown in panel (A). The violin plot in (B) shows the number of EdU-positive cells in the 'injured' area and 'outside' represents two adjacent somites surrounding the injury area (n=22 control, n=9 hrNAMPT treated, n=6 NAMPT402-491, and n=8 NAMPT422-491). The thick black line and the dashed black line in the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tuckey's multiple comparison test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0104] Detailed Description of the Invention The invention disclosed and defined herein will be understood to extend to all different combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which constitute various different aspects of the invention.

[0105] 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 it is not intended 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 invention as defined by the claims.

[0106] Those skilled in the art will recognize many methods and materials that can be used in the practice of the present invention, which are similar or equivalent to the methods and materials described herein.The present invention is in no way limited to the methods and materials described.It will be understood that the invention disclosed and defined herein extends to all different combinations of two or more individual features mentioned or evident from the text or figures.All of these different combinations constitute various different aspects of the present invention.

[0107] All patents and publications referenced herein are incorporated by reference in their entirety.

[0108] For the purposes of this specification, terms used in the singular will also include the plural and vice versa.

[0109] General chemical terms used in the formulas herein have their usual meanings.

[0110] The present invention is based on the surprisingly direct and important role of a specific macrophage subset identified to regulate tissue regeneration in vivo, demonstrating that a portion of wound-attracted macrophages form a transient stem cell niche with tissue-resident stem cells and induce their activation. The elimination of this niche-specific macrophage subset leads to a severe reduction in the number of proliferative progenitor cells present in the injury site, and consequent regeneration failure. In this specification, the term injury refers to any wound, whether exogenously or endogenously inflicted or present, in which tissue regeneration is required to replace lost tissue or to reconstruct or regenerate functional tissue lost through any process, such as disease process, infection or trauma, aging, poor diet and lack of exercise.

[0111] An obligate satellite cell-macrophage niche has been identified that directs efficient skeletal muscle regeneration and injury repair in real time and within wounds. This demonstrates that a portion of wound-attracted macrophages form a transient stem cell niche and are pro-myogenic. Removal of this niche-specific macrophage leads to a severe reduction in the number of myogenic progenitor cells present within the injury site and consequent muscle regeneration failure.

[0112] Thus, along with their well-described ability to regulate pro- and anti-inflammatory phenomena, certain macrophage populations also provide a transient stem cell activation niche, where the cells are spatially fixed together and directly interact with muscle tissue. The stem cells may be tissue stem cells, such as muscle stem cells. For example, the stem cells are skeletal muscle stem cells (satellite cells). Other muscle stem cells include cardiac tissue stem cells or non-striated muscle cells.

[0113] Thus, the macrophage-derived factors detailed herein are contemplated for use in regulating stem cell activity, directing activation of quiescent tissue stem cells, and regenerating tissue. One of the macrophage-derived factors described herein is nicotinamide phosphoribosyltransferase (NAMPT, also known as visfatin and PBEF (pre-B cell enhancing factor)). The inventors have identified that NAMPT is upregulated and produced by macrophages that reside at the site of injury. As described and exemplified herein, certain derivatives of NAMPT, particularly truncated forms of the cif motif and various fusion polypeptides, have been developed that induce proliferation of muscle stem cells.

[0114] Thus, NAMPT polypeptides and fusion proteins are contemplated for use in stimulating wound healing and improving the quality of healing to promote full restoration of tissue function, i.e., productive tissue repair and regeneration.

[0115] Reference to polypeptides and fusion proteins includes variants that activate quiescent tissue stem cells and derivatives thereof, including adaptations known in the art that are suitable for production and clinical or commercial use, such as enhanced delivery to tissues or improved signaling functionality. The terms include orthologs and isoforms.

[0116] Reference to "regeneration" in relation to muscle is used in a broad context herein and includes indirect effects on muscle and muscle-associated tissues as a direct result of muscle stem cell (also called satellite cell) activation. That is, regeneration includes muscle wound repair and muscle maintenance, growth, repair, and increased ability of muscle cells to productively proliferate and form functional tissue. The term includes muscle tissue generation and repair of damaged muscle and relates to the process of muscle regeneration (myogenesis), which begins with activation and proliferation of muscle stem cells, proliferation of myoblasts, early differentiation into muscle cells, and terminal differentiation into muscle fibers. In certain embodiments, regeneration is associated with minimal fibrosis, which allows for the establishment of native structures or regenerated tissues with normal or near-normal biological properties than fibrotic or wasted tissues. Muscle functional properties may be determined by standard tests of muscle contractile function, including strength (e.g., eccentric muscle contraction), force, and endurance, as well as physical length and volume. The term also includes growth of muscle tissue in commercially available cultures.

[0117] In certain embodiments, treatment of muscle with the C-terminal fragments of NAMPT described herein was associated with little or minimal fibrosis in a clinically relevant volumetric wound model.

[0118] The present invention finds particular use in treating subjects with myopathies in which inflammation is particularly undesirable, or in regenerating muscle tissue in said subjects. For example, the present invention provides a method of stimulating regeneration of muscle tissue in a subject in which inflammation is particularly undesirable, said method comprising administering to muscle an effective amount of a polypeptide, fusion protein, composition, or cell described herein, wherein the polypeptide, fusion protein, composition, or cell described herein thereby stimulates regeneration of muscle tissue in the subject.

[0119] In one embodiment, the unwanted inflammation is inflammation mediated by TLR activation, preferably TLR4 activation.

[0120] In some embodiments, the subject may be diagnosed with inflammatory myopathy. Exemplary inflammatory myopathy includes polymyositis, dermatomyositis, inclusion body myositis, and necrotizing autoimmune myopathy. Other inflammatory myopathies, their clinical characteristics, and methods for diagnosing them are described in (Dalakas, 2015, N Engl J Med 2015;372:1734-47).

[0121] The present invention provides a method of treating an inflammatory myopathy in a subject, the method comprising administering an effective amount of a polypeptide, fusion protein, composition, or cell described herein to a muscle of the subject, thereby treating the inflammatory myopathy. Preferably, the inflammatory myopathy is polymyositis, dermatomyositis, inclusion body myositis, or necrotizing autoimmune myopathy.

[0122] In one embodiment, the present application provides a pharma- ceutical or physiologically active regenerative composition, comprising: a polypeptide, fusion protein, nucleic acid, vector, or cell described herein, Satellite cells or their precursor cells or their progeny Macrophages or their precursor cells or their progeny Scaffolding or support Components that enhance tissue delivery The present invention allows for a composition comprising one, or two, or three, or four, or five of the above.

[0123] In certain embodiments, as described elsewhere herein, the monomeric or dimeric form of the polypeptide or fusion protein is modified to create a drug suitable for attachment to a biological carrier or extracellular matrix. In addition, the drug is modified to enhance signaling through the CCR5 receptor by adding a moiety that binds to a co-receptor of heparin sulphate proteoglycan (such as syndecan).

[0124] In certain embodiments, the polypeptides, fusion proteins, nucleic acids, vectors, cells, or compositions described herein are for use in stimulating muscle regeneration in vitro, ex vivo, or in vitro, or for use in the manufacture of a composition for such use.

[0125] In certain embodiments, the compositions described herein are for or are used in the production of artificial muscles (such as fish, birds, or other non-human animal muscles for direct or indirect consumption). For example, supplementation of the growth medium allows for scalability and more efficient muscle growth.

[0126] In certain embodiments, the compositions described herein are for or are used in stem cell therapy, i.e., the compositions support proliferation in vitro and / or are included in grafts (or as pre-treatments) to promote proliferation and tissue integration in vivo.

[0127] In one embodiment, the present application provides a method for stimulating tissue regeneration, comprising administering to isolated or tissue-resident tissue stem cells or their progenitors an effective amount of a composition comprising or encoding a polypeptide or fusion protein as described herein, and optionally a component that enhances delivery to tissue, wherein the polypeptide or fusion protein binds to tissue stem cells or their progenitors and stimulates (activates) the proliferation of quiescent tissue stem cells and tissue regeneration. In one embodiment, the polypeptide or fusion protein comprises a component or moiety that enhances delivery to target tissue.

[0128] In some embodiments, the compositions described herein are for use in, or for use in, the manufacture of a composition for the treatment of muscular, neuromuscular, or musculoskeletal defects, disorders, or injuries. Muscular, neuromuscular, or musculoskeletal defects, disorders, or injuries are known in the art. Defects and disorders are found, for example and without limitation, in sarcopenia, cachexia, and muscular dystrophy, muscle atrophy, muscle pseudohypertrophy, or muscular dystrophic conditions and myopathies. All appropriate formats are encompassed, including Swiss-style use, method of treatment, and / or EPC2000 style claims.

[0129] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found, or to the progeny of such a cell. The cell may have been cultured in vitro, e.g., in the presence of other cells, and the cell may be destined for subsequent introduction into a second organism or for reintroduction into the organism from which it (or the cells from which it is derived) was isolated.

[0130] As used herein, terms such as "isolated cell population" refer to a population of cells that have been harvested and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched.

[0131] In one embodiment, the present application provides a pharma- ceutical or physiologically active regenerative composition comprising: (i) a polypeptide, fusion protein, nucleic acid, vector, or cell as defined herein; (ii) Tissue stem cells (such as satellite cells) or their progenitor cells or their descendants (iii) macrophages or their precursor cells or their progeny (iv) Scaffolding or support (v) components that enhance delivery to tissues The present invention allows for a composition comprising one, or two, or three, or four, or five of the above.

[0132] In some embodiments, the present application provides a cell composition comprising one or more of the stem cells, stromal cells, pre-satellite cells or satellite cells, pre-macrophages or macrophages, or macrophage-derived factors described herein. In some embodiments, multipotent "tissue stem cells" include pre-muscle cells or any pre-macrophage cells, which may be produced in a substantially native form or modified to express non-autologous or autologous factors. Similarly, the term multipotent "tissue stem cells" may include activated progeny of tissue stem cells.

[0133] Tissue stem cells, including muscle stem cells, may be isolated or derived (for ex vivo or in vitro or in vivo procedures).

[0134] Stem cells can be contacted with the medium or composition comprising polypeptide, fusion protein, nucleic acid, vector or cell for any time.For example, stem cells can be contacted with polypeptide, fusion protein, nucleic acid, vector or cell for 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more.Stem cells can be induced or stimulated to differentiate into cell lineage selected from the group consisting of mesodermal, endodermal, ectodermal, neural, mesenchymal and hematopoietic.

[0135] In some embodiments, the stem cells are human stem cells, multipotent adult stem cells, pluripotent adult stem cells, or embryonic stem cells.

[0136] Human adult stem cells undergo mitosis, and typically one of the daughter cells remains a stem cell. Adult tissues contain one or more resident committed progenitor or stem cells that occupy specific niches in those tissues and actively sense and respond to their local environment. Each tissue typically has its own resident committed stem cells that are committed to produce progeny cells that differentiate into a specific range of cell types. Muscle tissue contains satellite cells that are committed to produce myoblasts. Other well-studied stem cells of this type are mesenchymal stem cells (MSCs), which produce many different cell types, including muscle, cartilage, bone, and fat, and hematopoietic stem cells (HSCs), which produce blood cells and the hematopoietic system, and neural stem cells (NSCs). All tissues, including the heart, intestine, and liver, contain a population of resident stem cells. Adult stem cells are typically multipotent, which refers to cells that can differentiate into some, but not all, cells derived from all three germ layers. That is, multipotent cells are partially differentiated cells. MSCs can be obtained by a number of methods well known in the art, for example, see USPN 5,486,358; 6,387,367; and USPN 7,592,174, and USPN 2003 / 0211602. MSCs may be derived from bone, fat, and other tissues in which they reside. "Derived from" does not refer to a direct origin, but merely an indication of where it was originally derived from.

[0137] In certain embodiments, the stem cells are non-embryonic or adult multipotent stem cells.

[0138] In certain embodiments, the stem cells are HSCs or MSCs.

[0139] Adult stem cells expressing CCR5 can be stimulated to differentiate by exposure to a polypeptide, fusion protein, nucleic acid, vector, or cell described herein, and the cells are monitored for changes in expression of, for example, myogenic regulatory factors known in the art.

[0140] The cells may be cultured in a standard medium or in a specifically defined medium.

[0141] The expression of the cells may be modified by techniques known in the art.

