Peptides and Methods for Use in the Treatment of Pain

Abstract

The present invention relates to a set of peptides, compositions and kits comprising or enabling the expression of said peptides, and their use in the prevention or treatment of pain in a subject in particular in need thereof.

Description

Technical Field

[0001] The present invention generally relates to the field of pain management. More specifically, the present invention relates to a set of peptides, compositions and kits comprising or enabling the expression of said peptides, and their use, specifically for preventing or treating pain in a subject in need thereof. The set of peptides is a specific fragment of the human TAFA-4 protein or an equivalent fragment thereof that can be used as an active ingredient for the prevention or treatment of pain (specifically, acute, subacute or chronic pain caused by neuropathic pain, postoperative pain or inflammatory pain) or for the prevention or treatment of hyperalgesia or allodynia caused by injury-induced pain.

Background Art

[0002] Pain is generally classified as acute or chronic. Acute pain is transient and essential for maintaining the integrity of the body, while chronic pain persists beyond the normal healing period and has adverse effects on health. Chronic inflammatory pain, neuropathic pain, or postoperative pain cause long-term sensory abnormalities such as hyperalgesia (extreme pain caused by noxious stimuli) and mechanical allodynia (pain caused by innocuous mechanical stimuli). These pain categories differ in terms of etiology and clinical features but share some common mechanisms such as changes in neuroimmune interactions and neuronal sensitization in both the peripheral and central nervous systems (Costigan et al., 2009). There is increasing evidence suggesting that loss of inhibition may be an important mechanism underlying chronic pain (Bourane et al., 2015a; Bourane et al., 2015b; Boyle et al., 2019; Coull et al., 2005; Duan et al., 2014; Peirs et al., 2015; Petitjean et al., 2019; Petitjean et al., 2015; Zeilhofer et al., 2012; Zhang et al., 2018, Yoo et al., 2021). However, despite extensive knowledge of the mechanisms and circuits underlying chronic pain in rodents, translating these findings into effective treatments for human chronic pain remains inadequate (Colloca et al., 2017). In fact, non-steroidal anti-inflammatory drugs (NSAIDs) have limited effectiveness against chronic pain, and opioids have multiple side effects such as potentially fatal respiratory depression, nausea, constipation, hyperalgesia, tolerance, physical dependence, and psychological dependence (Benyamin et al., 2008). Therefore, efforts should be encouraged to identify new targets with analgesic or antalgic potential for the treatment of chronic pain.

[0003] In recent years, the inventors have discovered remarkable features of the secreted protein TAFA-4, suggesting that it may be an interesting agent for the treatment of chronic pain (International Publication No. WO 2014 / 180853). TAFA-4 belongs to a family of five highly conserved secreted neuropeptides (Sarver et al., 2021). TAFA-4 contains a signal peptide and a highly conserved core region that includes 10 subsequent cysteine residues, and the highly conserved core region contains a CC-chemokine motif, making it cytokine-like (Tom Tang et al., 2004).

[0004] The inventors have also already shown that in mice lacking the TAFA-4 gene, mechanical hypersensitivity induced by nerve injury lasts much longer than in wild-type (WT) mice (Delfini et al., 2013). These phenotypes are reversed by intrathecal injection of recombinant TAFA-4.

[0005] As described in International Publication No. 2014 / 180853, the inventors have already reported experimental evidence showing that TAFA-4 has a strong effect on pathologically increased mechanical pain. In both male and female mice, intrathecal and subcutaneous administration of human recombinant TAFA-4 has been shown to reverse inflammatory, postoperative, and nerve injury-induced mechanical hypersensitivity. The inventors have also shown that TAFA-4 can reverse nerve injury-induced neuronal sensitization of spinal cord layer II interneurons, which has been reported to be the cause of changes in mechanical thresholds. However, since TAFA-4 is a cysteine-rich protein (a total of 10 cysteines), the production of recombinant mature TAFA-4 protein can be difficult. In fact, refolding can result in non-native higher-order structures or inappropriate disulfide bridge patterns, which can greatly affect the activity of the protein. Furthermore, several purification steps required to obtain high-purity recombinant TAFA-4 protein, along with the problem of possible aggregation, can limit the yield of properly folded protein. Additionally, chemical synthesis of the TAFA-4 protein has been performed, but this is also difficult due to the length of the protein and requires the assembly of at least four (protected or partially protected) peptide segments (starting from the N-terminus and ending with the most C-terminal peptide). Three ligation purification steps are required to generate full-length TAFA-4, leading to a low yield that is not suitable for industrial-scale production. Therefore, there is a strong need for alternative therapeutic agents and alternative treatment methods that can be used effectively to prevent or treat pain (specifically, acute, subacute, or chronic pain), and there would be great advantages in identifying novel compounds with activity equal to or greater than that of full-length TAFA-4 protein that can be produced on an industrial scale and in a cost-effective manner.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

