ES2763349B2 - Myticin peptide and its use in cell regeneration - Google Patents

Description

DESCRIPTION

Myticin peptide and its use in cell regeneration

The present invention relates to peptides comprising the sequence SEQ ID NO: 2 and their therapeutic uses, specifically in cell and/or tissue regeneration.

BACKGROUND OF THE INVENTION

Antimicrobial peptides (AMPs) are innate immune effectors and have a wide range of actions against many microorganisms. In marine invertebrates, which live in environments with an abundance of potentially pathogenic microorganisms, AMPs are the main elements of the immune response. For example, in Mediterranean mussels ( Mytilus galloprovincialis), several types of AMP have been described, including myticin C.

Myticin C (Myt-C) is a cysteine-rich antimicrobial peptide isolated from hemocytes of the mussel Mytilus galloprovincialis whose antibacterial and chemotactic activity has been proven in vitro (Martinez-Lopez et al., Mar Drugs. 11(7): 2328 46 (2013)). Its antiviral effect has also been demonstrated not only against mollusc viruses, such as the oyster herpervirus OsHV-1, but also against human herpes simplex (HSV-1 and HSV-2) (Novoa B., et al., J Virol. 90(17):7692-702 (2016)) and against fish pathogenic rhabdoviruses (Balseiro P. et al., PLoS One.

6(8):e23140 (2011)).

During the life of any animal, injuries inevitably occur, and can result from a wide variety of events, such as contact with sharp or hot objects, or appear in certain clinical disorders, such as diabetes. Another important clinical example is the development of decubitus ulcers, that is, injuries caused by unmitigated pressure on any part of the body, especially portions of bone or cartilaginous areas. Although fully treatable if discovered early, pressure ulcers, like any other wound, can be fatal without medical care. In the case of poor general health, the closure (healing) of wounds may be delayed, and may lead to other problems, such as Infections, inflammation, tissue necrosis, and ineffective wound closure are once again life-threatening.

The wounds usually occur on the skin. The skin is a vital organ that performs multiple functions, such as sensitive functions, protective functions against external aggressions, as well as immunological, metabolic or thermoregulatory functions. These roles are possible due to the complex structure that associates various tissues. The skin consists of three distinct overlapping layers: the epidermis, the dermis, and the hypodermis. The epidermis is a lining epithelium, which constitutes the external structure of the skin and provides its protective function. This function is provided by the cohesion of the epithelial cells and by the production of a tough, filamentous protein, keratin.

In general, wound healing is described as consisting of three phases, namely, the inflammatory phase, the proliferative phase, and the maturation phase (referred to as the acute inflammatory phase, collagen and extracellular matrix synthesis, and remodeling). The healing process sequence begins during an acute inflammatory phase with provisional tissue deposition. This is followed by re-epithelialization, collagen synthesis and deposition, fibroblast proliferation, and neovascularization, all of which ultimately define the remodeling phase.

The inflammatory phase is characterized by hemostasis and inflammation. Collagen exposed during wound formation activates the coagulation cascade (both the intrinsic and extrinsic pathways), initiating the inflammatory phase. Platelets, the first responder cell, release multiple chemokines that help stabilize the wound through clot formation. These mediators act to control bleeding and limit the extent of injury. The second responsive cell to migrate into the wound, neutrophils, are responsible for debris uptake, complement-mediated opsonization of bacteria, and bacterial destruction via oxidative burst mechanisms (i.e., superoxide and peroxide formation). of hydrogen). Macrophages are essential for wound healing. Numerous enzymes and cytokines are secreted by macrophages, which marks the transition to the process of tissue reconstruction, that is, the proliferative phase.

During the proliferative phase, epithelialization, angiogenesis, granulation tissue formation, and collagen deposition are the main steps in wound healing. Epithelialization appears at an early stage of wound repair. Angiogenesis, stimulated, for example, by TNF-alpha, is noted by endothelial cell migration and capillary formation. The new capillaries transport nutrients to the wound and help maintain the granulation tissue bed. The final part of the proliferative phase is the formation of granulation tissue. Fibroblasts differentiate and produce ground substance and then collagen. Basal substance is deposited in the wound bed; collagen is then deposited as the wound undergoes the final phase of repair. Many different cytokines, including PDGF, insulin-like growth factor (IGF), and EGF, are involved in the proliferative phase of wound repair. During the maturation phase, the wound undergoes contraction which ultimately results in less scar tissue being seen.

From the foregoing, it is obvious that proper wound healing involves a complex interaction of cells and substances, such as cytokines, acting in concert. Based on basic research, many drugs, substances, and treatment methods have been proposed to promote wound healing.

Many of these substances provide the patient with partial relief from wounds, require a long healing time and fail to show the optimal response to treatment. For example, patent EP0575484 discloses a pharmaceutical composition for the regeneration and repair of mammalian tissues, which includes PDGF and dexamethasone.

Therefore, it is necessary to provide new substances and compositions with a high power in cell regeneration, leading to healing and repairing tissue damage effectively.

DESCRIPTION OF THE INVENTION

The inventors of the present invention have observed that myticin-C (QSVACRSYYCSKFCGSAGCSLYGCYLLHPGKICYCLHCSR, SEQ ID NO: 1) is expressed in response to wounds or tissue damage, thus enhancing cell regeneration and tissue repair after damage. The role of myticin-C in cell regeneration was demonstrated by an in vitro assay using human keratinocyte cells (HaCat line), and by an in vivo assay using zebrafish ( Danio rerio) larvae. In both cases, the wound healed faster in the presence of myticin-C than in its absence, thus promoting healing and repairing tissue damage (see Example 1, Figures 1 and 2).

Next, the inventors designed a library of overlapping short small peptides from the amino acid sequence of miticin-C (see Table 1), in order to identify the minimum sequence necessary for miticin-C to play its role. in cell and/or tissue regeneration. Thus, through in vitro and in vivo tests, the inventors observed that some peptides called P1 to P4 were capable of repairing the wound or damage generated. These peptides have the consensus sequence SYYCSK (SEQ ID NO: 2), this being therefore the amino acid sequence responsible for carrying out the best regeneration rates at 48 hours. Particularly if amino acids F and C are added to the carboxyl terminus, they show an improvement in total wound healing at 72 h, SYYCSKFC (SEQ ID NO: 3), as can be seen in Figure 3 with the peptides called P2b, P3 and P4.