[0142] Induced or partially induced pluripotent stem cells are a convenient source of stem cells. They are derived from differentiated adult cells, such as human foreskin.

[0143] Human iPS cells can be generated by introducing a specific set of reprogramming factors into non-pluripotent cells, which can include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5), and / or related transcription factors such as NANOG, LIN28, and / or Glis1. For example, the reprogramming factors can be introduced into the cells using one or more plasmids, lentiviral vectors, or retroviral vectors. In some cases, the vectors can be integrated into the genome and removed after reprogramming is complete. In some cases, the vectors are non-integrating (e.g., positive-strand based, single-stranded RNA species derived from the non-infectious (non-packaged) self-replicating Venezuelan equine encephalitis (VEE) virus, Simplicon RNA Reprogramming Kit, Millipore, SCR549 and SCR550). Simplicon's RNA replicons are synthetic in vitro transcribed RNAs that express all four reprogramming factors (OKG-iG; Oct4, Klf4, Sox2, and Glis1) in polycystronic transcripts that can self-replicate for a limited number of cell divisions. Induced human pluripotent stem cells produced using the Simplicon kit are referred to as "integration-free" and "footprint-free". Human iPS cells can also be generated, for example, by the use of miRNAs, small molecules that mimic the action of transcription factors, or lineage specifiers. Human iPS cells are characterized by their ability to differentiate into cells of any of the three vertebrate germ layers, e.g., endoderm, ectoderm, or mesoderm. Human iPS cells are also characterized by their ability to grow indefinitely in suitable in vitro culture conditions.Human iPS cells express alkaline phosphatase, SOX-2, OCT-4, Nanog and Tra-1-60 markers.

[0144] The terms "naive" and "primed" identify different pluripotency states of human iPS cells. The characteristics of naive and primed iPS cells are described in the art. Naive human iPS cells exhibit a pluripotency state similar to that of ES cells of the inner cell mass of preimplantation embryos. Such naive cells are not primed for lineage specification and commitment. Female naive iPS cells are characterized by two active X chromosomes. In culture, the self-renewal of naive human iPS cells depends on leukemia inhibitory factor (LIF) and other inhibitors. Cultured naive human iPS cells exhibit clonal morphology, characterized by rounded dome-shaped colonies and lack of apical-basal polarity. Cultured naive cells may further exhibit one or more pluripotency markers described elsewhere herein. The doubling time of naive human iPS cells cultured under appropriate conditions can be between 16 and 24 hours.

[0145] Primed human iPSCs express a pluripotent state similar to that of post-implantation epiblast cells. Such cells are primed toward lineage specification and commitment. Female primed iPSCs are characterized by one active X chromosome and one inactive X chromosome. In culture, the self-renewal of primed human iPSCs depends on factors such as fibroblast growth factor (FGF) and activin. Cultured primed human iPSCs exhibit a clonal morphology characterized by an epithelial monolayer and exhibit apical-basal polarity. The doubling time of primed human iPSCs cultured under appropriate conditions can be 24 hours or more, depending on the level from the adult cells they were derived.

[0146] Embryonic stem cells (ESCs) are characteristically pluripotent. That is, the cells have the ability to differentiate into all cell types characteristic of the three germ layers (endoderm, mesoderm and ectoderm) under different conditions. Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers. In some embodiments, pluripotent cells are undifferentiated cells. Pluripotent cells may also have the potential to divide in vitro for more than one year, or for more than 30 generations.

[0147] ESCs are typically pluripotent stem cells of the inner cell mass of blastocysts (see U.S. Patent Nos. 5,843,780 and 6,200,806). Such cells can also be obtained from the inner cell mass of somatic cell nuclear transfer-derived blastocysts (see U.S. Patent Nos. 5,945,577, 5,994,619 and 6,235,970). Exemplary characteristics that distinguish embryonic stem cells include, but are not limited to, gene expression profiles, proliferation potential, differentiation potential, karyotype, and responsiveness to specific culture conditions.

[0148] In certain embodiments, the stem cells are adult.

[0149] In certain embodiments, the stem cells are autologous or non-autologous to the subject.

[0150] In certain embodiments, the stem cells are mammalian or human.

[0151] Stem cells, including macrophages and satellite cells, may be prepared using art-recognized methods and methods described herein, including the use of iPSCs and optionally gene editing procedures.

[0152] In some embodiments, isolated macrophages or stem cell-derived macrophages are modified to express a polypeptide or fusion protein as described herein. Generally, M2 type macrophages are selected or provided.

[0153] In certain embodiments, stem cells are contacted in vitro, ex vivo, or in vivo with a polypeptide, fusion protein, nucleic acid, vector, or cell described herein to induce activation and proliferation. In vitro or ex vivo treated stem cells may be introduced to a wound site to effect repair, or may be administered systemically to effect regeneration of damaged tissue or to treat or ameliorate muscle-related conditions described herein.

[0154] The polypeptides, fusion proteins, nucleic acids, vectors, or cells expressing said polypeptides or fusion proteins may be administered in the form of functionalized hydrogels, either alone or together with cells for transplantation, such hydrogels or similar biomaterials or scaffolds providing enhanced engraftment efficiency at the wound site.

[0155] The hydrogel may be ECM-based, such as fibrin-based. Alternatively, the hydrogel may be non-ECM-based, such as acrylamide-based using RAFT technology (see Chiefari et al Macromol. 31:5559-5526, 1998 and Fairbanks et al Advanced Drug Delivery Reviews 91: 141-152, 2015). Suitable materials control release kinetics and have the desired mechanical and physical properties for tissue regeneration, as known in the art.

[0156] In one embodiment, the satellite cells are encapsulated in a CCR5-functionalized hydrogel or other biomaterial.

[0157] Kits are also provided that include the cell compositions and / or drugs described herein. Kits suitable for muscle repair or regeneration are particularly contemplated. The polypeptides, fusion proteins, nucleic acids, vectors, or cells may be preformulated for administration, or the components of the formulation may be provided in the kit. The polypeptides, fusion proteins, nucleic acids, vectors, or cells may be formulated, for example, in hydrogels or other support media for topical application. The polypeptides, fusion proteins, nucleic acids, vectors, or cells may be, for example, lyophilized or liquid.

[0158] The terms "protein," "polypeptide," and "peptide," as used interchangeably herein, include polymeric forms of amino acids of any length, unless a length is defined, including coded and non-coded amino acids, and amino acids that have been chemically or biochemically modified or derivatized. The terms also include modified polymers, such as polypeptides having modified peptide backbones.

[0159] Proteins are referred to as having or being "N-terminal" and as having or being "C-terminal". The term "N-terminus" refers to the beginning of a protein or polypeptide, which terminates in an amino acid having a free amine group (--NH2). The term "C-terminus" refers to the end of a chain of amino acids (protein or polypeptide), which naturally terminates in a free carboxy group (--COOH). In this application, references to C-terminus and N-terminal fragments broadly describe the region of the full-length molecule from which the selected portion is derived, and exclude the full-length or native molecule. A C-terminal fragment may, but does not include, all of the amino acids at the C-terminus, and an N-terminal fragment may, but does not include, all of the amino acids at the N-terminus.

[0160] The present application discloses and enables the use of various polypeptides, fusion proteins, nucleic acids, vectors or cells based on initial findings described in the examples.

[0161] Numerous peptide modifications are known in the art and are included to stabilize peptides against serum proteases or to facilitate intracellular positioning. Some of these modified peptides can be expressed from nucleic acid in cells, while others are synthetically produced. Similarly, when it is desired to target the polypeptides or fusion proteins described herein to one or more specific cell types, this can be achieved by either ex vivo manipulation of target cells, or by incorporating targeting moieties that specifically bind to target cells or tissues, such as ECM binding moieties, as known in the art.

[0162] "Conservative amino acid substitution" refers to a substitution in which a naturally occurring or non-naturally occurring amino acid residue is replaced with a naturally occurring or non-naturally occurring amino acid with a similar side chain. Families of amino acid residues with 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), non-polar 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). That is, predicted non-essential amino acid residues of CCR5 may be replaced, for example, with another amino acid residue from the same side chain family. Other examples of permissible substitutions are those based on isosteric considerations (e.g., norleucine for methionine) or other properties (e.g., 2-thienylalanine for phenylalanine). All amino acid subclassifications are presented in Table 2, and exemplary substitutions are presented in Table 3. [Table 2] [Table 3]

[0163] In some embodiments, a Trp residue is substituted.

[0164] The polypeptides or fusion proteins described herein may contain modifications known to alter the pharmacokinetic characteristics of the peptide, such as by improving protease resistance in vivo.

[0165] In some embodiments, the peptide comprises one or more linkers or spacers, modified or non-natural or non-proteinogenic amino acids, modified side chains, modified backbones, terminal modified groups, such as GGS or repeats of GGS and variants known in the art, or comprises modified spatial constraints, or is a D-retro-inverso peptide. In some embodiments, the peptide is a pseudopeptide, pseudopeptide, peptoid, azapeptide, cyclized peptide, stapled peptide, ether peptide, or lactam peptide, or comprises spatial constraints.

[0166] In certain embodiments, the polypeptides or fusion proteins described herein are conjugated or otherwise attached / bound / expressed with lipids, carbohydrates, polymers, proteins, nanoparticles, peptides, proteoglycans, antibodies or fragments or antigen-binding forms thereof, aptamers, or nucleic acids, as appropriate.

[0167] In certain embodiments, a polypeptide or fusion protein described herein specifically binds to muscle cells or muscle cell tissue or associated structures, such as ECM.

[0168] In certain embodiments, the polypeptides or fusion proteins described herein include physiologically or pharma- ceutically acceptable salts, hydrates, stereoisomers, and prodrugs.

[0169] In certain embodiments, non-essential amino acids may be altered. Reference to a "non-essential" amino acid residue means a residue that may be altered from the wild-type sequence of a polypeptide without eliminating or substantially altering its ability to bind to endogenous or non-autologous CCR5.

[0170] In one embodiment, the polypeptides or fusion proteins described herein comprise or encode amino acid sequences having one, two, three, four, five, or six conservative amino acid substitutions (e.g., those outlined in Table 3 above) or non-conservative amino acid substitutions, deletions, or additions relative to the above sequences, but retain the activity of interacting with CCR5.

[0171] In certain embodiments, the present invention also provides nucleic acid molecules capable of expressing the polypeptides or fusion proteins described herein. Nucleotide sequences encoding the polypeptides or fusion proteins described herein are disclosed herein.

[0172] In certain embodiments, the nucleic acid molecule is RNA or DNA or RNA:DNA, or chemically modified forms thereof.

[0173] In certain embodiments, a proportion of at least one type of nucleotide (eg, cysteine ​​and / or uracil) is chemically modified to increase its stability in vivo.

[0174] In certain embodiments, the nucleic acid is in the form of a viral or non-viral vector.

[0175] In some embodiments, the polypeptides, fusion proteins, nucleic acids, vectors, cells, or compositions described herein are administered to cells ex vivo. The present invention encompasses the use of genetically modified cell depots (e.g., CAR T cells, TCRs, genetically modified macrophages, etc.).

[0176] In certain embodiments, the polypeptide or fusion protein comprises an antibody or antibody fragment that specifically targets the drug to a target cell, such as a muscle stem cell.

[0177] In certain embodiments, the present application provides a pharmaceutical or physiological composition comprising a polypeptide, a fusion protein, a nucleic acid, a vector, or a cell as defined herein above.

[0178] The present application enables a method of treating muscle injury or a human having a reduced or suboptimal ability to repair or regenerate muscle, comprising administering an effective amount of a composition comprising a polypeptide, a fusion protein, a nucleic acid, a vector, or a cell, or a composition comprising a polypeptide, a fusion protein, a nucleic acid, a vector, or a cell, which is sufficient to stimulate muscle stem cell proliferation and muscle regeneration.

[0179] The compositions include physiologically or pharmacologically or pharma- ceutically acceptable carriers that are not biologically or otherwise undesirable. Pharmacologically acceptable salts, esters, prodrugs, or derivatives of the compounds described herein are salts, esters, prodrugs, or derivatives that are not biologically or otherwise undesirable.