[0007] [Non-Patent Document 1] Smith and Waterman, (1981) Adv. Appl. Math. 2: 482 [Non-Patent Document 2] Needleman and Wunsch, (1970) J. Mol. Biol. 48: 443 [Non-Patent Document 3] Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85: 2444 [Non-Patent Document 4] Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) [Non-Patent Document 5] Altschul et al., (1990), J. Mol. Biol. 215: 403 - 410 [Non-Patent Document 6] Altschul et al., (1977) Nucleic Acids Res. 3389 - 3402 [Non-Patent Document 7] Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915 [Non-Patent Document 8] Graham et al., Virology 52: 456 (1973) [Non-Patent Document 9] Sambrook, et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989) [Non-Patent Document 10] Davis, et al., Basic Methods in Molecular Biology, Elsevier (1986) [Non-Patent Document 11] Chu, et al., Gene 13:197 (1981) [Non-Patent Document 12] Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 [Non-Patent Document 13] Decosterd and Woolf, 2000; Pain, Vol. 87, pp. 149 - 158 [Non-Patent Document 14] A peripheral mononeuropathy in rats that produces disorders of pain sensation like those in man. Pain, Vol. 33: pp. 87 - 107 [Summary of the Invention]

[0008] The present invention is based at least in part on the identification of peptides that exhibit antalgic activity or analgesic activity, particularly antalgic activity. The inventors have discovered that these peptides can advantageously reverse mechanical hypersensitivity induced by nerve injury, surgery, or injection of inflammatory substances such as carrageenan, i.e., they can treat neuropathic pain, postoperative pain, or inflammatory pain.

[0009] The peptide of interest is a specific fragment of the human TATF-4 protein or is derived from such a fragment. This peptide is effective against pain (especially against chronic pain, typically pain induced by injury) compared to the human TAFA-4 full-length protein (also identified herein as "TAFA-4 FL") and is easy to manufacture. Indeed, these peptides are short peptides that do not need to be linked to each other to obtain a therapeutic effect. The inventors provide herein a novel tool for patients that can reduce the problem of dependence on pain treatment.

[0010] The inventors specifically describe herein for the first time a set of products or compositions, specifically a set of peptides, nucleic acids encoding such peptides, vectors, and / or cells enabling the expression of such peptides, wherein the peptides comprise or consist of the first peptide of SEQ ID NO: 1 (CFPGQVAGTTRAQPSCVEASIVIQKWW) or a peptide having at least 90% identity to SEQ ID NO: 1 (also identified herein as a "variant", "peptide variant", or "variant of the peptide" of the sequence of SEQ ID NO: 1) and the second peptide of SEQ ID NO: 2 (CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR) or a peptide having at least 90% identity to SEQ ID NO: 2 (also identified herein as a "variant", "peptide variant", or "variant of the peptide" of the sequence of SEQ ID NO: 2), and said (at least two) peptides are not linked to each other. The variants are typically functional variants, i.e., variants having the same (therapeutic) effect as described herein by the inventors with respect to the peptides of SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] A composition comprising a peptide of the sequence of SEQ ID NO:1 or a variant thereof (i.e., a peptide having at least 90% identity to SEQ ID NO:1) and a peptide of the sequence of SEQ ID NO:2 or a variant thereof (i.e., a peptide having at least 90% identity to SEQ ID NO:2) is also described herein, and the (at least two) peptides are not linked to each other.