Thus, in one aspect, the present invention refers to a peptide that comprises at least 80% identity with the sequence SYYCSK (SEQ ID NO: 2), which preferably has a size between 6 to 99 amino acids, and which does not is myticin-C with sequence SEQ ID NO: 1, preferably the peptide comprises at least 80% identity with the sequence SYYCSKFC (SEQ ID NO: 3), which preferably has a size between 6 to 99 amino acids, and which it is not myticin-C with sequence SEQ ID NO: 1. Hereinafter, this peptide will be called "peptide of the invention".

The term "peptide", as used herein, refers to a molecule formed by the union, in a defined order, of alpha-amino acids by means of a peptide bond. It will be understood that the terms "peptide bond", "peptide ", "polypeptide" and protein are known to those skilled in the art. Hereinafter, "peptide" and "polypeptide" will be used interchangeably.

The peptide of the invention can be obtained by techniques widely known in the state of the art, such as chemical synthesis, genetic recombination, expression of the polynucleotide that encodes the polypeptide of the invention, etc. All of these techniques are routine practice for those skilled in the art.

Additionally, the carboxyl and amino termini of the peptide of the invention may be protected against proteolysis. For example, the amino terminus may be in the form of an acetyl group (Ac) and/or the carboxyl terminus may be in the form of an amide group (Am). It is also possible to carry out internal modifications of the peptides so that they are resistant to proteolysis. Examples of these internal modifications include, but are not limited to, modifications in which at least one -CONH- peptide bridge is modified and replaced by a reduced bond (CH2NH), a retroinverse bond (NHCO), an oxymethylene bond (CH2-O ), a thiomethylene bond (CH2-S), a ketomethylene bond (CO-CH2), a hydroxyethylene bond (CHOH-CH2), an (NN) bond, an E-alcene bond, or a -CH=CH- bond. Peptides can also be stabilized by intramolecular crosslinking, for example, by modification of at least two amino acid residues with olefin side chains, preferably C3-C8 alkenyl chains, preferably pentel-2-yl chains, followed by crosslinking. chains as described in the so-called "staple" technology (Walensky et al., 2004, Science 205: 1466-1470). All of these chemically modified peptides to resist proteolysis are also contemplated within the present invention.

Additional modifications to the peptide of the invention include covalent bonding to a polyethylene glycol (PEG) molecule at its terminal carboxyl end or to a lysine residue, in order to reduce its urinary elimination and therapeutic dose, and to increase the half-life of the peptide. peptide in blood plasma. The half-life of the peptide can also be increased by embedding the peptide in a biodegradable and biocompatible polymeric material to form microspheres that are used as a drug delivery system. Polymers and copolymers include, without limitation, poly(D,L-lactide-co-glycolic) or PLGA. The techniques and procedures for how to manufacture lipid microspheres or nanocapsules for use in the administration of drugs are widely known to those skilled in the art.

The peptide of the invention comprises at least 80% identity with the sequence SEQ ID NO: 2, preferably at least 80% identity with the sequence SEQ ID NO: 3. In the present invention, "identity" is understood as or "sequence identity" to the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the art, such as BLAST. BLAST programs, eg, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain at The National Center for Biotechnology Information (NCBI) website. In a particular embodiment, the peptide of the invention comprises an identity of at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 2 or SEQ ID NO: 3. In another even more particular embodiment, the peptide of the invention comprises a sequence identity of 100% with the sequence SEQ ID NO: 2 or SEQ ID NO: 3. As understood by those skilled in the art, all peptides that have an identity of at least 80% with the sequence SEQ ID NO: 2 or SEQ ID NO: 3, and that have the same function as the peptide SEQ ID NO: 2 or SEQ ID NO: 3, that is, they have the ability to enhance tissue regeneration and repair of tissue damage, are functionally equivalent variants of the peptide of the invention. Assays to identify peptides functionally equivalent to the peptide of the invention can be found in the Example of the present description.

Thus, in a particular embodiment, the peptide of the invention refers to a peptide that comprises the sequence SEQ ID NO: 2, preferably SEQ ID NO: 3, which can have a size between 6 to 99 amino acids, and which does not is myticin-C with the sequence SEQ ID NO: 1.

Another characteristic of the peptide of the invention is that it has a size between 6 and 99 amino acids, preferably between 6 and 50, more preferably between 6 and 40. In a more particular embodiment, it has a size between 6 and 25 amino acids, and in another even more particular embodiment, between 6 and 20 amino acids or between 6 and 15 amino acids. In another even more particular embodiment, the peptide of the invention It is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in size.

In another particular embodiment, the peptide of the invention refers to a peptide comprising the sequence QSVACRSYYCSK (SEQ ID NO: 4), VACRSYYCSKFC (SEQ ID NO: 5), CRSYYCSKFCGS (SEQ ID NO: 6) or SYYCSKFCGSAG (SEQ ID NO: 7). In another even more particular embodiment, the peptide of the invention refers to a peptide consisting of the sequence SYYCSK (SEQ ID NO: 2), SYYCSKFC (SEQ ID NO: 3), QSVACRSYYCSK (SEQ ID NO: 4), VACRSYYCSKFC (SEQ ID NO: 5), CRSYYCSKFCGS (SEQ ID NO: 6) or SYYCSKFCGSAG (SEQ ID NO: 7). More preferably the peptide consists of the sequence VACRSYYCSKFC (SEQ ID NO: 5), CRSYYCSKFCGS (SEQ ID NO: 6) or SYYCSKFCGSAG (SEQ ID NO: 7). As mentioned above, the peptides of the invention may be protected against proteolysis, particularly the peptide of the invention refers to a peptide comprising the sequence SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO: 11 or SEQ ID NO: 12.

The peptide of the invention can be linked to other amino acid sequences to give rise to a fusion protein. Therefore, in another aspect, the present invention relates to a fusion protein comprising the peptide of the invention, hereinafter "fusion protein of the invention".

The term "fusion protein", as used herein, refers to an artificially designed polypeptide chain comprising two or more amino acid sequences of different origins, natural and/or artificial. The fusion protein, by definition, is never found in nature as such.

The fusion protein of the invention can be obtained by any suitable technique that allows peptides to be obtained in a single polynucleotide chain. Such techniques include recombinant techniques, where a gene construct encoding the fusion protein is introduced into a suitable vector for expression in a suitable expression system, and protein ligation techniques involving the formation of a peptide bond between two polypeptides, such as native chemical ligation or ligation of expressed proteins.