[0180] In some embodiments, the polypeptide or fusion protein is modified. The activity of the polypeptide and fusion protein is tolerant of additional moieties, adjacent residues, and substitutions within the defined boundaries. Similarly, backbone modifications and replacements, side chain modifications, and N- and C-terminal modifications are commonly used in the art. Generally, modifications are for enhancing stability or pharmacological profile, targeting / delivery. For example, cyclization or stapling of peptides is commonly used to enhance peptide stability. In another embodiment, the peptide or drug is in the form of a microparticle or nanoparticle or foam, gel, liposome, conjugate, or fusion protein, which includes a moiety adapted for stability, delivery, or specificity to target tissue.

[0181] In certain embodiments, the drugs or nucleic acids encoding them are assembled in liposomes, hydrogels, emulsions, viral vectors, virus-like particles, or virosomes, as appropriate.

[0182] In certain embodiments, specific binding moieties such as antibodies or antibody fragments or mimics are used to target the polypeptide or fusion protein to the muscle environment.

[0183] In certain embodiments, the polypeptide or fusion protein is delivered by in vivo biosynthesis, such as via delivery of mRNA, gene editing, such as CRISPR components, or through bacteria or cells.

[0184] The compositions generally comprise a polypeptide or fusion protein, a peptidomimetic or an encoding nucleic acid, where appropriate, and a pharma- ceutically acceptable carrier and / or diluent. In certain embodiments, the carrier may be a nanocarrier.

[0185] In some embodiments, the polypeptides or fusion proteins of the present disclosure are not naturally occurring molecules, but are modified forms of naturally occurring molecules that do not have certain characteristics or functions of the full-length naturally occurring molecules, for example, they may lack the enzymatic activity of NAMPT.

[0186] In another embodiment, the polypeptides or fusion proteins described herein are immobilized by means of a linker covalently attached to at least two amino acids in the peptide. Various cyclization strategies to improve stability and cell permeability are known in the art.

[0187] In some embodiments, the polypeptide or fusion protein described herein is delivered in the form of a nucleic acid molecule encoding the same or a prodrug thereof, or in the form of a vector containing a nucleic acid molecule encoding the same or a prodrug thereof. In some embodiments, the nucleic acid is an mRNA. The polypeptide or fusion protein described herein may bind to the surface of, or function within, muscle stem cells to stimulate signaling and proliferation.

[0188] In another embodiment, the polypeptides, fusion proteins, nucleic acids, vectors, or cells described herein are combined with amphiphilic agents, such as lipids that exist in aqueous solution as aggregates, such as micelles, insoluble monolayers, liquid crystals, or lamellar layers, in addition to other pharma- ceutically acceptable carriers.

[0189] In certain embodiments, the present disclosure enables compositions for use as pharmaceuticals or for use in therapy that comprise a polypeptide or fusion protein described herein that interacts with endogenous CCR5.

[0190] In another aspect, the disclosure provides a composition for stimulating proliferation of muscle stem cells comprising a polypeptide or fusion protein described herein, or a nucleic acid molecule capable of expressing said peptide.

[0191] In certain embodiments, the subject compositions are co-administered with a second physiologically active therapeutic or prophylactic or regenerative agent. Exemplary cytokines include, but are not limited to, one or more of IGF-1, TGF-β, GDF-5, bFGF, PDGF-b3, IL-4.

[0192] In another aspect, the disclosure provides for the use of a polypeptide, fusion protein, nucleic acid, vector, or cell described herein in the manufacture of a medicament for stimulating muscle regeneration, or in stem cell therapy.

[0193] In one embodiment, the present application provides a screening assay for drugs that interact with CCR5 as described herein, the assay comprising evaluating the ability of the drug to induce muscle stem cell proliferation and muscle regeneration, or an indicator thereof.

[0194] Peptide-based therapeutics provide useful molecules because they are known to be potent and selective against biological targets that are difficult to engineer with small molecules. Modified peptides have been successfully developed to improve the pharmacokinetic properties of linear peptides.

[0195] The peptides of the present disclosure comprise amino acids. Reference to "amino acids" includes naturally occurring amino acids and non-naturally occurring amino acids.

[0196] Peptide compositions can generally and routinely be modified within understood parameters by the addition of moieties, adjacent peptide residues, and substitutions. Peptides can further include backbone, side chain, peptide bond replacement, and terminal modifications routinely modified using standard peptide chemistry.

[0197] The amino acids incorporated into the amino acid sequences 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. Unnatural amino acids may not be recognized by proteases, thereby altering their half-life. In certain embodiments, D-retro inversion sequences are employed.

[0198] Non-naturally occurring amino acids include chemical analogs of the corresponding naturally occurring amino acids. Examples of non-naturally occurring amino acids and derivatives include, but are not limited to, 4-aminobutyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienylalanine, and / or D-isomers of amino acids.

[0199] In some embodiments, peptides are modified using art-recognized modifications to enhance their pharmacodynamic properties. Peptides may be substituted, such as alanine substitutions, or may be substituted and / or linked with crosslinkable moieties. Suitable residues may include additional alpha carbon substitutions selected from hetero-lower alkyl, hetero-methyl, ethyl, propyl, and butyl. Replacements of peptide bonds, such as trifluoroethylamine, are used to produce more stable and active peptide mimetics.

[0200] Thus, cyclic or stapled peptides, peptoids, peptomers, and peptidomimetic forms of peptides are included.

[0201] Backbone-fixed peptidomimetics and cyclic peptides are protected from exopeptidases. Peptides can be cyclized by coupling the N-terminus to the C-terminus after cleavage. This can be achieved by direct coupling or by the introduction of specific functional groups that allow for targeted cyclization by bioorthogonal reactions. Exemplary modifications include side chain modifications to include linkers that form Cys-Cys disulfide bridges, macrolactam peptides, thioether peptides, or stapled peptides, etc. Click variants are particularly useful for peptide cyclization. Another approach is by using 2-amino-d, 1-dodecanoic acid (Laa) coupled to the N-terminus and replacing Asn with lipoamine.

[0202] A more defined structure can be obtained through the use of heterocycles, N-methylated amine linkages, or more rigid backbones with methylated alpha carbon atoms.

[0203] Among the techniques used for peptide stapling, the two-component double Cu-catalysed azide-alkyne cycloaddition (CuAAC) strategy locks peptides into biologically active conformations and simultaneously improves pharmacokinetic properties. Moreover, this strategy uses unnatural azide amino acids that can be easily synthesized and facilitate the functionalization of staples, fluorescently labeled tags, and photo-switchable linkers. Independent functionalization of the staples is particularly useful because complex functionality is added to the staples rather than to the N- or C-terminus of the peptide. In addition, this approach requires only one linear peptide to generate various functionalized staple peptides, facilitating the exploration of various functionalities of the linkers and thus the properties of the entire peptide.

[0204] Azapeptides are peptide analogs in which one or more amino residues are replaced by semicarbazide. This nitrogen to α-carbon substitution results in conformational fixation, bending the peptide from a linear shape at the aza amino acid residue. The resulting azapeptide turn conformation has been observed by X-ray crystallography and spectroscopy, and predicted based on computational models. Aza substitutions in biologically active peptide analogs have led to improved properties such as enhanced activity and selectivity, as well as extended duration of action and metabolic stability.

[0205] Half-life may also be enhanced by acylation or amidation of the terminus. Peptoids are produced with N-alkylated oligoglycine side chains. In some embodiments, peptides may be acetylated, acylated (e.g., lipopeptides), formylated, amidated, phosphorylated (on Ser, Thr, and / or Tyr), sulfated, or glycosylated.

[0206] The term "macrocyclization reagent" or "macrocycle-forming reagent" as used herein refers to any reagent that may be used to prepare a peptidomimetic macrocycle by mediating a reaction between two reactive groups. The reactive groups may be, for example, azides and alkynes, in which case the macrocyclization reagents include, but are not limited to, Cu reagents such as CuBr, CuI, or CuOTf, which provide reactive Cu(I) species, as well as Cu(CO2CH3)2, CuSO4, and CuCl2, which can be converted in situ to active Cu(I) reagents by the addition of a reducing agent, such as ascorbic acid or sodium ascorbate. The macrocyclization reagent is, for example, Cp * RuCl(PPh3)2, [Cp * RuCl] 4、or other Ru reagents known in the art that may provide reactive Ru(II) species. In other cases, the reactive group is a terminal olefin. In such embodiments, the macrocyclization or macrocycle-forming reagent is a metathesis catalyst, including but not limited to a stabilized late transition metal carbine complex catalyst, such as a Group VIII transition metal carbene catalyst. For example, such a catalyst is a Ru and Os metal center with an oxidation state of +2, an electron count of 16, and penta-coordinated. Additional catalysts are disclosed in (Grubbs et al., "Ring Closing Metathesis and Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28, 446-452) and U.S. Pat. No. 5,811,515. In yet another case, the reactive group is a thiol group. In such embodiments, the macrocyclization reagent is a linker functionalized with two thiol-reactive groups, such as, for example, halogen groups.

[0207] In certain embodiments, the peptidomimetic macrocycle exhibits improved biological properties, such as increased structural stability, increased affinity for a target, resistance to proteolytic degradation, etc., when compared to the corresponding non-macrocyclic polypeptide. In another embodiment, the peptidomimetic macrocycle contains one or more α-helices in aqueous solution and / or exhibits an increased degree of α-helicity when compared to the corresponding non-macrocyclic polypeptide.

[0208] For example, the sequence of the peptide can be analyzed and the azide- and alkyne-containing amino acid analogs of the invention can be substituted at the appropriate positions. The appropriate positions are determined by ascertaining which faces of the secondary structure of the molecule are required for biological activity, i.e., across which other faces the macrocycle-forming linker of the invention can form a macrocycle without sterically blocking the faces required for biological activity. Such determinations are made using methods such as X-ray crystallography of the complex between the secondary structure and a natural binding partner to visualize the residues (and faces) important for activity; by sequential mutagenesis of residues in the secondary structure to functionally identify the residues (and faces) important for activity; or by other methods. Upon such determination, the appropriate amino acids are substituted with the amino acid analogs and the macrocycle-forming linkers of the invention. For example, for a helical secondary structure, one of the faces of the helix (e.g., the face of the molecule that extends longitudinally along the axis of the helix and radially at 45-135° about the axis of the helix) may be required to contact another biomolecule in vivo or in vitro for biological activity. In such cases, a macrocycle-forming linker is designed to extend along the longitudinal axis of the face of the helix and connect two carbons in the portion of the face not directly required for activity.

[0209] The peptidomimetic macrocycle may comprise a helix in aqueous solution. For example, the peptidomimetic macrocycle may exhibit an increased helical structure compared to the corresponding non-macrocyclic polypeptide. In some embodiments, the peptidomimetic macrocycle exhibits improved thermal stability compared to the corresponding non-macrocyclic peptide. In other embodiments, the peptidomimetic macrocycle exhibits improved biological activity compared to the corresponding non-macrocyclic polypeptide. In yet other embodiments, the peptidomimetic macrocycle exhibits improved resistance to proteolytic degradation compared to the corresponding non-macrocyclic polypeptide. In still other embodiments, the peptidomimetic macrocycle has improved ability to penetrate living cells compared to the corresponding non-macrocyclic polypeptide.

[0210] The term "amino acid analog" refers to a molecule that is structurally similar to a naturally occurring amino acid and that may be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, compounds that are structurally identical to the amino acids defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxy groups, or replacement of the amino or carboxy groups with a group of similar reactivity (e.g., replacement of a primary amine with a secondary or tertiary amine, or replacement of a carboxy group with an ester).

[0211] The peptide may include an acetyl, formyl, myristoyl, palmitoyl, carboxy, 2-furanosyl group at the N-terminus, and / or a hydroxy, amide, ester, or thioester group at the C-terminus. In some embodiments, the peptide is acetylated at the N-terminus and amidated at the C-terminus. In some embodiments, a chelating agent, such as DOTA, DPTA, etc., is introduced. The peptide may be modified, for example, by pegylation, lipidation, xtenylation, pasylation, and other approaches, to extend the half-life of the peptide in vivo or in vivo. In some embodiments, pegylation is used to improve the solubility and bioavailability of the peptide. Various forms of PEG are known in the art and include HiPeg, branched and forked PEG, releasable PEG, heterobifunctional PEG with terminal NHS ester, maleimide, vinyl sulphone, pyridyl disulphide, amine, and carboxylic acid. An example of a therapeutic PEGylated peptide includes pegfilagrastin (Neulasta) made by Amgen.