[0012] In certain embodiments, similar to the peptides of SEQ ID NO:1 and SEQ ID NO:2, the variants described herein also modulate the excitability of spinal interneurons (preferably, spinal lamina IIi interneurons).

[0013] In a preferred embodiment, the glutamine (Q) residue at position 13 in the peptide of SEQ ID NO:1 (based on the position described in SEQ ID NO:1) remains unchanged in the variant of the peptide of SEQ ID NO:1. Similarly, the tyrosine (Y) residue at position 18 in the peptide of SEQ ID NO:2 (based on the position described in SEQ ID NO:2) remains unchanged in the variant of the peptide of SEQ ID NO:2 or is replaced by serine (S).

[0014] The inventors herein describe, specifically, a set of nucleic acids that enable the expression of (at least two) peptides that are not linked to each other, wherein the (first) nucleic acid comprises a sequence encoding a peptide of SEQ ID NO:1 or a peptide having at least 90% identity to SEQ ID NO:1, and the (second) nucleic acid comprises a sequence encoding a peptide of SEQ ID NO:2 or a peptide having at least 90% identity to SEQ ID NO:2, and a composition comprising such a set of nucleic acids.

[0015] A set of vectors that enables the expression of (at least two kinds of) peptides that are not linked to each other, wherein the (first) vector contains a sequence encoding the peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, and the (second) vector contains a sequence encoding the peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2. Also disclosed herein are a set of such vectors and a composition containing such a set of vectors.

[0016] Cells that enable the expression of the peptides (or their variants) described herein that are not linked to each other (at least two kinds), wherein the cells i) contain a (first) nucleic acid sequence or vector encoding the peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, and a (second) nucleic acid sequence or vector encoding the peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2. Also disclosed herein are a composition containing such cells and an acceptable support.

[0017] Also described herein are the sets, compositions, or cells that are disclosed herein for the first time with respect to use as an effective therapeutic or prophylactic ingredient (typically with respect to use as a pharmaceutical composition, drug, or medicine).

[0018] All of the compositions described herein are typically pharmaceutical compositions, therapeutic compositions, veterinary compositions, or nutritional / dietary compositions that contain a pharmaceutically and / or nutritionally / dietarily acceptable support in addition to (at least two kinds of) peptides.

[0019] In certain embodiments, the composition may further contain at least one additional (distinct) active compound / drug, preferably may further contain an active agent effective against pain (acute pain, subacute pain, or chronic pain), and even more preferably may further contain a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID), or an opioid drug.

[0020] The present invention also relates to the set or cells described herein for use as an [active ingredient] for preventing or treating pain in a subject in need thereof, typically a set of peptides, a set of nucleic acids encoding such peptides, or a set of vectors enabling the expression of such peptides, or cells comprising or expressing such a set. The present invention also relates to the use of such a peptide or such a set for the manufacture of a pharmaceutical composition, a drug, or a medicament for preventing or treating pain in a subject in need thereof. Specifically, the present invention can be used to treat chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia, allodynia, or acute pain. Similarly, the present invention can be used to treat arthralgia, pain associated with joint injury, pain associated with Ehlers-Danlos syndrome, pain due to sunburn, or pain due to ice burn.

[0021] The present invention also relates to a method for preventing or treating pain in a subject in need thereof, comprising administering a (therapeutically effective) amount of a product such as the set and / or composition described herein, preferably a set of peptides comprising the peptide of SEQ ID NO: 1, or a variant of this peptide having at least 90% identity to SEQ ID NO: 1, and the peptide of SEQ ID NO: 2, or a variant of this peptide having at least 90% identity to SEQ ID NO: 2, or administering a (therapeutically effective) amount of a composition comprising such a set.

[0022] The products described herein (specifically, peptides, sets, and compositions) can be administered to a subject by any route, for example, intramuscularly, intravenously, intraperitoneally, (per os), anal, cutaneous, subcutaneous, topical, dermically, transdermically, or intrathecally. Preferably, the products described herein (e.g., peptides or compositions) are administered to the subject subcutaneously, orally, or intravenously, and even more preferably subcutaneously or orally to the subject.

[0023] Each of the products (e.g., peptides or compositions) described herein can be part of a kit.