The fusion protein of the invention may additionally comprise a domain N-terminal signal peptide, which allows processing, eg, extracellular secretion, in a suitable host cell. Preferably, the N-terminal signal peptide domain comprises a protease, eg, a signal peptidase cleavage site, and thus may be removed after or during expression to obtain the mature protein.

Additionally, the fusion protein may comprise a recognition/purification domain, eg, a Strep tag domain and/or a poly-His domain, which may be located at the N-terminus or the C-terminus. Likewise, the fusion protein of the invention may additionally comprise a C-terminal flexible element, which has a length of, for example, 1-50, preferably 10-30 amino acids that may include and/or connect to a recognition domain/ purification as described herein.

The peptide of the invention can be linked to other amino acid sequences through a spacer (linker), which can be flexible.

Both the peptide and the protein of the invention can be contained in a nanoparticle for better administration, conservation, etc. Thus, in another aspect, the present invention relates to a nanoparticle comprising the peptide or fusion protein of the invention.

The term "nanoparticle", as used herein, refers to any material having dimensions in the range between 1 and 1,000 nm. In some embodiments, the nanoparticles have dimensions in the range of 2-200nm, preferably in the range of 2-150nm, and even more preferably in the range of 2-100nm. Nanoparticles can help preserve the integrity of the peptide or fusion protein in biological fluids until it reaches the target organ. Lastly, the nanoparticles can also be modified to target the nanoparticle to an organ of interest, by adding ligands without compromising the ability of the nanoparticles to deliver their peptide payloads. It is contemplated that this will allow delivery to specific cells, tissues and organs. The targeting specificity of ligand-based delivery systems is based on the distribution of ligand receptors in different cell types. The targeting ligand may be non-covalently associated or covalently with a nanoparticle, and can be conjugated to the nanoparticles by a variety of methods.

Suitable nanoparticles that can be used in the context of the present invention include nanoscale materials such as lipid-based nanoparticle, superparamagnetic nanoparticle, nanohelix, semiconductor nanocrystal, quantum dot, polymer-based nanoparticle, silicone-based nanoparticle , a silica-based nanoparticle, a metal-based nanoparticle, a fullerene, and a nanotube.

As the person skilled in the art understands, the peptide of the invention or the protein of the invention can also be contained within a particle. Therefore, in another aspect, the invention relates to a virus-like particle comprising the peptide or fusion protein.

The term "virus-like particle", also referred to as "VLP", refers to non-infectious virus-like particles that do not contain any viral genetic material. VLPs are the result of the expression of viral structural proteins, such as capsid proteins, and their self-assembly.

In a particular embodiment, the VLP may comprise, or alternatively consist of, structural proteins from Parvovirus, Rotavirus; Norwalk virus structural proteins; alphavirus structural proteins; structural proteins of foot-and-mouth disease virus; measles virus structural proteins, Sindbis virus structural proteins, Hepatitis B virus retrovirus structural proteins (eg, an HBcAg); structural proteins of tobacco mosaic virus; FlockHouse virus structural proteins; structural proteins of human papillomavirus; structural proteins of polyoma virus; bacteriophage structural proteins, RNA phage structural proteins.

In a particular embodiment, the peptide or fusion protein of the invention is coupled or bound to the capsid of the virus-like particle. The attachment of the peptide or fusion protein to the capsid may be by a covalent or non-covalent bond.

The peptide of the invention can be obtained by techniques widely known in the state of the art, such as the expression in a cell of the polynucleotide that encodes the polypeptide of the invention, or the protein of the invention, and its subsequent isolation. Therefore, in another aspect, the invention relates to a polynucleotide, hereinafter "polynucleotide of the invention", that encodes the peptide of the invention or the fusion protein of the invention.

The terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides can have any three-dimensional structure and can perform any function, known or unknown. The term "polynucleotide" includes, for example, single-stranded, double-stranded, and triplex molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. In addition to a native nucleic acid molecule, a nucleic acid molecule of the present invention may also comprise modified nucleic acid molecules. As used herein, mRNA refers to an RNA that can be translated in a cell.

In a preferred embodiment, the polynucleotide of the invention is an mRNA. The mRNA can be chemically synthesized, can be obtained by in vitro transcription, or can be synthesized in vivo in the target cell. The nucleotide sequences that form the nucleic acid encoding the conjugate or fusion protein of the invention are in the same correct reading frame for expression thereof.

The polynucleotide encoding the peptide or fusion protein of the invention may have various modifications made to the coding region as long as the amino acid sequence of the peptide or fusion protein does not change due to codon degeneracy. , or taking into consideration the codons preferred by the organism in which they are to be expressed, and various modifications or alterations may be introduced even in regions other than the coding region, as long as they have no influence on the expression of the gene.

As the person skilled in the art understands, the polynucleotide encoding the peptide of the invention or the fusion protein of the invention may be included within a gene construct or within an expression vector. In general, an expression vector comprises, in addition to the polynucleotide encoding the peptide of the invention or the fusion protein of the invention, a promoter that directs its transcription (for example, pT7, plac, ptrc, ptac, pBAD, ret, etc.), to which it is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription start and termination signals (tlt2, etc. ), polyadenylation signal, origin of replication, ribosome binding sequences (RBS), coding sequences for transcriptional regulators, enhancers, transcriptional silencers (silencers), repressors, etc. Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. which may also contain markers that can be used to select the cells transfected or transformed with the gene or genes of interest. The choice of vector will depend on the host cell and the type of use to be made. For example, said vector may be a viral vector (adenoviruses, adenovirus-associated viruses as well as retroviruses and, in particular, lentiviruses) or a non-viral vector (pcDNA3, pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl). Said vectors can be administered directly to the subject by conventional methods. Alternatively, said vectors can be used to transform, transfect or infect cells, for example mammalian cells, ex vivo, and subsequently implant them in the animal body to obtain the desired therapeutic effect. For administration to the receiving subject, said cells will be formulated in a suitable medium that does not adversely affect the viability of said cells. Obtaining said vector can be carried out by conventional methods known to those skilled in the art, just as for the transformation of microorganisms and eukaryotic cells, different widely known methods can be used.

In another aspect, the invention relates to a gene construct, hereinafter "gene construct of the invention", comprising the polynucleotide of the invention.