[0212] The linker or spacer may be an amino acid or nucleic acid or other atomic structure known in the art, typically 2 to 10 amino acids or nucleotides in length. The spacer should be sufficiently flexible to allow correct orientation of the construct that interacts with CCR5, such as the nanoparticles, antibody fragments, liposomes, constructs that include cell penetrating and / or intracellular delivery moieties described herein. One form of spacer is the hinge region of IgG, suitable for use when the construct includes an antigen binding moiety for cell targeting.

[0213] Antigen-binding molecules include, for example, extracellular receptors, antibodies or antibody fragments (including molecules such as ScFv). A signal peptide may be present at the N-terminus. Bispecific antibodies capable of specifically binding to two or more epitopes are known in the art and can be used in the CCR5-interacting drugs of the present invention, for example, to bind to muscle environment or other substrates.

[0214] In certain embodiments, the peptide is conjugated or otherwise associated (covalently or non-covalently) with a delivery agent, hi certain embodiments, the delivery agent delivers the peptide to a tissue, target cell, or cell population.

[0215] Derivatives of the polypeptides or fusion proteins described herein include biologically active fragments thereof, including those containing the described structures or orthologs. Systematic truncation, or alanine scanning, or modeling around conserved motifs can be routinely performed to identify minimal peptides with CCR5 agonistic activity.

[0216] Derivatives also include molecules having percent amino acid or polynucleotide sequence identity over a window of comparison after optimal alignment. In certain embodiments, the percent identity is at least 80%-99%, including any number between 80 and 99.

[0217] Suitable assays for the biological activity of a peptide or drug are known to those of skill in the art and are described herein.

[0218] In some embodiments, markers of peptide activity include upregulation of satellite cell signaling (eg, MAPK), stem cell and myoblast proliferation and differentiation.

[0219] In some embodiments, the polypeptide or fusion protein is modified with a moiety that is not a naturally occurring amino acid residue. The moiety may be selected from the group consisting of a detectable label, a non-naturally occurring amino acid as described herein, a reactive group, a fatty acid, cholesterol, a lipid, a biologically active carbohydrate, a nanoparticle, a small molecule drug, and a polynucleotide. In certain embodiments, the moiety is a detectable tag label. In some examples, the detectable label is selected from the group consisting of a fluorophore, a fluorogenic substrate, a luminogenic substrate, and biotin. Art-recognized tags or labels include affinity agents, and moieties for detection include fluorescent and luminescent compounds, metals, and dyes. Other useful moieties include affinity tags, biotin, lectins, chelators, lanthanides, fluorescent dyes, and FRET acceptors / donors.

[0220] In some embodiments, the polypeptide or fusion protein, which may include a detectable label, is accompanied in a kit by a modified control version of a drug, in which conserved residues of the polypeptide or fusion protein are replaced, for example, with alanine. The kit containing the drug is intended for sale and may be used for screening or therapeutic purposes.

[0221] Peptides of this type may be obtained by use of recombinant nucleic acid techniques such as, for example, (Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), especially sections 16 and 17); (Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), especially chapters 10 and 16); and (Coligan et al. CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), especially chapters 1, 5, and 6).

[0222] Alternatively, peptides of this type may be synthesized using conventional liquid phase synthesis methods or the increasingly used solid phase synthesis methods, for example, initial reference may be made to solution synthesis or to solid phase synthesis as described, for example, by Atherton and Sheppard (SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989), see especially chapter 9) or by Roberge et al. (1995 Science 269: 202).

[0223] Azapeptide synthesis was previously hampered by cumbersome solution-phase synthetic routes for selective hydrazine functionalization. Recently, submonomer procedures for azapeptide synthesis have enabled the addition of diverse side chains to a common semicarbazone intermediate, providing the means to construct azapeptide libraries by solution- and solid-phase chemistry. Briefly, aza residues are introduced into the peptide chain using a submonomer strategy by incorporation of semicarbazone, deprotonation, N-alkylation, and orthogonal deprotection. Aminoacylation and elongation of the resulting semicarbazide leads to the desired azapeptide. Furthermore, many of the chemical transformations have utilized orthogonal chemistry of the semicarbazide residue (e.g., Michael addition and N-arylation). In addition, oxidation of the azaglycine residue has led to azopeptides that react in pericyclic reactions (e.g., Diels-Alder and Alder-ene chemistry). Many of these transformations of azaglycine residues were developed by the Lubell laboratory, which has utilized such chemistry in the synthesis of ligands with promising biological activity for the treatment of diseases such as cancer and age-related macular degeneration. Azapeptide analogs of growth hormone releasing peptide 6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH2, GHRP-6) have been pursued, for example, as ligands for the cluster of differentiation 36 (CD36) receptor and have shown promising activity for the development of treatments for age-related macular degeneration as well as diseases related to angiogenesis such as atherosclerosis. Azapeptides have also been employed to create a series of conformationally locked second mitochondria-derived activator of caspase (Smac) mimetics that exhibit promising apoptosis-inducing activity in cancer cells.The synthesis of cyclic azapeptide derivatives was used to perform aza scans to study the conformation-activity relationship of the anticancer drug cilengitide, cyclo(RGDf-N(Me)V), and its parent compound cyclo(RGDfV), which exhibit efficacy against human tumor metastasis and tumor-induced angiogenesis. Innovations in the synthesis and utilization of azapeptides are described in (Acc Chem Res. 2017 Jul 18;50(7):1541-1556).

[0224] Alternatively, peptides can be produced by digesting the adaptor polypeptide with proteinases such as endoLys-C, endoArg-C, endoGlu-C and Staphylococcus aureus V8-protease. The digested fragments can be purified, for example, by high performance liquid chromatography (HPLC) techniques. Steps that may be taken to optimize the pharmacodynamic parameters of peptides and peptide analogues are described by (Werle M. et al (2006) Strategies to improve plasma half-life time of peptide and protein drugs amino Acids 30(4):351-367; and Di L (2014) Strategic approaches to optimising peptide ADME properties AAPS J 1-10).

[0225] The polypeptide or fusion protein may be stabilized as known in the art, for example, via nanoparticles, liposomes, micelles, or, for example, PEG. Methods for forming liposomes are described in (Prescott, Ed. Methods in Cell Biology, Volume XIV, Academic Press, New York, NY (1976), p. 33 et seq.), the contents of which are incorporated herein by reference. Polymeric nanoparticles ideally use surfactants that are non-toxic and do not physically adsorb to the nanoparticles. In some embodiments, biodegradable surfmers are used. For example, biodegradable, poly(ethylene glycol) (PEG)-modified N-(2-hydroxypropyl) methacrylamide (HPMA)-based surfmers have been synthesized and used to stabilize lipophilic NPs. In particular, the core of the NP is made from a macromonomer containing poly(lactic acid) (PLA) chains functionalized with HPMA double bonds. The nanoparticle-forming polymer chains are then constructed with a homogeneous poly(HPMA) backbone that is biocompatible and water-soluble, and hydrolysable PEG and PLA pendants that ensure complete degradability of the polymer. The stability offered by the synthesized surfumers is studied in both cases of emulsion and solution free radical polymerization followed by flash nanoprecipitation of the resulting amphiphilic copolymers.

[0226] Other stabilizing or non-autologous moieties include NMEG, ECM binding, syndecan-binding albumin, albumin binding proteins, immunoglobulin Fc domains.

[0227] While traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, various artificial Fc domains have been designed as asymmetric interaction pairs, as described by Spiess et al. (2015) Molecular Immunology 67(2A): 95-106. Fc conjugates may contain amino acid sequences derived from the Fc domains of IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1, IgA2), IgE, or IgM immunoglobulins. Such immunoglobulin domains may contain one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote hetero- or homodimer or multimer amyloid formation in host cells.

[0228] In some embodiments, nanoparticles comprising a polypeptide, a fusion protein, a nucleic acid, a vector, or a cell can be further modified by conjugation of a tissue type specific binding agent, an antibody or a fragment thereof known in the art.

[0229] Other suitable binding agents are known in the art and include antigen-binding constructs such as affimers, aptamers, or suitable ligands (receptors) or portions thereof.

[0230] Antibodies, such as monoclonal antibodies, or derivatives or analogs thereof, include, but are not limited to, Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, humanized antibodies and antibody fragments, camelized antibodies and antibody fragments, and multivalent versions of the above. Multivalent binding reagents may also be used as appropriate, including, but are not limited to, monospecific or bispecific antibodies, disulfide stabilized Fv fragments, scFv tandem (scFv) fragments, diabodies, tribodies or tetrabodies, which are typically covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments.

[0231] The term "antibody fragment" as used herein includes any portion of an antibody that retains the ability to bind to the epitope recognized by the full-length antibody. Examples of antibody fragments include Fab, Fab' and F(ab')2, Fd, single chain Fvs (scFv), disulfide-linked Fvs (dsFv), and VFv. L or V H Antigen-binding fragments of antibodies may include, but are not limited to, fragments that contain any of the regions. Antigen-binding fragments of antibodies may include various regions alone or in combination with a portion of the hinge region, CH1, CH2, CH3, or a combination thereof. Fragments that contain fewer than all six CDRs may also be functional, but preferably, antibody fragments contain all six CDRs of the entire antibody.

[0232] A "single-chain Fv" ("scFv") is a compound that encodes the light chain variable region (V L ) and the heavy chain variable region (V H) but lacking some or all of the constant domains of an antibody. The link between the VH and VL can be achieved through a short, flexible peptide selected to ensure that proper three-dimensional folding of the VL and VH regions occurs and that the target molecule binding specificity of the entire antibody from which the scFv is derived is maintained. scFvs lack some or all of the constant domains of an antibody.

[0233] Methods for making receptor-specific binding agents, including antibodies and their derivatives and analogs and aptamers, generally based on natural ligands, are known in the art. Polyclonal antibodies can be generated by immunization of animals. Monoclonal antibodies can be prepared according to standard (hybridoma) methodologies. Antibody derivatives and analogs, including humanized antibodies, can be prepared recombinantly by isolating DNA fragments of DNA encoding the monoclonal antibodies and subcloning the appropriate V regions into appropriate expression vectors according to standard methods. Phage display and aptamer technologies have been described in the literature and allow for the in vitro clonal amplification of target-specific binding reagents with very high affinity and low cross-reactivity. Phage display reagents and systems are commercially available and include the Recombinant Phage Antibody System (RPAS) available from Amersham Pharmacia Biotech, Inc., Piscataway, NJ, and the pSKAN Phagemid Display System available from MoBiTec, LLC, Marco Island, FL. Aptamer technology is described, for example, but not limited to, U.S. Patent Nos. 5,270,163; 5,475,096; 5,840,867, and 6,544,776.

[0234] Optionally, one or more amino acid residues are selected from the group consisting of glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, and amino acids conjugated to lipid moieties and organic derivatizing agents. CCR5-interacting peptides may include at least one N-linked sugar and may include two or more N-linked sugars. The peptide may also include an O-linked sugar. CCR5-interacting peptides or drugs may be produced in a variety of cell lines that glycosylate proteins in a manner appropriate for patient use, including artificial insect or yeast cells, and mammalian cells such as COS, CHO, HEK, and NSO cells. In some embodiments, the CCR5 peptide is glycosylated and has a glycosylation pattern obtained from a Chinese Hamster Ovary cell line. In most embodiments, CCR5-interacting drugs are synthesized and have component moieties added using techniques known in the art.

[0235] In some embodiments, the subject polypeptides or fusion proteins described herein have a half-life in a mammal (e.g., mouse or human) of about 0.5, 1, 2, 3, 4, 6, 12, 24, 36, 48, or 72 hours. Alternatively, they may exhibit a half-life in a mammal (e.g., mouse or human) of about 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 20, 25, or 30 days, depending on the characteristics of the conjugate and carrier and the mode of administration. In some embodiments, the peptide is modified to maximize retention in muscle tissue and avoid or minimize systemic circulation. Drugs may be administered in various retention-enhancing compositions known in the art, such as gels, foams, glues, hydrogels, patches, and films.