[0024] Certain kits of the invention include: i) the first peptide of SEQ ID NO: 1, a peptide having at least 90% identity to SEQ ID NO: 1, or a nucleic acid sequence encoding such a peptide; ii) the second peptide of SEQ ID NO: 2, a peptide having at least 90% identity to SEQ ID NO: 2, or a nucleic acid sequence encoding such a peptide; and optionally iii) written instructions for using the kit, wherein the first and second peptides, or the nucleic acids encoding said peptides, are in separate containers. The kits described herein are typically used in connection with the prevention or treatment of pain.

[0025] In certain aspects, the products of the invention can also be used in connection with research. Using a nucleic acid encoding a peptide or variant thereof described herein, a vector that permits its expression, or a cell modified using such a nucleic acid or such a vector, the peptide of SEQ ID NO: 1, or a functional variant of this peptide having at least 90% identity to SEQ ID NO: 1, and / or the peptide of SEQ ID NO: 2, or a functional variant of this peptide having at least 90% identity to SEQ ID NO: 2, can be expressed in vitro or ex vivo, or the expression can be regulated in vitro or ex vivo. In another specific aspect, the invention also relates to a transgenic animal modified to express at least two distinct nucleic acids described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

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Mode for Carrying Out the Invention

[0027] Definitions The terms used in this specification generally have their ordinary meaning in the context of the present invention and in the specific context in which each term is used. In the description of the methods of the present invention and their use, certain terms are discussed below or elsewhere in this specification to provide further guidance to the skilled reader. Further, it will be understood that the same matter can be described in multiple ways. Accordingly, alternative languages and synonyms may be used with respect to any one or more of the terms discussed herein. Certain synonyms are provided. The elaboration of one or more synonyms does not exclude the use of other synonyms.

[0028] The terms "peptide", "peptide variant", "protein fragment", and "protein segment" are used synonymously to refer to a sequence or polymer of amino acid residues. Such a polymer of amino acid residues can include natural or non-natural amino acid residues. The term also includes post-expression modifications of the peptide (e.g., glycosylation, sialylation, acetylation, amidation, phosphorylation, carbamylation, and the like). Further, in the context of the present invention, a "peptide" can optionally include one or several modifications (generally, essentially conservative), such as deletions, additions, and substitutions with respect to the native (wild-type) sequence, as long as the peptide maintains the desired activity (i.e., prevention or treatment of pain), and can include such post-expression modifications or combinations of such modifications. Such modifications are preferably intentional mutations, obtained, for example, by site-directed mutagenesis.

[0029] The term "isolated peptide" refers to a peptide that has been removed from its original environment (i.e., the natural environment if it exists naturally). A peptide that exists naturally in a natural system (e.g., in a living animal) should be distinguished from the same peptide that has been separated from all or some of the substances coexisting in said natural system. The separated peptide is referred to herein as an "isolated peptide". In other words, in the context of the present invention, an isolated peptide is a fragment of the TAFA-4 protein that does not exist naturally as such.

[0030] The term "synthetic peptide" refers to a peptide obtained by chemical synthesis.

[0031] The term "set of peptides" or "peptide set", as used herein, refers to at least two peptides that are used in combination as (the set's) active ingredients, which are mixed in a composition but not linked to each other. If there are two peptides, the peptides of this set are not linked to each other by any bond, for example, by a bond / linkage consisting of a peptide (amino acid sequence), or by a bond / linkage of chemical nature or both organic and chemical nature. (If there are more than two peptides in this set, these peptides are not linked to each other.) The peptides of this set are isolated peptides, synthetic peptides, or mixtures thereof. This set can exist in the form of a dry powder, solution, suspension, or colloid, and can be homogeneous or heterogeneous. The set of peptides is generally part of a composition. This composition also typically includes a pharmaceutically and / or nutritionally acceptable support, carrier, excipient, or vehicle. Typically, the excipient is ascorbic acid and / or an ascorbate. Ascorbic acid or L-ascorbic acid (IUPAC name: (5R)-5-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one) is a naturally occurring water-soluble vitamin commonly known as vitamin C. Examples of ascorbates include, but are not limited to, magnesium ascorbate, sodium ascorbate, calcium ascorbate, and ascorbyl palmitate. L-ascorbic acid and its sodium salt are antioxidants and are useful as preservatives or stabilizers when used in combination with a set of peptides. Other antioxidants (e.g., methionine) can also be used.