Preferably, the gene construct of the invention comprises the polynucleotide of the invention operably linked to expression-regulating sequences of the polynucleotide of the invention. In principle, any promoter can be used in the gene constructs of the present invention, as long as said promoter is compatible with the cells in which it is desired to express the polynucleotide.

A promoter, or promoter region, is a nucleotide sequence that controls the transcription of a given gene (nucleotide sequences). In the present invention it refers to a nucleotide sequence that controls the transcription of the polynucleotide of the invention. Promoter sequences can be unidirectional or bidirectional. A unidirectional promoter is one that controls the transcription of one gene or more genes that are located in tandem with the first. "In tandem" refers to the 3' end of the first gene being followed, either consecutively or separated by a certain nucleotide sequence, by the 5' end of the second gene. A bidirectional promoter refers to the promoter region that controls transcription. in two opposite directions, that is, a bidirectional promoter directs the transcription of two divergently located genes, that is, in opposite directions, the 5' end of both nucleotide sequences being closer to each other than the 3' end. In the present invention, the terms "promoter" and "promoter region" are used interchangeably. Furthermore, the promoters in the present invention may be constitutive or inducible. The term "inducible", as used in the present description, refers to the possibility that the promoter has a control element that allows activating or deactivating (repressing) the transcription of the gene that it regulates, in the presence of a factor. external to the promoter.

Additionally, the gene construct of the invention may contain markers or labels that allow the isolation of the peptide of the invention once it is synthesized in the cell.

On the other hand, the polynucleotide or the gene construct of the invention may be part of a vector. Thus, in another aspect, the invention relates to a vector, hereinafter "vector of the invention", comprising the polynucleotide or gene construct of the invention.

The term "vector", as used herein, refers to a nucleic acid sequence comprising the necessary sequences such that after transcribing and translating said sequences in a cell a polypeptide encoded by the nucleic acid of the invention. Said sequence is operably linked to additional segments that provide for its autonomous replication in a host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector which, in addition to regions of autonomous replication in a host cell, contains regions operably linked to the nucleic acid of the invention and which are capable of enhancing the expression of the nucleic acid products according to the invention. The vectors of the invention can be obtained by means of techniques widely known in the state of the art.

The person skilled in the art will appreciate that there is no limitation as to the type of vector that can be used, since said vector can be a cloning vector suitable for propagation or an expression vector. Thus, suitable vectors according to the present invention include, without limitation, (i) prokaryotic expression vectors such as pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, CoIEl, pCRl, RP4, phage and shuttle vectors such as pSA3 and pAT28, (ii) yeast expression vectors such as 2 micron plasmid-like vectors, integrating plasmids, YEP vectors, centromeric plasmids and the like, (iii) cell expression vectors insect vectors such as pAC-series and pVL-series vectors, (iv) plant expression vectors such as pIBI-series vectors, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like and (v) higher eukaryotic cell expression vectors either based on viral vectors, including but not limited to adenoviruses, adenovirus-associated viruses as well as retroviruses and lentiviruses, as well as non-viral vectors including, not limited to, pSilencer 4 .1-CMV (Ambion), pcDNA3, pcDNA3.1/hygpHCMV/Zeo, pCR3.1, pEFVHis, pIND/GS, pRc/HCMV2, pSV40/Ze02, pTRACER HCMV, pUB6N5-His, pVAXl, pZeoSV2, pCl, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl.

The vector of the invention can be used to transform, transfect or infect cells capable of being transformed, transfected or infected by said vector.

Said cells can be prokaryotic or eukaryotic. As an example, the vector where said DNA sequence is introduced can be a plasmid or a vector that, when introduced into a host cell, integrates into the genome of said cell and replicates together with the chromosome(s) into which it has been integrated. Obtaining said vector can be carried out by conventional methods known to those skilled in the art.

Therefore, in another aspect, the invention relates to a cell, hereinafter "cell of the invention", comprising a peptide, a fusion protein, a polynucleotide, a gene construct or a vector according to the present invention , for which said cell could have been transformed, transfected or infected with the construction or the vector provided by this invention Transformed, transfected or infected cells can be obtained by conventional methods known to those skilled in the art, including, without limitation a, electroporation, protoplast fusion, coprecipitation with calcium phosphate (CaPO4) and precipitation with calcium chloride (CaCl2).

In a particular embodiment, said host cell is an animal cell, preferably a mammalian cell and more preferably a human cell, transfected or infected with an appropriate vector.

Suitable host cells for expression of the polypeptide of the invention include, without limitation, mammalian cells, plant cells, insect cells, fungal cells, and bacterial cells. Mammalian cells suitable for use in the present invention include epithelial cell lines (porcine, human, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc. .), glial cells (murine, human, etc.), liver cell lines (monkey, etc.), CHO (ChineseHamsterOvary) cells, COS cells, BHK cells, HeLa cells, 911, ATI080, A549, 293 or PER. C6, human ECCs 5 NTERA-2 cells, D3 cells of the mESC line, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3, 293T, REH and MCF-7 cells and hMSCs (human mesenchymalstemcells ), and GliNS2 cells (glioma stem cells).

In another aspect, the invention relates to a composition, preferably a pharmaceutical or cosmetic composition, comprising an amount therapeutically or cosmetically effective peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector or cell of the invention, and optionally a pharmaceutically or cosmetically acceptable carrier or excipient.

The peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector and cell of the invention have been previously defined.

As used herein, the term "pharmaceutical composition" refers to a formulation that has been adapted to deliver a predetermined dose of one or more useful therapeutic agents to a cell, group of cells, organ, tissue, or an animal in which there is damage or injury and requires tissue and/or cell regeneration.

As used herein, the term "cosmetic composition" refers to a formulation that has been adapted to deliver a predetermined dose of one or more useful cosmetic agents to a cell, group of cells, organ, tissue, or an animal in which there is cosmetic damage or injury and requires tissue and/or cell regeneration.

The expression "therapeutically or cosmetically effective amount", as used in this document, is understood as an amount capable of providing a therapeutic or cosmetic effect, and that can be determined by those skilled in the art by commonly used means. The amount of peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector or cell of the invention that can be included in the pharmaceutical or cosmetic compositions according to the invention will vary depending on the subject and the particular mode of application. management.