[0236] The size of a peptide may be modified to alter its hydrodynamic radium and renal clearance. PEGylation and lipidation, often with a linker, are well-established modifications that increase the serum half-life of drugs by reducing clearance and protecting them from proteases. Second-generation PEGylation processes introduced branched structures as well as the use of alternative chemistries for PEG attachment. In particular, PEGs with cysteine-reactive groups, such as maleimide or iodoacetamide, allow PEGylation to be targeted to a single residue in the peptide, reducing the heterogeneity of the final product. In addition, biodegradable hydrophilic amino acid polymers that are functional analogs of PEG have been developed, including XTEN (see US20190083577) and PAS, which are uniform and easily produced. The chemical conjugation of antibodies and peptides developed by ConX shows a range of hybrid peptide half-life extension methods that promise to overcome many of the disadvantages of previous methods.

[0237] Oral and injectable solution solubilizing excipients include water-soluble organic solvents (polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide), non-ionic surfactants (Cremophor EL, Cremophor RH40, Cremophor RH60, d-α-tocopherol polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, Solutol HS15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire 44 / 14, Softigen, etc.). 767, and mono- and di-fatty acid esters of PEG 300, 450, or 1750), water insoluble lipids (castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium chain triglycerides of coconut and palm seed oils), organic liquids / semi-solids (beeswax, d-α-tocopherol, oleic acid, medium chain mono- and diglycerides), various cyclodextrins (α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutylether-β-cyclodextrin), and phospholipids (hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, L-α-dimyristoylphosphatidylcholine, L-α-dimyristoylphosphatidylglycerol). Chemical approaches to solubilize drugs for oral and injectable administration include pH adjustment, cosolvents, complexation, microemulsions, self-emulsifying drug delivery systems, micelles, liposomes, and emulsions.

[0238] Constructs / vectors A construct or vector for expressing a polypeptide or fusion protein described herein from a recipient cell may include one or more DNA regions including a promoter operably linked to a nucleic acid sequence encoding the peptide. The promoter may be inducible or constitutive. Examples of suitable constitutive promoters include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter, the Elongation Growth Factor-1a (EF-1a) gene promoter, the Simian Virus 40 (SV40) early promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobulin promoter, and the creatine kinase promoter. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0239] The expression construct may be produced by any suitable method, including recombinant or synthetic techniques, utilizing various vectors known and available in the art, such as plasmids, bacteriophages, baculoviruses, mammalian viruses, artificial chromosomes, among others. The expression construct may be circular or linear, and should be suitable for replication and integration into eukaryotes. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Numerous virus-based systems have been developed to transfer genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene is inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's stem cells. Numerous retroviral systems are known in the art.

[0240] In certain embodiments of the invention in which the peptide is provided as a nucleic acid encoding said peptide, the nucleic acid may be administered in vivo to promote expression of the protein it encodes by constructing it as part of a suitable nucleic acid expression vector and administering it so that it enters the cell (e.g., by use of a retroviral vector, by direct administration, by microparticle bombardment, by coating it with lipids or cell surface receptors, or by administering it in conjunction with a homeobox-like peptide or other intracellular targeting moiety). Alternatively, the nucleic acid may be introduced intracellularly for expression and integrated into the DNA of the host cell.

[0241] 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 that contain purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0242] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react to make oligonucleotides in such a way that the 5' phosphate of the pentose ring of one mononucleotide is attached unidirectionally to the 3' oxygen of a nearby mononucleotide via a phosphodiester bond. An end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of the pentose ring of a mononucleotide. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to the 5' phosphate of the pentose ring of another mononucleotide. Nucleic acid sequences, even if internal to a larger oligonucleotide, may also be said to have 5' and 3' ends. In either a linear or circular DNA molecule, distant elements are referred to as being "upstream" or 5' relative to the "downstream" or 3' elements.

[0243] "Codon optimization" may be used and generally includes the process of modifying a nucleic acid sequence, particularly for enhanced expression in a host cell, by replacing at least one codon of the original sequence with a codon that is more or most frequently used in the host cell's genes while maintaining the original amino acid sequence. For example, a nucleic acid sequence encoding a Cas protein may be modified to replace codons that have a higher frequency of usage compared to the naturally occurring nucleic acid sequence in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell. Codon usage tables are readily available, for example in the "Codon Usage Database." These tables can be adapted in a number of ways. See (Nakamura et al. (2000) Nucleic Acids Research 28:292), which is incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available for codon optimization of a particular sequence for expression in a particular host (see Gene Forge).

[0244] The nucleic acid molecules described herein may be 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 molecules and chemically synthesized molecules, and modified forms thereof. Nucleic acid molecules may be single-stranded or double-stranded, and linear or covalently closed to form a circle. RNA may be modified by stabilizing sequences, caps, and polyadenylation. RNA or DNA may be delivered as a plasmid to express peptides. RNA-based approaches are routinely available.

[0245] The term "RNA" refers to a molecule that includes ribonucleotide residues, and preferably, is composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxy group at the 2' position of a β-D-ribofuranosyl group. The term includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and RNA that differs from naturally occurring RNA, modified by the addition, deletion, substitution and / or modification of one or more bases. Such modifications may include the addition of non-nucleotide material, such as at the end or within the RNA, for example, at one or more nucleotides of the RNA. The nucleotides of the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0246] An optimized mRNA-based composition may include 5' and 3' untranslated regions (5'-UTR, 3'-UTR) that optimize translation efficiency and intracellular stability as known in the art. In certain embodiments, removal of uncapped 5'-triphosphates may be achieved by treating the RNA with phosphatase. The RNA may have modified ribonucleotides to improve its stability and / or reduce cytotoxicity. For example, in certain embodiments, 5'-methylcytidines in the RNA are partially or completely replaced with cytidines. In certain embodiments, the term "modification" refers to providing a 5'-cap or 5'-cap analog to the RNA. The term "5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule and generally consists of an mRNA guanosine nucleotide that is attached to the mRNA via an unconventional 5'-5' triphosphate linkage. In certain embodiments, the guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to the naturally occurring RNA 5'-cap, preferably the 7-methylguanosine cap. The term "5'-cap" includes 5'-cap analogs that resemble an RNA cap structure and have been modified to have the ability to stabilize RNA and / or enhance transcription of RNA. Providing an RNA with a 5'-cap or 5'-cap analog may be accomplished by in vitro transcribing a DNA template in the presence of the 5'-cap or 5'-cap analog, where the 5'-cap is co-transcriptionally incorporated into the generated RNA strand, or an RNA may be generated, for example, by in vitro transcription, and a 5'-cap may be attached to the RNA post-transcriptionally using a capping enzyme, such as a bacterial capping enzyme.

[0247] Further modification of the RNA may be the extension or truncation of the naturally occurring poly(A) tail, or the introduction of a UTR not related to the coding region of said RNA, for example, the modification of the 5'- or 3'-untranslated region (UTR), such as replacing or inserting one or more, preferably two copies of an existing 3'-UTR with a globin-derived 3'-UTR, such as alpha2-globin, alphaI-globin, β-globin, etc. RNA with unmasked poly-A sequences is translated more efficiently than RNA with masked poly-A sequences. To improve the stability and / or expression of the RNA, the RNA may be modified to be present with a poly-A sequence, preferably with a length of 10-500, more preferably 30-300, even more preferably 65-200, and especially 100-150 adenosine residues. To improve the expression of the RNA, the RNA may be modified to increase the GC content within the coding region to improve the stability of the mRNA and to perform codon optimization and thereby enhance translation in the cell. The modified mRNA may be enzymatically synthesized and packaged into nanoparticles, such as lipid nanoparticles, and administered, for example, intramuscularly.

[0248] The nucleic acid molecules can be encapsulated in microcapsules, colloidal drug delivery systems (e.g., liposomes, microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions prepared, for example, by coacervation techniques or interfacial polymerization. Such techniques are known in the art and are disclosed in (Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, J., ed. (2000)). Targeted delivery of drugs to specific cell subsets can enhance the therapeutic index. Antibody-targeted drugs bind to cells that contain the antigen recognized by the antibody or its binding fragment. This includes, for example, maleimide-functionalized PEG-PLGA polymer nanoparticles, or simply incorporating a polypeptide or fusion protein that interacts with CCR5 into a composition that includes a delivery moiety or shuttle agent.

[0249] Ex vivo approaches contemplate administering gene editing components such as CRISPR to modify cells to contain or express the polypeptides or fusion proteins described herein.

[0250] Administration According to the present disclosure, compositions or medicaments comprising or encoding the polypeptides or fusion proteins described herein may be administered to a patient for wound healing or to slow, maintain, or regenerate muscle in various conditions associated with muscle loss or reduced ability to regenerate functionality.

[0251] The composition may be delivered by injection, by topical or mucosal application, by inhalation, or via oral route, including controlled release, over a long period of time, and in an amount effective for stimulating muscle regeneration levels in subjects.Administration may be local or systemic (e.g., parenteral administration, for example, via intravenous, intraperitoneal, intradermal, subcutaneous, or intramuscular route), or may be targeted.In some embodiments, administration of the drug that interacts with CCR5 is systemic or directly to wound.Subcutaneous or intramuscular route may be directly to affected muscle tissue.

[0252] The polypeptide, fusion protein, nucleic acid, vector, or cell described herein can be formulated into a preparation suitable for ointment, cream, patch, powder, or other topical preparation.Low molecular weight polypeptide or fusion protein formulations can deliver drugs through the skin to deeper muscle tissue.Therefore, such formulations may contain one or more drugs that enhance the penetration of active ingredients through the skin.For topical application, the polypeptide, fusion protein, nucleic acid, or vector can be included in a wound covering composition and / or a skin coating composition.

[0253] The amount of drug to be administered may be determined by one of ordinary skill in the art by standard clinical procedures. In addition, in vitro assays may be employed in some cases to help identify optimal dosage ranges. The exact dose to be employed will also depend on the nature of the drug and other clinical factors (condition, weight, age, other conditions of the subject, route of administration, and type of composition (cell, scaffolded, hydrogel-based, or oral formulation)). The exact dosage that is therapeutically or prophylactically effective and not harmful can be determined by one of ordinary skill in the art. Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, (Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, J., ed. (2000)) and later editions.

[0254] Reference to an effective amount includes a therapeutically or prophylactically or regeneratively effective amount. As used herein, a "therapeutically effective amount" refers to an amount of a composition that contains chemokine receptor agonist activity, which is effective to produce some desired therapeutic effect in at least some subpopulation of cells of an animal, with a reasonable benefit / risk ratio applicable to any medical treatment. For example, the amount of polypeptide or fusion protein administered to a subject is sufficient to produce statistically significant, measurable muscle repair or regeneration. The determination of a therapeutically effective amount is well within the capabilities of a person skilled in the art. In general, a therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, and the severity and type of the condition in the subject, as well as the administration of other pharmacologic active drugs.

[0255] The term "administration" as used herein refers to placing the composition in a subject by a method or route that results in at least partial localization of the composition at a desired site so that the desired effect occurs.The route of administration suitable for fast-acting compositions depends on its format and includes both local and systemic administration.In general, local administration results in more polypeptide or fusion protein or cells treated with polypeptide or fusion protein being delivered to a specific site compared to the whole body of the subject.On the other hand, systemic administration results in delivery to substantially the whole body of the subject.One method of local administration is intramuscular injection.

[0256] According to the present invention, the term "administration" also includes transplantation of cells into a subject. The term "transplantation" as used herein refers to the process of implanting or transferring at least one cell into a subject. The term "transplantation" includes, for example, autologous transplantation (taking cells from one location in a patient and transferring them to the same location or to another location in the same patient), allogeneic transplantation (transplantation between members of the same species), and xenogeneic transplantation (transplantation between members of different species). Those skilled in the art will be well aware of methods of implantation or transplantation of stem cells for muscle repair and regeneration that are applicable to the present invention. See, for example, U.S. Patent No. 7,592,174 and U.S. Patent Publication No. 2005 / 0249731, the contents of both of which are incorporated herein by reference.

[0257] As described herein, the regeneration of muscle tissue in the method may be associated with minimal fibrosis.In particular, the methods and drugs described herein may reduce and / or inhibit scar-like tissue formation in damaged, non-regenerating, or atrophied muscle tissue.Thus, in some embodiments, scar-like tissue formation in damaged muscle tissue is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% compared to controls not treated with the drug of the present invention.Fat deposits may be reduced as well.

[0258] However, suitable dosage ranges for intravenous administration of the polypeptides or fusion proteins described herein are generally about 1.25-5 micrograms of active compound per kilogram (Kg) body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg / kg body weight to 1 mg / kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral compositions preferably contain 10% to 95% active ingredient.