[0032] The terms "sequence identity", "a sequence having at least X% identity", and "a sequence that is X% identical" (where X is, for example, 90) are used interchangeably to refer to the degree to which sequences are identical in nucleotide or amino acid units over the entire comparison window. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over the entire comparison window, and a portion of the polynucleotide sequence or polypeptide sequence within the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences. This percentage can be calculated by determining the number of positions in which the same nucleic acid bases (e.g., A, T, C, G, U) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present in both sequences to obtain the number of matching positions, dividing this number of matching positions by the total number of positions within the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, this percentage can be calculated by determining the number of positions in which the same nucleic acid bases or amino acid residues are present in both sequences or in which the nucleic acid bases or amino acid residues are aligned with gaps to obtain the number of matching positions, dividing this number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Preferably, sequence identity is determined over the entire length of the reference sequence (here, SEQ ID NO: 1 or SEQ ID NO: 2).

[0033] Optimal alignment of arrays for comparison can be carried out, for example, by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA), or by visual inspection (generally, see Current Protocols in Molecular Biology, F. M. Ausubel et al., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)).

[0034] Examples of algorithms suitable for determining percent sequence identity and similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990), J. Mol. Biol. 215: 403-410 and Altschul et al., (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length "W" in the query sequence, which when aligned with words of the same length in the database sequence, either match or satisfy some positive-valued threshold score "T". T is referred to as the neighborhood word score threshold (Altschul et al., supra). This initial neighborhood word hit functions as a seed to initiate a search to find longer HSPs that contain it. Then, word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using the parameters "M" (reward score for pairs of matching residues; always >0) and "N" (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hit in each direction is stopped when the cumulative alignment score decreases from its maximum value by an amount "X", when the cumulative score becomes less than or equal to 0 due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value ("E") of 10, M = 5, N = -4, and a comparison of both strands.In the case of amino acid sequences, the BLASTP program by default uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0035] The percent identity of amino acid sequences can also be obtained by counting the number of identical matches in the alignment, dividing the number of such identical matches by the length of the reference sequence, and subsequently using the following default ClustalW parameters: gap opening penalty: 10; gap extension penalty: 0.10, protein weight matrix: Gonnet series; DNA weight matrix: IUB, slow / fast pairwise alignment switching = slow or complete alignment to achieve a slow / accurate pairwise optimal alignment by ClustalW analysis (version W 1.8).

[0036] As used herein, the terms "nucleic acid" or "polynucleotide" refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. For this reason, these terms include single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, but are not limited to these. The backbone of a polynucleotide can include sugars and phosphate groups (as can typically be found in RNA or DNA), or sugars or phosphate groups that have been modified or substituted. Alternatively, the backbone of a polynucleotide can include polymers of synthetic subunits such as phosphoramidates, and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing this strand under appropriate conditions, or by newly synthesizing the complementary strand using DNA polymerase with an appropriate primer.

[0037] As used herein, the terms "expression cassette", "nucleic acid construction", or "nucleic acid construct" are used synonymously and refer to a nucleic acid that includes at least one transcriptional promoter operably linked (in the sense understood by those skilled in the art) to one or more (coding) sequences of interest, or typically to an operon that includes several coding sequences of interest whose expression product contributes to the occurrence of the function of interest in the cell, or to a nucleic acid that further includes activation sequences and / or transcriptional terminators.

[0038] The term "vector" refers to i) a DNA molecule or an RNA molecule that contains a polynucleotide sequence encoding a peptide, polypeptide, or protein (e.g., an expression cassette or a transgene), or ii) a product that encapsulates such a DNA molecule or RNA molecule. A vector generally contains regulatory elements capable of inducing the expression of the encoded polynucleotide sequence (referred to as a transgene) in a cell into which the nucleic acid molecule has been introduced. The term "transgene" refers to a polynucleotide that can be introduced into a cell, transcribed into RNA, optionally translated, and / or expressed under appropriate conditions. In certain embodiments, the transgene confers a desired property to the cell into which it is introduced or otherwise provides a desired technical effect (typically a therapeutic effect herein). A transgene can contain a sequence encoding one or more proteins or one or more protein fragments. In certain embodiments, the vector is a plasmid. Alternatively, the vector is any known vehicle capable of artificially harboring an exogenous nucleic acid sequence for the purposes described herein, e.g., a viral vector, cosmid, phage, or double lipid membrane (e.g., liposome) that can be used to encapsulate the product described herein and deliver it to the desired site.