The adequate dose of the active principle or principles within the pharmaceutical or cosmetic composition will depend on the type of tissue damage to be treated, the severity and course of the disease or damage, if the composition is administered for cosmetic, preventive or therapeutic purposes, previous therapy , patient medical history and response to the peptide or polypeptide, and the discretion of the treating physician. The amount of peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector or cell of the invention is suitably administered to the patient or subject at one time or over a series of treatments. Depending on the type and severity of the disease or injury, an appropriate dose level will generally be from about 0.1 to about 10 mg/kg; more preferably from about 0.5 to about 5 mg/kg, even more preferably from about 0.75 to about 2.5 mg/kg, even more preferably from about 1 mg/kg, which may be administered in single or multiple doses. The compounds can be administered in a regimen of 1 to several times per day or for two, three, four, five, six or seven days, preferably once every other day. The pharmaceutical or cosmetic composition can be administered for at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 or more days, preferably for 14 days. The pharmaceutical or cosmetic composition can preferably be administered once every other day for 14 days.

The pharmaceutical compositions of the invention also contain one or more additional pharmaceutically acceptable excipients. By "pharmaceutically acceptable excipient" is meant a therapeutically inactive substance that is used to incorporate the active ingredient and that is acceptable to the patient from a pharmacological/toxicological point of view and to the pharmaceutical chemist who manufactures it from a physical/chemical point of view. regarding composition, formulation, stability, patient acceptance and bioavailability. Carrier or carrier also includes any substance that serves to enhance the delivery and efficacy of the active ingredient within the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include one or more from the list comprising: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases it will be preferable to include isotonic agents, eg sugars, polyols such as mannitol, sorbitol or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, that enhance the shelf life or efficacy of the fusion protein or compositions that form part of pharmaceutical compositions. Examples of suitable carriers are well known in the literature. Examples of carriers without limitation are a series of saccharides such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; a number of starches such as corn starch, wheat starch, rice starch and potato starch; a series of cellulose such as cellulose, methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl cellulose; and a series of fillers such as gelatin and polyvinylpyrrolidone. In some cases, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added.

The cosmetic composition may also comprise adjuvants, excipients and other usual additives in the cosmetic and dermatological field, such as, for example and without limitation, stabilizers, preservatives, antioxidants, solvents, perfumes, chelating agents, odor absorbers, chemical or mineral filters. , mineral pigments, organic solvents, silicones, thickeners, softeners, emulsifiers, antifoaming agents, moisturizers, fragrances, surfactants, anionic, cationic, nonionic or amphoteric polymers or mixtures thereof, sequestering agents, propellants, acidifying or basifying agents, dyes, colorants , pigments or nanopigments, or any other ingredient normally used in cosmetic products and compositions.

The composition of the invention may further comprise any substance that can be delivered to a wound, tissue injury or site of inflammation. For example, the disclosed composition may further comprise one or more classes of antibiotics (eg aminoglycosides, cephalosporins, chloramphenicol, clindamycin, erythromycins, fluoroquinolones, macrolides, azolides, metronidazole, penicillins, tetracyclines, trimethoprim-sulfamethoxazole, vancomycin), steroids (eg andranes (eg testosterone), cholestanes (eg cholesterol), cholic acids (eg cholic acid), corticosteroids (eg dexamethasone), estranes (eg estradiol), pregnanes (eg progesterone), narcotic and non-narcotic analgesics (for example morphine, codema, heroma, hydromorphone, levorphanol, meperidine, methadone, oxycodone, propoxyphene, fentanyl, methadone, naloxone, buprenorphine, butorphanol, nalbuphine, pentazocine), chemotherapy (for example anticancer drugs such as, but not limited to, altretamine, asparaginase, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, diethylstilbesterol, ethinyl estradiol, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, goserelin, hydroxyurea, idarubicin, ifosfamide, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, paclitaxel, pentostatin, pipobroman, plicamycin, prednisone, procarbazine, streptozocin, tamoxifen, teniposide, vinblastine, vincristine), anti-inflammatory agents (eg alclofenac; alclometasone dipropionate; algestone acetonide; alpha amylase; amcinafal; amcinafide; amfenac sodium; amiprilose hydrochloride; anakinra; anirolac; anitrazafen; apazone; balsalazide disodium; bendazac; benoxaprofen; benzydamine hydrochloride; bromelam; broperamol; budesonide; carprofen; cycloprofen; Cintazone; clipprofen; clobetasol propionate ; clobetasone butyrate; clopirac; cloticasone propionate; cormetasone acetate; Cortodoxone; decanoate; deflazacort; delatestril; depo-testosterone; desonide; desoxymetasone; dexamethasone dipropionate; diclofenac potassium; diclofenac sodium; diflorasone diacetate; diflumidone sodium; diflunisal; difluprednate, diphthalone, dimethylsulfoxide; drocinonide; endrysone; enlimomab; sodium enolicam; epirizole; etodolac; etofenamate; felbinac; fenomol; fenbufen; Fenclofenac; fenchlorac; fendosal; fenpipalone; fentiazaco; flazalone; fluazacort; flufenamic acid; flumizole; flunisolide acetate; flunixin; flunixin meglumine; fluocortibutyl; fluorometholone acetate; flucuazone; flurbiprofen; fluretofen; Fluticasone Propionate; furaprofen; furobufen; halcinonide; Halobetasol Propionate; Halopredone Acetate; ibufenac; ibuprofen; aluminum ibuprofen; ibuprofen piconol; ilonidap; indomethacin; indomethacin sodium; indoprofen; indoxol; intrazole; Isoflupredone Acetate; isoxepac; isoxicam; ketoprofen; Lofemizole Hydrochloride; lomoxicam; loteprednol etabonate; sodium meclofenamate; meclofenamic acid; mechlorisone dibutyrate; mefenamic acid; mesalamine; meseclazone; mesterolone; methandrostenolone; methenolone; methenolone acetate; Methylprednisolone Suleptanate; momiflumate; nabumetone; nandrolone; naproxen; sodium naproxen; naproxol; nimazone; olsalazine sodium; orgothem; orpanoxine; Oxandrolone; oxaprozin; oxyphenbutazone; oxymetholone; paraniline hydrochloride; sodium pentosan polysulfate; sodium fenbutazone glycerate; pirfenidone; piroxicam; Piroxicam Cinnamate; piroxicam olamine; pirprofen; prednazate; prifelone; prodolic acid; provuazone; proxazole; Proxazole Citrate; rimexolone; romazarite; salcolex; salnacedin; salsalate; bloodroot chloride; seclazone; sermethacin; Stanozolol; sudoxicam; sulindac; suprofen; talmethacin; talniflumate; talosalate; tebufelone; hadp; sodium tendap; tenoxicam; tesicam; tesimide; testosterone; testosterone combinations; tetridamine; thiopinac; tixocortol pivalate; tolmetin; tolmetin sodium; triclonide; triflumidate; zidomethacin; zomepirac sodium), or antihistamine agents (for example, ethanolamines (such as diphenhydramine carbinoxamine), ethylenediamine (such as tripelenamine pyrilamine), alkylamine (such as chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine), other antihistamines such as astemizole, loratadine, fexofenadine, bropheniramine, clemastine, acetaminophen, pseudoephedrine, triprolidine).