[0259] By "derivative" is meant a drug or active derived from a polypeptide or fusion protein by modification of the amino acid sequence or, for example, conjugation or complexation with other chemical moieties or expression (e.g., as a fusion protein) or other post-translational modification techniques as would be understood in the art. The term "derivative" includes within its scope alterations made to the parent sequence, including additions or deletions, that result in functionally equivalent or functionally enhanced molecules.

[0260] By "isolated" is meant material that is substantially or substantially free from components that normally accompany it in its natural state.

[0261] The term "subject" includes patients and refers to any subject of medical or veterinary interest. The subject may be a vertebrate subject, such as a mammalian subject (e.g., cows, pigs, dogs, cats, horses, llamas, camels, etc.), non-mammals, reptiles, birds, fish, etc. The subject includes a human for whom prevention or treatment is desired. The subject may be in need of prevention or treatment of a pathology, disease, disorder, or condition associated with cancer, wound care, sarcopenia, or other tissue degeneration.

[0262] As used herein, the term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, cDNA, or DNA. The term refers to oligonucleotides typically longer than 30 nucleotides.

[0263] The term sequence "identity" as used herein refers to the degree of sequence identity on a nucleotide-nucleotide basis or amino acid-amino acid basis over a window of comparison.That is, "percent sequence identity" is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions where the same nucleic acid base {e.g., A, T, C, G, U} or the same 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 obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., window size), and multiplying the result by 100 to obtain the percent sequence identity. For the purposes of the present invention, "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, Calif., USA) using the standard default settings used with reference to the 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), part of the European Molecular Biology Laboratory. This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings including Gap Open: 10.0 and Gap Extend 0.5 are used. The default matrix "Blosum62" is used for amino acid sequences and the default matrix.

[0264] The term sequence "identity" refers to the percentage of amino acids that are identical or that constitute conservative amino acid substitutions as defined in Table 3 above. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12: 387-395). In this method, sequences that are similar to or substantially different in length from the sequences exemplified herein are compared by inserting gaps in the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. Methods including conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in Molecular Cloning: A Laboratory Manual, 3 rded., vol. 1-3, ed. Sambrook et al.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, (1992) (regularly updated). Immunological techniques are generally known in the art and can be found in Current Protocols in Immunology, ed. Coligan et al.; Greene Publishing and Wiley-Interscience, New York, (1992) (regularly updated); 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, 6 thed., 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, NY, (2014). Conventional methods of gene transfer and gene therapy may be adapted for use in the present invention. For example, Gene Therapy: Principles and Applications, ed. T. Blankenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. PD 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 282010.

[0265] The invention disclosed and defined herein will be understood to extend to all the different combinations of two or more individual features mentioned or apparent from the text or figures, all of which constitute various different aspects of the invention. EXAMPLES

[0266] Example 1 - Materials and Methods Assessment of muscle volume loss injury and regeneration in mice Injury: Male C57BL / 6J mice between 10-12 weeks of age were anesthetized and the left hind limb was shaved. An approximately 1 cm unilateral incision was made to expose the underlying fascia. The left hind limb was extended and the surrounding tissue was retracted to expose it through the incision site. A full thickness segment of 3x4 mm rectus femoris muscle was removed. Immediately after, the injury site was filled with fibrin hydrogel with or without 200 ng or 500 ng of hrNAMPT(1) (hydrogel components; 40 μl, 8 mg / ml human fibrinogen (FIB3, Enzyme Research Laboratories), 4 U / ml bovine thrombin, (T4648, Sigma), 5 mM CaCl2, 17 μg / ml aprotinin (ab146286, Abcam)), which polymerized in the defect. The soft tissue was then sutured closed.

[0267] Histology: After 10 days of treatment, animals were sacrificed and wounds were harvested for histological analysis. The defect site and associated proximal and distal segments of the quadriceps (including rectus femoris, vastus medialis, and vastus lateralis) were excised and embedded. Histological analysis was performed on serial paraffin sections (4 μm sections harvested through the center of the wound). Sections were stained with Masson's trichrome (to detect collagen deposition) and the degree of fibrosis (represented by blue staining) was measured by histomorphometric analysis using ImageJ software (version 1.51h, National Institutes of Health, USA). To maintain sample uniformity, the length of the vastus medialis taken at multiple depths ranging from 1.0 mm to 3.0 mm served as a reference between tissue sections to determine the depth of sectioning. For quantification of fibrosis, the average muscle fibrosis area at each depth was scored and normalized with the area of ​​the rectus femoris. The total muscle area was determined by calculating the average area of ​​the rectus femoris muscle at each depth.

[0268] Immune cell profiling and quantification of PAX7+ cells by flow cytometry: Four, six, or eight days after treatment with 0.5 μg hrNAMPT(1) delivered by fibrin hydrogel or control fibrin hydrogel alone, mice were euthanized by CO2 asphyxiation. The defect site and associated proximal and distal segments of quadriceps were isolated and transferred into 890 μl complete RPMI (with 10% FBS and 2 mM Glutamax, Life Technologies). Tissues were minced with surgical scissors and 100 μl 10 mg / ml collagenase II (Sigma-Aldrich) and 10 μl 10 mg / ml DNAse I (Biolabs), and 100 μl of dispase II (10 mg / ml) was added to the digest to obtain PAX7. The mixture was vortexed and incubated at 37° C. for 45 min. Collagenase was inactivated with 500 μl ice-cold PBS, 5% FBS, 5 mM EDTA. The mixture was then filtered through 70 μm and 40 μm filters. The cell suspension was diluted with 1 ml complete RPMI and centrifuged at 300Xg for 10 min. The supernatant was discarded and the pellet was resuspended in 250 μl complete RPMI and aliquoted into 96-well U-bottom plates for antibody staining. The cell solution was centrifuged, the supernatant discarded, and washed with PBS. The cell viability stain used was 100 μl Zombie Aqua (Biolegend) Live-Dead dye diluted in PBS (1:400 dilution) and stained for 30 min at 4° C. The cells were then blocked with FcX (anti-CD16 / 32 antibody, Biolegend, 1 μg / ml) flow cytometry buffer and centrifuged. Primary surface antibody staining was performed on two separate stains with 100 μl of anti-mouse antibody cocktail (Biolegend) diluted in flow cytometry buffer: T cell staining with 2 μg / ml anti-CD4 (clone RM4.5, #100516), anti-CD8 (clone 53-6.7, #100738), and anti-CD3 (clone 17A2, #100220).Neutrophil and macrophage staining was performed with 2 μg / ml anti-CD-11b (clone M1 / 70, #101208), 1 μg / ml anti-Ly6G (clone 1A8, #127628), 4 μg / ml anti-F4 / 80 (clone BM8, #123147), 10 μg / ml anti-CD80 (clone 16-10A1, #104714), and 2.6 μg / ml anti-CD206 (clone C068C2, #141720). Cells were stained for 30 min on ice and washed as above. For internal Foxp3 staining in the T cell panel, cells were fixed for 35 min with 100 μl of fixation / permeabilization solution (42080, Biolegend). Cells were then washed and resuspended in 100 μl of flow cytometry buffer containing 0.5% saponin and 5 μg / ml anti-Foxp3 (clone 3G3, #35-5773-U100) for 45 min. Cells were then resuspended in flow cytometry buffer (100 μl) and acquired on a Fortessa x20 (Beckman Coulter). Satellite cell flow cytometry staining was performed with 200 μl of the following antibody cocktail (Biolegend) diluted in flow cytometry buffer: anti-VCAM / CD106 biotin (clone 429 (MVCAM.A), #105703) at 5 μg / ml, anti-streptavidin (#405250) at 2.5 μg / ml, anti-CD45 (clone 30-F11, #103114) at 2 μg / ml, anti-CD11b (clone M1 / 70, #101208), anti-Ly6G (clone 1A8, #127607), and anti-CD31 (clone MEC13.3, #102507) at 1 μg / ml. Cells were also stained for 1 h on ice with 200 μl of flow cytometry buffer containing 0.5% saponin and intracellular antibody cocktail: 1 μg / ml anti-Ki67 (clone 16A8, #652411) from Biolegend, 10 μg / ml anti-Pax7 (clone Pax7 / 497, #NBP2-34706AF488) from Novus Biologicals.Cells were then resuspended in 25 μl of flow cytometry buffer (275 μl) with Invitrogen Count Bright Absolute Counting Beads (25,000 beads, #C36950) and acquired on a Fortessa x20 (Beckman Coulter). All events were acquired and the number of PAX7+ cells per 10,000 wound cells was calculated using the following formula: PAX7+ number in wound = 10,000xPAX7+ cell count / [(1 / 25,000)xbead countx(percent viable cells / 100)xtotal cell count after digestion]. To quantify the number of proliferative PAX7+ cells, the same calculation was performed using PAX7 and Ki67 double positive cell counts.

[0269] Immunofluorescence on frozen sections: Immunostaining was performed on 10 μm frozen sections using standard protocols and antigen retrieval (10% sodium citrate, 0.05% Tween 20, pH 6.0). To minimize nonspecific binding of mouse antibodies to mouse tissues, sections were blocked with 2% BSA, 5% normal goat serum, 0.3% Triton-X, and AffiniPure Fab fragment goat anti-mouse IgG (H+L) (Jackson Immuno Research Laboratories) in PBS. Antibodies: Mouse anti-mouse Pax7 (2 μg / ml, Developmental Studies Hybridoma Bank) and Alexa Fluor-conjugated secondary antibodies (Thermo Fisher). Muscle sarcolemma was visualized with rhodamine-labeled wheat germ agglutinin (WGA) (Vector Laboratories) and nuclei were visualized by staining with DAPI (Sigma-Aldrich).

[0270] Quantification of centralized myofibers: Hematoxylin and eosin (H&E) staining was performed on 4 μm paraffin-embedded sections. Nuclear centralisation within myofibers was counted by histomorphometric analysis using ImageJ software (version 1.51h, National Institutes of Health, USA) from five consecutive sections per sample. To maintain sample uniformity, the length of the vastus medialis taken at multiple depths ranging from 1 to 3 mm served as a reference between tissue sections to determine the sectioning depth. For fibrosis quantification, the average muscle fibrosis area at each depth was scored and normalised with the area of ​​the rectus femoris. The total muscle area was determined by calculating the average area of ​​the rectus femoris at each depth.

[0271] Competitive binding of NAMPT to CCR5 (ELISA) Competitive binding of hrNAMPT to mrCCR5: ELISA plates (Medium binding, Greiner Bio-One) were coated overnight at 4°C with PBS containing 1% BSA or 20 nM recombinant mouse CCR (MyBioSource). The wells were then blocked with PBS containing 0.05% Tween-20 (PBS-T) with 1% BSA for 1 h at room temperature. The wells were washed three times with PBS-T and further incubated with hNAMPTcif at various concentrations (0 nM to 100 nM) for 1 h in PBS-T with 0.1% BSA containing 100 nM hrNAMPT(1) (Peprotech). Bound hrNAMPT(1) molecules were detected using biotinylated NAMPT antibody and HRP-streptavidin (human PBEF / visfatin DuoSet ELISA, R&D Systems). To obtain specific binding values, the signal obtained from BSA-coated cells for each hrNAMPT concentration was used to remove non-specific binding. To obtain the half maximal inhibitory concentration (IC50) of hNAMPTnif, the specific binding data were fitted by non-linear regression in Prism7 using A450nm = A450nmMin + (A450nmMax - A450nmMin) / (1+10^(X-LogIC50)).

[0272] cell culture Mouse muscle cell line C2C12 (Yaffe, D. & Saxel, O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725 (1977)) was cultured in Growth Medium (Dulbecco's Modified Eagle Medium (4.5 g / l D-glucose, no L-glutamine, no sodium pyruvate (Gibco)) + 20% fetal bovine solution - One Shot (Gibco) + 1% Glut Max 100x (Gibco)). Cells were maintained at 37°C and 5% CO2. 70% confluent, passage 8 cells were extracted from a T75 flask with 0.025% Trypsin (Tryspin) EDTA (Gibco), neutralized with Growth Medium, and spun at 180Xg for 5 minutes to pellet the cells. Cells were then resuspended in 10 ml of fresh Growth Medium. 500 μl of cells were plated at 1×103 cells / ml in 8 wells on a cover slip II (Sarstedt) chamber slide. For readhesion, cells were left at 37° C. for 4 hours. For drug treatment, the medium was supplemented with the appropriate dose and incubated for 6 hours.