[0039] The term "gene therapy" refers to the treatment of a subject involving the delivery of a gene / nucleic acid into the cells of the individual for the purpose of preventing or treating a disease.

[0040] The term "gene transfer" refers to the uptake of exogenous polynucleotides by cells such as prokaryotic or eukaryotic cells. When an exogenous polynucleotide is introduced into a cell, this cell is identified as "gene - transferred". Many gene transfer techniques are generally known in the art. See, for example, Graham et al., Virology 52:456 (1973), Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989), Davis et al., Basic Methods in Molecular Biology, Elsevier (1986), and Chu et al., Gene 13:197 (1981). Using such techniques, one or more exogenous nucleic acids can be introduced into a suitable host cell.

[0041] The term "transduction" refers to the delivery of a nucleic acid molecule to a recipient host cell by a gene delivery vector (e.g., a recombinant viral vector, specifically, a retrovirus, an adenovirus, a recombinant adeno - associated virus (AAV), a herpes simplex virus, or a lentivirus). For example, by transduction of a target cell with rAAV viral particles, the rAAV vector contained in these viral particles is introduced into the transduced cell.

[0042] Recombinant "adeno - associated virus (AAV)" is a small Dependoparvovirus that has a single - stranded linear DNA genome, lacks pathogenicity, and has low immunogenicity, and is artificially produced using recombinant methods. Recombinant AAV (rAAV) preferably has tissue / cell - specific targeting ability, and thus, the transgene of rAAV is specifically or preferentially delivered to one or more predetermined tissues / cells. The AAV capsid, as well as the types of regulatory regions and administration routes, are important factors in determining these tissue - specific targeting abilities.

[0043] The term "pain", as used in relation to the present invention, refers to any pain or sensitivity associated with tissue damage. Preferably, the term "pain" as used herein is understood as abnormal sensitivity, i.e., typically, sensitivity mediated by abnormally increased activity of all types of sensory neurons, including nociceptors and non-nociceptors. The term "pain" includes any pain selected from nociceptor-mediated pain (also referred to herein as "nociceptive pain"), neuropathic pain, inflammatory pain, pathological pain, acute pain, subacute pain, chronic pain, mechanical pain, chemical pain, somatic pain, visceral pain, deep somatic pain, superficial somatic pain, somatic-referred pain, allodynia, hyperalgesia, or pain associated with nerve injury.

[0044] "Nociceptive" pain or "nociceptor-mediated" pain occurs in response to activation of a specific subset of peripheral sensory neurons (nociceptors) by strong or noxious stimuli. Examples of nociceptive pain according to the present invention include mechanical pain (compression, laceration, etc.) and chemical pain (iodine in a cut, chili powder in the eye). Examples of nociceptive pain include, but are not limited to, traumatic or postoperative pain, labor pain, sprains, fractures, burns, bruises, boils, injections, dental procedures, skin biopsies, and constrictions. Nociceptive pain includes visceral pain and somatic pain, particularly deep somatic pain and superficial somatic pain.

[0045] "Visceral pain" is diffuse, difficult to localize, and often refers to distal and usually superficial structures. It may be accompanied by nausea and vomiting and may be described as sick, deep, squeezing, and / or listless. Deep somatic pain is caused by stimulation of nociceptors in ligaments, tendons, bones, blood vessels, fascia, and muscles and is dull, aching, and poorly localized. Examples of deep somatic pain include sprains and fractures. Superficial pain results from activation of nociceptors in the skin or other surface tissues and is sharp, well-defined, and clearly localized. Examples of injuries that cause superficial somatic pain include minor trauma and mild (first-degree) burns.