The pharmaceutical or cosmetic compositions of the invention can be administered by any type of suitable route, such as orally, nasally, ocularly, topically, intradermally, intracranially, or intravenously. The preferred route of administration of said pharmaceutical compositions is the oral, nasal, ocular, topical, intracranial or intradermal route.

"Oral route" is understood as the pharmaceutical composition incorporated into the body after swallowing.

By "nasal route" is meant the administration of the pharmaceutical composition insufflated through the nose.

"Ocular route" is understood as the topical administration of the pharmaceutical composition by instillation directly into the eye.

The "topical route" is understood as the application to the exterior of the body, such as, for example, the skin, the scalp and the nails; and also the application to mucous membranes such as, without limitation, buccal, nasal or rectal mucosa.

"Intradermal route" is understood as the administration of the pharmaceutical composition by injection into the dermis.

"Intracranial route" is understood as the administration of the pharmaceutical composition within the skull.

"Intravenous route" is understood as the administration of the pharmaceutical composition by injection into the blood stream.

Preferably, the cosmetic composition of the present invention can be presented, for example and without limitation, in the form of an aqueous, hydroalcoholic or oily solution, an emulsion of oil in water or water in oil, or multiple, of an aqueous or oily gel, of an anhydrous liquid product, of a dispersion of oil in an aqueous phase with the aid of spheroids (such as nanospheres, nanocapsules and lipid vesicles), silicone emulsions in water, microemulsions, pastes , milks, balms, foams, lotions, creams, soaps, hydroalcoholic solutions, hydroglycolic solutions, liniments, sera, serums, polysaccharide films, ointments, fatty ointments, mousses, ointments, gels, cream gels, foam gels, emulsion-gels and solutions.

Materials comprising the compositions disclosed herein (eg, peptides, fusion proteins, nucleic acids, vectors, cells, polynucleotides, etc.) are also contemplated by the present invention. For example, materials used to treat wounds are disclosed, where the materials are coated with the peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, vector, cell, or composition of the invention. Illustrative, non-limiting examples of materials used to treat wounds include dressings, surgical tape, sutures, staples, or grafts (eg, skin grafts).

For example, the material (bandage, surgical tape, suture, staple, graft) can be soaked in the disclosed peptide at a suitable concentration. The material can then be dried and sealed in a sterile container. The peptide can also be incorporated into a crosslinkable hydrogel system, such as poly(lactic-coglycolic acid) (PLGA) or polyurethane, which can then be designed as wound care materials (eg, bandage, surgical tape, suture, staple, etc.). graft). Thus, hydrogel-peptide composites are disclosed.

Medical implants coated with the peptide of the invention prior to implantation in a subject are also disclosed. For example, a common problem in such implant surgeries is the formation of a contraction capsule around the implant from the formation of scar tissue leading to undue hardening, shrinkage, and ultimately deformation of the tissue of interest. The use of the present polypeptides in or on the implant can reduce or prevent this distortion. Non-limiting examples of medical implants include: limb prostheses, breast implants, penile implants, testicular implants, artificial eyes, facial implants, artificial joints, heart valve prostheses, vascular prostheses, dental prostheses, facial prostheses, tilted disc valve , valve caged ball, hearing aids, prosthetic noses, pacemakers, cochlear implants, and skin substitutes (eg, porcine/pigskin heterograft, BIOBRANE, cultured keratinocytes).

The peptide according to the invention is coated on a medical implant or wound treatment material. Therefore, another aspect of the invention relates to a material or implant comprising the peptide, the fusion protein, the nanoparticle, the virus-like particle, the polynucleotide, the gene construct, the vector, the cell or the composition. described in the present invention.

Another aspect of the present invention relates to the peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, cell, composition, material or implant of the present invention for use as a medicament. Alternatively, this aspect of the invention relates to the use of the peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, cell, composition, material or method. implant for the manufacture of a drug.

The present invention further provides a method of promoting wound healing following tissue injury in a subject, comprising administering to the subject one or more of the compositions of the present invention (eg, peptides, polynucleotides, or vectors) in a vehicle. pharmaceutically acceptable. Further disclosed is a method of treating a subject with tissue injury, comprising administering to the subject one or more of the compositions disclosed herein (eg, peptides, polynucleotides, or vectors) in a pharmaceutically acceptable carrier.

"Promoting,""promoting," and "promoting" refer to an increase in an activity, response, condition, disease, or other biological parameter. This may include but is not limited to the onset of the activity, response, condition, or disease. This may also include, for example, a 10% increase in activity, response, condition, or disease compared to native or control, thus the increase may be 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100%, or any amount of increase in between compared to native or control levels.

By "treating" or "treatment" is meant a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the underlying cause of the disease or condition itself, rather than just the symptoms. Treatment may be any reduction in native levels and may be but is not limited to complete suppression of the disease, condition or symptoms of the disease or condition. For example, a disclosed method of promoting wound healing is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject with the disease compared to native levels in the same subject or control subjects. . Thus, the reduction can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between compared to native levels. or control.

As used herein, "subject" includes, but is not limited to, animals or plants. The subject may be a vertebrate, more specifically a mammal (eg, a human, horse, pig, rabbit, dog, etc.). sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, a bird, or a reptile or amphibian. The subject may be an invertebrate, more specifically an arthropod (eg, insects and crustaceans). The term does not indicate a particular age or sex, therefore, adult and newborn subjects, as well as fetuses, both male and female, are intended to be covered.

Another aspect of the present invention relates to the fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, cell, composition, material or implant of the present invention for its use in cell and/or tissue regeneration, in particular for its use in the treatment of a wound, preferably the wound is selected from the group consisting of a skin wound, a decubitus ulcer and/or an external wound or internal.