[0273] For isolation of primary mouse myoblasts, skeletal muscles from E17.5 C57BL / 6J mouse limbs were minced and digested with 0.125% trypsin for 20 min at 37°C. Myoblasts were depleted by plating cells onto 10 cm2 tissue culture dishes (2 embryos per dish) in growth medium (DMEM + 20% FBS) for 1 h. Cultures with non-adherent cells were replated onto gelatin-coated 10 cm2 tissue culture dishes in growth medium for 24 h. Myoblasts were depleted for fibroblasts before co-culture on gelatin-coated 48-well plates in DMEM + 20% FBS + 10% L929 supplemented medium. Per well, 100,000 myoblasts were plated together with either 7,500 MafB / c-Maf-deficient (Maf-DKO) macrophages (Aziz, A., Soucie, E., Sarrazin, S. & Sieweke, MH MafB / c-Maf deficiency enables self-renewal of differentiated functional macrophages. Science 326, 867-871 (2009)) or 1,000 3T3 cells. For drug treatment, the medium was supplemented with the appropriate dose and incubated for 24 h.

[0274] Cell surface CCR5 receptor concentration The mouse muscle cell line C2C12 was cultured as previously described (see above). The mouse macrophage cell line Raw264.7 (ATCC) was cultured in growth medium (Dulbecco's modified Eagle's medium + 10% FBS). At 70-80% confluence, cells were detached using a cell scraper and membrane proteins were isolated using an extraction kit (Plasma Membrane Protein Extraction Kit, abcam). The CCR5 concentration in the membrane extract was then measured by ELISA (Mouse Ccr5 ELISA Kit, Biorbyt), and the amount of CCR5 per cell was then calculated using [CCR5](molecules / cell)=[CCR5](ng / cell) / CCR5mw*10^-9*N0, where N0=Avogadro's constant.

[0275] Generation of truncated hNAMPTcif variants and proliferation assays Human NAMPTcif (hNAMPTcif) and truncated variants of hNAMPTcif were designed by stepwise removal of the N-terminal region containing positively charged amino acids. Four truncated forms were produced, numbered T1 to T4. Recombinant proteins were produced using a bacterial expression system. hNAMPTcif variants were purified by FPLC using His-tag affinity purification. Cell proliferation assays were performed using C2C12 mouse myoblasts treated with purified recombinant hNAMPTcif variants as well as full-length NAMPT. Cells were treated with 10 nM recombinant protein for 48 hours in DMEM supplemented with 2% fetal bovine serum at 37°C and 5% CO2. PBS treatment was used as a negative control. 10% fetal bovine serum was used as a positive control. Proliferation quantification was performed using a commercially available CyQuant Proliferation Assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions and read on a Synergy H1 plate reader (Bio Tek). Data were expressed as percentage increase versus.

[0276] Generation of PlGF-NAMPTcif fusion protein and proliferation assay The heparin-binding sequence of placental growth factor 2 (PlGF2) was fused to the N-terminus of NAMPTcif and produced using a bacterial expression system. PlGF-NAMPTcif was purified using His-tag affinity purification followed by size-exclusion chromatography on an FPLC. Cell proliferation assays were performed using C2C12 mouse myoblasts treated with the recombinant NAMPTcif and full-length NAMPT produced. Cells were treated with recombinant protein at concentrations of 2 nM, 10 nM, and 20 nM for 48 hours in DMEM supplemented with 2% fetal bovine serum at 37°C and 5% CO2. PBS treatment was used as a negative control. 10% fetal bovine serum was used as a positive control. Quantification of proliferation was performed using a commercially available CyQuant Proliferation Assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions and read on a Synergy H1 plate reader (Bio Tek). Data were expressed as a percentage increase relative to the negative control. An unpaired two-tailed t test was used to determine statistical significance.

[0277] TLR4 signaling assay Assays for TLR4 receptor activation were performed using the HEK-Blue TLR4 reporter cell line (InvivoGen). In this cell line, TLR4 and downstream NFκB signaling induce expression and secretion of alkaline phosphatase, which can be quantified via a colorimetric assay. To ensure no TLR4 activation by traces of endotoxin, HEK-Blue cells were treated with 19 nM NAMPTcif or PlGF-NAMPTcif plus 5 ug / ml polymyxin B for 24 hours. TLR4 activity was reported as the equivalent amount of activation by bacterial lipopolysaccharide (LPS). Thus, a standard curve was generated by serial dilutions of LPS from 0.01 to 20 ng / mL. Detection of alkaline phosphatase activity was performed using the QUANTI-Blue kit (InvivoGen) according to the manufacturer's instructions, and plates were read using a Synergy H1 plate reader (BioTek). Data were expressed in ng / ml as the equivalent LPS concentration. One-way analysis of variance with Dunnett's multiple comparison test was performed to determine statistical significance.

[0278] Larval zebrafish muscle injury and EdU pulse chase Zebrafish larvae (4 dpf) were anesthetized in Ringer's solution with 0.01% tricaine (MS-222) (Sigma-Aldrich). A needlestick injury was performed in the dorsal myotome with a 30-gauge needle puncture, generating a large lesion with many damaged muscle fibers. Nampt fragment treatment was performed by incubating 4 dpf needle-injured larvae in Ringer's solution with 57 nM Nampt immediately after injury. 6 dpf (2 dpi) needle-injured larvae were transferred to Ringer's solution containing 50 μg ml-1 EdU (Thermo Fisher Scientific) for 1 h and chased for an additional 1.5 h before fixation. Samples were developed using Click-iT EdU Alexa Fluor 647 Imaging Kit (Thermo Fisher Scientific) according to the manufacturer's instructions, followed by phalloidin immunostaining (Thermo Fisher Scientific). EdU+ cells in the area encompassing two myotomes on either side of the injury were quantified as the number of EdU+ cells outside the injury area. EdU+ cells in the tail hematopoietic tissue were excluded from the analysis. Statistical analysis was performed using two-way ANOVA with Tuckey's multiple comparison test.

[0279] Example 2 - Exogenous NAMPT supplementation accelerates regeneration in a mouse model of muscle volume loss Muscle volume loss is a paradigm of injury that is normally resistant to repair processes mediated by endogenous stem cells and an area of ​​unmet medical need. Herein, it is shown that the addition of exogenously applied NAMPT can accelerate regeneration in a mouse model of muscle volume loss. Surprisingly, delivery of hrNAMPT to muscle defects via fibrin hydrogel was able to fully restore muscle architecture when applied to the wound site, whereas a fibrin-only control hydrogel could not (Figure 1A-D). On average, treatment with a single dose of hrNAMPT (0.5 μg) at the injury site reduced the mean muscle area by 3.276 ± 0.4926 mm2. 2Addition of NAMPT in the VML injury model resulted in a significant increase in both the total number and percentage of proliferating PAX7+ satellite cells (Figure 1E-G), and a significant increase in the number of centrally nucleated (de novo) myofibers (Figure 1H-I).

[0280] These findings suggest that exogenously supplemented NAMPT protein stimulates muscle regeneration in the context of acute injury in adult mammalian muscle.

[0281] Example 3 - Selective signaling of NAMPT through the CCR5 receptor is required to induce myoblast proliferation. CCR5 receptors were found in C2C12 myoblasts at a concentration of 2,470 ± 441 molecules / cell (n = 6), consistent with previously reported physiologically meaningful levels of CCR5. To determine the physiological significance of NAMPT-CCR5 interaction, two human recombinant NAMPT protein sources (hrNAMPT(1) and hrNAMPT(2)) were applied to C2C12 myoblasts and proliferation assays were performed using EdU incorporation as a measure. Both sources of NAMPT resulted in a comparable and significant dose-dependent increase in myoblast proliferation (Figure 2A). To analyze the intracellular and extracellular roles of NAMPT during proliferation, these myoblasts were treated with GMX1778, a highly specific and potent inhibitor of NAMPT enzymatic function. Drug treatment had no negative effect on the basal level of proliferation of C2C12 in culture, nor did it affect the increase in myoblast proliferation that resulted after exogenous NAMPT supplementation (Figure 2A). This emphasizes that the growth-promoting role of NAMPT is not due to its intracellular enzymatic function. The enhanced growth response observed after NAMPT supplementation could be reproduced in C2C12 cells by addition of the classical CCR5 ligands CCL8 / MCP-2 and CCL4 / MIP-1β, but not by the CCR2 ligand CCL2 / MCP-1 (Fig. 2A). In addition, this growth response was blocked by the CCR2 / CCR5 dual antagonist cenicriviroc (CVC) and the CCR5-selective antagonist maraviroc (MVC), but not in the presence of the CCR2-selective antagonist PF-4136309 (PF) (Fig. 2A).

[0282] Taken together, the data highlight the requirement for selective signaling of NAMPT through the CCR5 receptor to induce myoblast proliferation.

[0283] NAMPT is a large homodimeric intracellular enzyme that acts as a cytokine when released into the extracellular environment. However, the domain of NAMPT involved in cytokine activity is unknown. By reviewing the crystal structure of NAMPT, it was determined that the C-terminal end structure of NAMPT is very similar in size and structure (C-terminal α-helix and β-sheet) to classical CCR-binding chemokines (such as CCL2) (Figure 2B). Furthermore, the domain extends out from the core structure of the protein and may facilitate receptor binding. Thus, we recombinantly reproduced the C-terminus of NAMPT and tested its ability to compete with NAMPT binding to CCR5 and stimulate satellite cell proliferation. Surprisingly, this fragment, referred to herein as "cytokine finger" (cif), inhibits NAMPT binding to CCR5 (IC50=21.5 nM, Figure 2C) and stimulates satellite cell proliferation in a dose-dependent manner, inducing myoblast proliferation (Figure 3F).

[0284] Taken together, these data demonstrate that the C-terminal cif domain is involved in the muscle cytokine activity of NAMPT.

[0285] Example 4 - Truncation variants of hrNAMPTcif To further explore the active fragments of NAMPT cytokine fingers, we produced four truncated forms of NAMPTcif, numbered T1 to T4 (Figure 3B-E), and evaluated the ability of the variants to stimulate satellite cell proliferation. Surprisingly, hNAMPTcif and variants T1-T3 demonstrated enhanced stimulation of satellite cell proliferation compared to full-length hNAMPT, even though all molecules contained the cytokine fingers (Figure 3F).

[0286] Taken together, these data unexpectedly demonstrated that truncated forms of NAMPTcif have improved satellite cell proliferation capabilities compared to full-length NAMPT.

[0287] Example 5 - NAMPT cytokine derivatives NAMPTcif fused to ECM-binding / syndecan domain The addition of ECM and / or syndecan binding motifs is used as one of the preferred approaches to optimize delivery and increase sustained signaling at CCR5 (see Mochizuki et al Nat. Biomed Engineering 2019). Several proteins, such as laminin, bind to syndecans. One particular syndecan binding moiety is the globular domain (SB) of the laminin alpha chain, which has the sequence RKRLQVQLSIRT. The addition of binding molecules may be done by synthetic means or using recombinant approaches, as known in the art. Exemplary, non-limiting ECM binding moieties include RGD, or YGISR, YIGSR, GFOGER, IKVAV, and GEFYFDLRLKGK.

[0288] Advantageously, the fusion of the ECM-binding moiety PlGF2 to the N-terminus of NAMPTcif retained the growth-promoting activity of unfused NAMPTcf in vitro (Figure 4). Since there is no ECM to bind in vitro, it is predicted that increased activity of the fusion protein will be achieved in vivo, where the ECM-binding moiety PlGF2 will aid in delivery to the cell membrane and increase sustained signaling at CCR5 in myoblasts.