[0046] The term "injury-induced pain" encompasses, in the context of the present invention, neuropathic pain, inflammatory pain, and postoperative pain.

[0047] "Inflammatory pain" is pain that occurs in the presence of tissue injury or inflammation, and includes postoperative pain, post-traumatic pain, arthritis (rheumatoid or osteoarthritis) pain, pain associated with autoimmune diseases (e.g., psoriasis), and pain associated with injury to joints, muscles, and tendons such as axial low back pain. Inflammation causes sensitization of peripheral sensory neurons, induces spontaneous pain, and abolishes pain hypoalgesia. Acute or chronic pathological tissue inflammation strongly affects pain perception by sensitizing peripheral sensory neurons and causing local and intolerable pain hyperalgesia. Inflammatory mediators are known to enhance the excitability of nociceptive primary afferent fibers by altering the expression and / or function of ion channels present at nerve endings.

[0048] "Neuropathic pain" is a common type of chronic non-malignant pain that results from damage or dysfunction of the peripheral or central nervous system. Neuropathic pain has various etiologies, such as trauma, surgery, intervertebral disc herniation, spinal cord injury, diabetes, herpes zoster (shingles) infection, HIV / AIDS, terminal cancer, amputation (including mastectomy), carpal tunnel syndrome, chronic alcohol intake, radiation exposure, and may occur as an unintended side effect of neurotoxic therapeutic agents such as certain anti-HIV drugs and chemotherapeutic drugs. A specific type of neuropathic pain is "chemotherapy-induced peripheral neuropathic pain" (CIPN) or "chemotherapy-induced neuropathic pain" (CINP). CINP or CIPN is one of the most severe side effects of anti-cancer agents such as platinum and taxane-derived drugs (oxaliplatin, cisplatin, carboplatin, and paclitaxel). CINP can be a factor in treatment interruption and may even increase the risk of death. Neuropathic pain is often characterized by or caused by the appearance of chronic allodynia (defined as pain caused by stimuli that normally do not cause a pain response, such as light touch) and / or hyperalgesia (defined as heightened sensitivity to stimuli that normally are accompanied by pain), and may persist for months or years even after apparent healing of any damaged tissue. Cancer patients may also experience pain, which can be due to multiple causes such as inflammation, compression, invasion, and metastasis to bone or other tissues. Pain also includes migraine and headache associated with activation of sensory fibers innervating the meninges of the brain.

[0049] The products of the present invention (specifically, peptides, sets, cells, or compositions) can be used for the prevention or treatment of chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia, allodynia, or acute pain.

[0050] In another specific embodiment, the products of the present invention (specifically, peptides, sets, cells, or compositions) can be used for the prevention or treatment of joint pain, pain associated with joint injury, pain associated with Ehlers-Danlos syndrome, pain due to sunburn, or pain due to frostbite.

[0051] In particular, the product of the present invention (specifically, a peptide, a set, a cell, or a composition) can be used for the prevention or treatment of pain associated with Ehlers-Danlos syndrome including chronic muscle pain and / or bone pain and / or joint pain, and can be particularly used for treatment. By using the product of the present invention (specifically, a peptide, a set, a cell, or a composition), pain caused by sunburn (i.e., caused by excessive exposure to ultraviolet rays) or pain caused by frostbite (also called freezer burn or freeze-burn) can also be prevented or treated, and can be particularly used for treatment.

[0052] Preferably, the product of the present invention (specifically, a peptide, a set, a cell, or a composition) is used for the prevention or treatment of injury-induced pain. More preferably, the product of the present invention (specifically, a peptide, a set, a cell, or a composition) is used for the prevention or treatment of neuropathic pain (e.g., chemotherapy-induced neuropathic pain or chemotherapy-induced peripheral neuropathic pain), postoperative pain, and / or inflammatory pain.

[0053] Typically, the product of the present invention (specifically, a peptide, a set, a cell, or a composition) is used for the prevention or treatment of chronic injury-induced pain. Typically, the product of the present invention is used for the prevention or treatment of chronic neuropathic pain (e.g., chronic chemotherapy-induced peripheral neuropathy (CIPN), chronic chemotherapy-induced neuropathic pain (CINP), or chronic nerve injury-induced pain; chronic postoperative pain; and / or chronic inflammatory pain; chronic pain associated with or resulting from severe burns).