Another aspect of the present invention relates to the cosmetic use of the fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, cell, composition, material or implant of the fusion protein. present invention as a skin healing and/or repairing agent, preferably the use it would be in dermocosmetic treatments as a healing and repairing agent for the skin or in dermocosmetic compositions for topical application.

Another aspect of the invention relates to a peptide comprising the sequence SEQ ID NO: 2, for use in cell regeneration, in particular for use in the treatment of a wound, preferably the wound is selected from the group consisting of a skin wound, a decubitus ulcer and/or an external or internal wound. More preferably, the peptide comprises the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. In a more preferred embodiment, the peptide consists of the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, more preferably the peptide consists of the sequence SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

In the present invention, the term "regeneration" refers to the process by which the structure and function of damaged organs or body parts are restored. The regeneration process can occur at multiple states, levels of biological organization, and the ability of different organisms to regenerate missing parts is highly variable. Regeneration can occur at the cellular level (cellular regeneration), tissue (tissue regeneration), organ, structure and even the entire body.

Non-limiting examples of injuries are:

- a burn wound, which is injury resulting from exposure to heat, electricity, radiation (for example, sunburn and laser surgery), or caustic chemicals,

- ulcers,

- wounds in diabetes mellitus, which are usually foot injuries due to numbness caused by nerve damage (diabetic neuropathy) and decreased blood flow to the legs and feet. The most serious injury is a foot ulcer. Diabetic foot ulcers have a very high risk of becoming infected, and sometimes cannot be cured. Foot ulcers that have not healed are a common cause of amputation in people with diabetes,

- decubitus wounds, that is, injuries caused by unmitigated pressure on any part of the body, especially portions on bone or cartilaginous areas,

- wounds due to an external force that damages the tissue,

- skin wounds due to aging or the environment. This includes, for example, cracks, dry skin, rough skin, and the like.

The wounds may be an internal wound, an oral wound, a skin wound, an external wound, an ulcer and/or a decubitus ulcer, damage to the eye, an eye injury, eg, conjunctiva. Other conditions that can be treated with the peptides according to the disclosure include oral ulcers, oral aphthous lesions, glossodynia, burning tongue, psoriasis, eczema, and hair loss.

These types of skin damage and lesions are known to those skilled in the art. An internal wound is a wound present in the body, for example due to a surgical incision. An oral wound is a wound present in the oral cavity. A skin wound is a wound present on the skin. An external wound should be understood as a wound that is visible and accessible from outside the body. An ulcer is a lesion on the surface of the skin or a mucosal surface. A pressure ulcer is a wound or ulcer caused by prolonged pressure on the skin and tissues when you are in one position for a long period of time, such as lying in bed. The bony areas of the body are the most frequently affected sites, which become ischemic under sustained and constant pressure. Skin aging is the change in the appearance of the skin due to time or exposure to the environment or the health status of an individual. Cellulite is the definition used in cosmetics in relation to a sagging or wavy appearance of the skin (orange peel in Dutch).

In a particular embodiment of the invention, the peptide of the invention is used for the treatment of a skin wound. Cutaneous wounds can be wounds in the epidermis or the dermis of the skin. There are several types of wounds where the skin or tissue may need repair: abrasions, lacerations, incisions, perforations and avulsions, and burns. The use of a peptide according to the invention can improve the general health condition of the skin. In another particular embodiment of the invention, the method is used for the treatment of an oral wound. Oral wounds are wounds anywhere in the oral cavity in which the oral mucosa is damaged. In another particular embodiment of the invention, the peptide of the invention is used for the treatment of an internal wound. Internal wounds are wounds in which cell layers of endodermal or mesodermal origin are damaged. Examples are injuries to arteries or veins, peritoneum or pericardium.

Within the present invention, the therapeutic or cosmetic use of all those embodiments that, directly or indirectly, comprise the peptide comprising the sequence SEQ ID NO: 2, for the treatment of a skin wound as previously defined is also contemplated in the present description. Examples of said embodiments refer to the fusion protein, the nanoparticle, the virus-like particle, the polynucleotide, the gene construct, the vector, the cell, the composition, the material or the implant that comprises the peptide that comprises the sequence SEQ ID NO: 2. All these terms have also been previously defined in the present description.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. Other objects, advantages and features of the invention will be apparent to those skilled in the art in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 (A and B) Effect of the entire myticin C peptide on wound healing in HaCat cells.

Figure 2. (A) Appearance of the tail of zebrafish larvae with the tail intact. (B) Fish after sectioning off a piece of tail. (C) Time course of tail tissue regeneration. (D) Comparison of tissue regeneration in myticin-treated and untreated fish after 4 days.

Figure 3. Evolution of wound healing in HaCat cells treated with the myticin-C peptides.

Figure 4. Effect of the complete peptide of myticin on the proliferation of human keratinocyte cells (HaCat).

Figure 5. Effect of peptides on cell viability of HaCat cells.

Figure 6. Comparison of regeneration in fish treated and untreated with myticin-C peptides after 4 days.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which reveal the effectiveness of the product of the invention.

Example 1: Peptides and their synthesis

The mature chain of myticin-C (Myt-C), composed of 40 amino acids with the sequence QSVACRSYYCSKFCGSAGCSLYGCYLLHPGKICYCLHCSR (SEQ ID NO: 1), was taken as a model for the synthesis of 14 dodecapeptides with a sequence shift of two amino acid residues and amidated at the carboxyl terminus and acetylated at the amino. The complete chain and the peptides thus designed were synthesized and purified by high performance liquid chromatography (HPLC) in the Proteomics service of the National Center for Biotechnology of the CSIC with >95% purity. Their sequences were verified by mass spectrometry.

Possible traces of trifluoroacetic acid (TFA) from the synthesis process were eliminated with three freeze-lyophilization cycles using 10 mM HCl as solvent. The content of the vial (3 mg of each peptide) was dissolved in 1 mL of serum-free DMEM aqueous culture medium supplemented with gentamicin and a commercial protease inhibitor cocktail (Protease Inhibitor Tablets, Thermo Scientific, Ref.

88265) leaving a final concentration of the stock solution of 3 mg/mL (676.8 pM) which was stored at -80°C before use.

Table 1: Synthetic peptides designed from the myticin-C sequence with their molecular weight (M.W.).