[0289] Dimerization of NAMPT cytokine fingers NAMPT is naturally homodimeric, and known CCR5-binding chemokines are dimeric, and can form multimers (trimers, tetramers, and more through oligomerization) that vary receptor binding affinity and signal transduction. In an embodiment, NAMPTcif and any polypeptide, fusion protein, or derivative described anywhere herein can form a dimer. A single cysteine ​​residue naturally present at the N-terminus of NAMPTcif is used to force its dimerization. The binding affinity of dimeric NAMPTcif to CCR5 can be verified by ELISA and surface plasmon resonance (SPR) assay. The activity of dimeric NAMPTcif to promote proliferation of mouse primary satellite cells would also suggest potential muscle regeneration ability. As an example, quiescent satellite cells are sorted from fresh muscle as used above, depending on negative or positive cell surface markers (CD31-, CD11b-, CD45-, TER119-, Sca1-, CD34+, CD106+) specific for the subset of satellite cells shown to have the highest self-renewal properties in vivo. The cells are cultured in vitro and the efficiency of NAMPT derivatives to stimulate the proliferation of these primary muscle stem cells is evaluated.

[0290] A model to examine various states of muscle loss In addition to the muscle volume loss model used in the examples above, there are different models in which muscle loss can be analyzed.

[0291] As an example, one suitable model for validating function derivatives is the established model of muscle damage induced by cardiotoxin. Cardiotoxin is a myonecrotic agent that kills muscle cells without destroying the muscle ECM, providing an important model for validating fusion proteins containing ECM-binding motifs. This is the most commonly used model for assaying muscle stem cell activation because it leaves the majority of stem cells intact. The standard model involves intramuscular injection of cardiotoxin into the tibialis anterior muscle and monitoring the recovery of lost fibers. Another model is the mdx mouse, which has also been established in ARMI. Since the mdx model exhibits chronic muscle fibrosis and muscle degeneration, the mdx model is used to validate both the potential ability of polypeptides, fusion proteins, and derivatives to activate stem cells, as well as their anti-fibrotic abilities. Muscle regeneration is validated at established time points using standard histological assays as described above.

[0292] Example 6 - Reduction of TLR4 receptor activation from NAMPTcif stimulation In muscle regeneration, anti-inflammatory responses are responsible for fibrosis and scar formation during healing. NAMPT has previously been shown to be a regulator of the inflammatory program through binding to inflammatory receptors such as TLR4. Furthermore, activation of TLR4 induces an excessive inflammatory response that leads to severe adverse events. This is problematic for a subset of vulnerable patients, for example those suffering from inflammatory myopathies.

[0293] Inflammatory myopathy is a group of muscle diseases in which immune system attacks differentiated muscle cells, resulting in muscle loss and chronic inflammation.Therefore, excessive inflammatory response is undesirable and can worsen the condition of patients.There is a need for an effective treatment that can restore the muscles of these vulnerable patients without triggering harmful inflammatory response.

[0294] Herein, it was confirmed that NAMPT activates TLR4 in vitro, and advantageously, NAMPTcif and PlGF2-NAMPTcif were demonstrated to inhibit TLR4 activity (Figure 5). These results suggest that NAMPTcif, polypeptides, fusion proteins, and derivatives described herein may improve muscle regeneration by avoiding inflammatory responses.

[0295] Example 7 - Human NAMPT variants stimulate proliferation of muscle progenitor cells To further elucidate the active fragments of the NAMPT cytokine finger, the inventors have generated additional truncated NAMPT 402-491 , NAMPT 414-491 , NAMPT 422-491 , NAMPT 430-491 , NAMPT 436-491 and NAMPT 422-471 (NAMPT variants, also referred to herein as hNAMPTcif, hNAMPTcif-T1, hNAMPTcif-T2, hNAMPTcif-T3, hNAMPTcif-T4 and hNAMPTcif-T5, respectively) were investigated. Amino acid numbers correspond to full-length NAMPT, e.g., SEQ ID NO: 19. The predicted structures of the NAMPT variants are shown in FIG. 6.

[0296] C2C12 mouse myoblasts were treated with 20 nM full-length NAMPT or NAMPT variants for 48 h and proliferation of muscle progenitor cells was assessed. 402-491 , NAMPT 414-491 , and NAMPT 430-491 All of the NAMPT variants tested showed enhanced cell proliferation compared to all other NAMPT variants tested (Figure 7). 422-491 There was a statistically significant increase in proliferation in muscle progenitor cells treated with NAMPT compared to full-length NAMPT. 436-491 and NAMPT 422-471 exhibited lower cell proliferation of muscle progenitor cells compared to full-length NAMPT and other NAMPT variants.

[0297] Example 9 - Human NAMPT variants stimulate proliferation of human satellite cells The inventors then synthesized full-length NAMPT, NAMPT 402-491 , and NAMPT 422-491 We aimed to compare the activity of NAMPT and NAMPT 1 on the proliferation of human satellite cells. Human primary satellite cells were cultured in the presence of 20 nM of full-length NAMPT or NAMPT 1. 402-491 and NAMPT 422-491 The subjects were treated with 48 h.

[0298] NAMPT 402-491 exhibited increased satellite cell proliferation compared to full-length NAMPT, 422-491 full-length NAMPT and NAMPT 402-491 The results demonstrated even stronger satellite cell proliferation compared to both the control and control groups (Figure 8).

[0299] Example 10 - Human NAMPT variants stimulate proliferation of human endothelial cells Similarly, the inventors then synthesized full-length NAMPT, NAMPT 402-491 , and NAMPT 422-491 We investigated the activity of on the proliferation of human endothelial cells, which were derived from umbilical vein and treated with 20 nM full-length NAMPT (NAMPT) or NAMPT variants for 48 h.

[0300] NAMPT 402-491 Full-length NAMPT and NAMPT 422-491 However, full-length NAMPT and NAMPT 422-491 Also stimulated endothelial cell proliferation more than the negative control (Figure 9).

[0301] Example 10 - A minimal version of the NAMPT protein enhances proliferation in response to muscle injury in zebrafish larvae Next, the inventors tested the ability of NAMPT variants to stimulate cell proliferation in an in vivo zebrafish muscle injury response model. 402-491 and NAMPT 422-491 Treatment with induced a significant increase in cell proliferation within the injured area (Figure 10). 422-491 ) stimulated cell proliferation at significantly higher levels compared to human recombinant NAMPT (hrNAMPT), especially in wounds.

[0302] Taken together, these findings suggest that exogenously supplemented NAMPT protein stimulates muscle regeneration in the context of acute injury in zebrafish larvae, similar to the results for adult mammalian muscle described above, and reinforce the finding that it is the secreted form of NAMPT that is active in both of these in vivo conditions.

Claims

1. A polypeptide comprising, consisting essentially of, or consisting of a C-terminal portion of NAMPT that includes a truncated cytokine finger (cif) motif.

2. the only amino acid sequence of said polypeptide that is derived from or has homology or identity to a NAMPT protein is a truncated cif motif; and / or the polypeptide binds to CCR5 and / or stimulates proliferation of muscle progenitor cells; and / or the only amino acid sequence of the polypeptide that binds to CCR5 and / or stimulates proliferation of muscle progenitor cells is a truncated cif motif; The polypeptide of claim 1.

3. the truncation of the cif motif is an N-terminal truncation or a C-terminal truncation, 3. The polypeptide of claim 1 or 2, wherein optionally the N-terminal and / or C-terminal truncation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids.

4. the truncation is a truncation of 1 to 12 residues at the N-terminus of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent to SEQ ID NO: 1; the truncation is a truncation of 1 to 20 residues at the N-terminus of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent to SEQ ID NO: 1; the truncation is a truncation of 1 to 28 residues at the N-terminus of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent to SEQ ID NO: 1; or the truncation is a truncation of 1 to 35 residues at the N-terminus of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent to SEQ ID NO: 1; The polypeptide of claim 1.

5. 2. A polypeptide according to claim 1, wherein the cif motif comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 2 or 3, preferably SEQ ID NO:

1.

6. The polypeptide of claim 1, comprising, consisting essentially of, or consisting of a sequence identical 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 of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11; and / or is equal to or less than about 110, about 109, about 108, about 107, about 106, about 105, about 104, about 103, about 102, about 101, about 100, about 99, about 98, about 97, about 96, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80, about 79, about 78, about 77, about 76, about 75, about 74, about 73, about 72, about 71, about 70, about 69, about 68, about 67, about 66, about 65, about 64, about 63, about 62, about 61, about 60, about 59, about 58, about 57, or about 56 amino acids in length; and / or an amino acid sequence comprising or consisting of any of the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11, or having 1, 2, 3, 4, 5, 6, 7, or 8 conservative or non-conservative amino acid substitutions, deletions, or additions relative to said sequence, and retaining the activity of interacting with CCR5 or tissue stem cells; Preferably, it comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11. More preferably, it consists of the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. Polypeptide.

7. The polypeptide of claim 1, which is in a monomeric, dimeric, or homodimeric form.

8. A method for treating a vascular endothelial cell comprising administering to a patient a therapeutically effective amount of ... Optionally, the moiety that enhances tissue delivery or retention is an extracellular matrix (ECM) binding moiety; Preferably, the ECM binding moiety is binds to any one or more of the following ECM moieties: collagen, fibronectin, tenascin-C, osteopontin, fibrinogen, and heparan sulfate; and / or derived from placental growth factor (PlGF), amphiregulin (Areg), collagenase or von Willebrand factor (vWF); and / or comprising, consisting essentially of, or consisting of positively charged amino acid residues, where preferably the positively charged amino acid residues comprise, consist essentially of, or consist of RRRPK, RKKK, KRRR, or SEQ ID NOs: 12 and 13; and / or comprising, consisting essentially of, or consisting of an amino acid sequence identical 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 of SEQ ID NOs: 12-16, wherein the ECM binding moiety binds to one or more ECM proteins with the same affinity, no significantly different affinity, or at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% affinity as the ECM binding moiety of any of SEQ ID NOs: 12-16 from which the ECM binding moiety is derived; and / or comprising, consisting essentially of, or consisting of the amino acid sequence of any of SEQ ID NOs: 12-16, optionally fused, linked or directly linked to the amino acid sequence of any of SEQ ID NOs: 4-11; and / or The cytokine finger motif of NAMPT comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1; Fusion proteins.

9. A nucleic acid encoding the polypeptide described in claim 1 or the fusion protein described in claim 8.

10. A vector comprising the nucleic acid described in claim 9.

11. A cell comprising the nucleic acid described in claim 9, or a vector containing the nucleic acid.

12. A composition comprising a polypeptide according to claim 1, a fusion protein according to claim 8, a nucleic acid encoding said polypeptide or said fusion protein, a vector comprising said nucleic acid, or a cell comprising said nucleic acid or said vector, and a pharmaceutically acceptable carrier, diluent, or excipient, and optionally further comprising one, two, or all of: (a) tissue stem cells (such as satellite cells), or precursor cells thereof, or descendant cells thereof; (b) macrophages, or precursor cells thereof, or descendant cells thereof; and (c) a scaffold or retaining material.

13. An in vitro method for stimulating stem cell proliferation, such as satellite cell proliferation, comprising administering to a cell an effective amount of a polypeptide described in claim 1, a fusion protein described in claim 8, a nucleic acid encoding the polypeptide or the fusion protein, a vector containing the nucleic acid, or a cell containing the nucleic acid or the vector, thereby stimulating stem cell proliferation.

14. A pharmaceutical composition comprising the polypeptide of claim 1, the fusion protein of claim 8, a nucleic acid encoding said polypeptide or said fusion protein, a vector comprising said nucleic acid, or a cell comprising said nucleic acid or said vector, stimulating regeneration of muscle tissue in a subject, wherein if the subject has unwanted inflammation, preferably said unwanted inflammation is mediated by activation of a TLR, preferably said TLR activation is activation of TLR4; stimulating stem cell proliferation in a subject; or Treating an inflammatory myopathy in a subject, wherein preferably said inflammatory myopathy is polymyositis, dermatomyositis, inclusion body myositis, or necrotizing autoimmune myopathy; A pharmaceutical composition for use in 。 15. A pharmaceutical composition for use in stimulating the regeneration of muscle tissue, comprising: The pharmaceutical composition comprises a polypeptide of claim 1, a fusion protein of claim 8, or a cell containing or expressing said polypeptide or said fusion protein, and is administered to muscle, and optionally comprises a component that enhances delivery to or retention in muscle, wherein said polypeptide or said fusion protein binds to satellite cells and stimulates myoblast proliferation and muscle regeneration; Preferably, said cells are macrophages, preferably said macrophages are isolated from tissue, preferably said macrophages are derived from stem cells such as bone marrow progenitor cells or iPSCs. Pharmaceutical compositions.

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