Example 2: Determination of the cytotoxicity of Myticin C in vitro.

The determination of the in vitro cytotoxicity of the mature chain of miticin C (SEQ ID NO: 1) and its fragments (Table 1) was evaluated in the HaCat line of keratinocytes humans, by the MTT assay in the concentration range 0-40 pM (Figures 4 and 5). As seen in Figures 4 and 5, the treatment with the peptides did not affect the viability of the HaCat cells.

Example 3: Determination of the wound repair activity of Myticin C in vitro.

The determination of the in vitro wound repair activity was carried out using monolayers of the HaCat line of human keratinocytes-.

A "wound" was made and photographed at different times to determine its degree of closure, in the presence and absence of myticin C peptides. First, the ability of the myticin C peptide (SEQ ID NO: 1) to stimulate wound healing in keratinocytes in vitro (Figure 1). Cells treated with a concentration of 5 pM showed faster wound healing than control cells. At 48 h the closure of the The wound was significantly larger in the treated cells and at 72 h the wound was fully repaired unlike the control cells.

This assay was repeated with the overlapping peptides based on the mature chain of myticin C from Table 1. As seen in Figure 3, peptides P1, P2a, P2b, P3 and P4 induced cell proliferation. When cells were wounded, significantly greater healing was observed in those that had been treated with peptides P1, P2a, P2b, P3 and P4. At 48 h of treatment, it is observed, as shown in Figure 3, that peptides P1, P2a, P2b, P3 and P4, unlike the control, present a lower percentage of wound opening and, therefore, present increased wound healing and damage repair. At 72 hours, peptides P2b, P3 and P4 had managed to completely close the wound (Figure 3). Due to problems in the synthesis process, peptides P6, P7 and P9 showed poor solubility and were not taken into account in the tests.

Example 4: Determination of the wound repair activity of Myticin C in vivo.

Once the experiments were carried out on the HaCat line of human keratinocytes, an in vivo assay was designed using zebrafish larvae ( Danio rhenio ). Larvae 3 days post-fertilization were used and their tails were cut. Subsequently, tissue regeneration was studied in larvae treated with the peptides of Table 1 of myticin C and in larvae not treated with myticin C, by means of photographs under magnification and image analysis using the imageJ program. Figures 2 and 6 show the results that show a significantly greater tissue regeneration of the tail in those larvae treated with the complete peptide of myticin C and with the peptides P2a, P2b, P3 and P4 overlapping the mature peptide of myticin. C. The presence of neutrophils in the wound area was evaluated using the transgenic fish line Tg(Mpx:GFP)i114, which have GFP-labeled neutrophils. In this way, the capacity of myticin C in the resolution of inflammation associated with the wound was determined.

Claims (26)

1. A peptide comprising the sequence SEQ ID NO: 2 characterized in that it comprises the sequence selected from the list consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and which is not myticin-C with sequence SEQ ID NO: 1. 2. The peptide according to claim 1, wherein the peptide is between 6 and 40 amino acids in size. 3. A peptide consisting of the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. 4. The peptide according to claim 3, consisting of the sequence SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 5. A fusion protein comprising the peptide according to any of claims 1 to 4. 6. A nanoparticle comprising the peptide according to any of claims 1 to 4, or the fusion protein according to claim 5. 7. A virus-like particle comprising the peptide according to any one of claims 1 to 4, or the fusion protein according to claim 5. 8. A polynucleotide encoding the peptide according to any one of claims 1 to 4, or the fusion protein according to claim 5. 9. A gene construct comprising the polynucleotide according to claim 8. 10. A vector comprising the polynucleotide according to claim 8 or the gene construct according to claim 9. 11. A cell comprising the peptide according to any of claims 1 to 4, the fusion protein according to claim 5, the polynucleotide according to claim 8, the gene construct according to claim 9, or the vector according to claim 10. 12. A composition comprising the peptide according to any of claims 1 to 4, the fusion protein according to claim 5, the nanoparticle according to claim 6, the virus-like particle according to claim 7, the polynucleotide according to claim 8, the gene construct according to claim 9, the vector according to claim 10, or the cell according to claim 11. 13. The composition according to claim 12, wherein the composition is a pharmaceutical composition or cosmetic composition. 14. The composition according to claim 13, further comprising a pharmaceutically or cosmetically acceptable excipient or vehicle. 15. Material comprising the peptide according to any of claims 1 to 4, the fusion protein according to claim 5, the nanoparticle according to claim 6, the virus-like particle according to claim 7, the polynucleotide according to claim 8, the gene construction according to claim 9, the vector according to claim 10, the cell according to claim 11 or the composition according to any of claims 12 to 14. 16. A fusion protein according to claim 5, a nanoparticle according to claim 6, a virus-like particle according to claim 7, a polynucleotide according to claim 8, a gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11, a composition according to any of claims 12 to 14 or a material according to claim 15, for use as a medicament. 17. A fusion protein according to claim 5, a nanoparticle according to claim 6, a virus-like particle according to claim 7, a polynucleotide according to claim 8, a gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11, a composition according to any of claims 12 to 14 or a material according to claim 15, for use in the treatment of wounds. 18. A fusion protein according to claim 5, a nanoparticle according to claim 6, a virus-like particle according to claim 7, a polynucleotide according to claim 8, a gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11, a composition according to any of claims 12 to 14 or a material according to claim 15, for use according to claim 17, wherein the wound is selected from the group consisting of a skin wound, a decubitus ulcer and/or or an external or internal wound. 19. Cosmetic use of a fusion protein according to claim 5, a nanoparticle according to claim 6, a virus-like particle according to claim 7, a polynucleotide according to claim 8, a gene construct according to claim 9, a vector according to claim 10, a cell according to claim 11, a composition according to any of claims 12 to 14 or a material according to claim 15, as a skin healing and/or repairing agent. 20. Peptide comprising the sequence SEQ ID NO: 2, for use in the treatment of a wound. 21. Peptide for use according to claim 20, wherein the wound is selected from the group consisting of a skin wound, a decubitus ulcer and/or an external or internal wound. 22. Peptide for use according to claim 21, comprising the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. 23. Peptide for use according to claim 22, consisting of the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. 24. Cosmetic use of the peptide comprising the sequence SEQ ID NO: 2 as a skin healing and/or repairing agent. 25. Use according to claim 24, wherein the peptide comprises the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. 26.

Use according to claim 25, where the peptide consists of the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7