[0054] In connection with the present invention, the term "treating" or "treatment" of a subject's pain refers to after exposure (i.e., application or administration) of the subject to an appropriate product or set of products of the present invention, specifically, i) a set of peptides (the peptides not being linked to each other), preferably peptides of the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 as defined herein or variants thereof, ii) a nucleic acid or set of nucleic acids enabling the expression of such a set of peptides not linked to each other, iii) an expression cassette or set of expression cassettes comprising the set of nucleic acids as defined herein, iv) a vector or set of vectors enabling the expression of such a set of peptides not linked to each other, v) a cell or set of cells enabling the expression of such a set of peptides not linked to each other, or vi) after exposure (i.e., application or administration) of the subject to a composition according to the present invention comprising such peptides, nucleic acids, expression cassettes, vectors, and / or cells, any form of pain of the subject as described herein, or any disease or condition associated with pain, specifically, acute, subacute, or chronic pain (specifically, any neuropathic state typically associated with chronic pain resulting from neuropathic pain, postoperative pain, or inflammatory pain), or delay, stabilization, curing, healing, alleviation, reduction, modification, relief, improvement, treatment, or effect on any symptom of such a disease or condition.

[0055] The term "treating" or "treatment" also refers to any indicator of success in the treatment of pain (which may be associated with any injury, pathology, or condition), including any objective or subjective parameter such as reduction of pain, remission, arrest or alleviation of progression or severity, stabilization, reduction of symptoms of pain, or making pain tolerable or more tolerable to the subject. The term "treating" pain also includes increasing pain tolerance and / or reducing perceived pain. In certain embodiments, the methods, compounds, and compositions of the invention are for increasing pain tolerance and / or reducing perceived pain. As used herein, the term "pain tolerance" refers to the amount of pain that a subject can perceive and tolerate before becoming emotionally and / or physically debilitated. Pain tolerance is different from the pain threshold (the minimum mechanical stimulus required to produce pain). As used herein, an "increase in pain tolerance" generally refers to a situation in which, for example, after administration to a subject of an appropriate peptide of the sequences of SEQ ID NO:1 and SEQ ID NO:2 or variants thereof, or a composition comprising said peptide or variant, the subject exhibits greater pain tolerance (i.e., a decrease in perceived pain) compared to a previous state.

[0056] In connection with the present invention, "prevent" or "prevention" related to the pain of interest refers to after the exposure (i.e., application or administration) of the product of the present invention to the subject, specifically, i) an appropriate set of peptides (the peptides are not linked to each other), preferably an appropriate set of peptides of the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 defined herein or variants thereof (typically, functional variants), ii) a nucleic acid or set of nucleic acids enabling the expression of such a set of peptides not linked to each other, iii) an expression cassette or set of expression cassettes containing the set of nucleic acids defined herein, iv) a vector or set of vectors enabling the expression of such a set of peptides not linked to each other, v) a cell or set of cells enabling the expression of such a set of peptides not linked to each other, or vi) at least a reduction in the likelihood (or susceptibility) of the subject experiencing any kind of pain after the exposure (i.e., application or administration) of the composition according to the present invention containing such peptides, nucleic acids, expression cassettes, vectors, and / or cells to the subject. For example, "prevent" includes preventing at least one of the clinical symptoms of pain from occurring in a subject who may be exposed to pain or have a predisposition to pain but has not yet experienced or presented the symptoms of pain.

[0057] In connection with the present invention, "subject" or "patient" refers to an animal (specifically, a mammal) in need of treatment for a disease or disorder or symptoms thereof. The subject can be a subject diagnosed with having a disease or disorder or a subject determined to be at risk of developing a disease or disorder, and the disease or disorder is known to cause the subject to feel pain. In certain examples, the subject is diagnosed with or suffering from pain, for example, acute pain, and / or subacute pain, or chronic pain (e.g., neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia, and / or allodynia).

[0058] In certain embodiments, the subject is a human.

[0059] In another specific embodiment, the subject is an animal, specifically a domestic animal or a breeding animal, specifically a horse, a dog, a cat, a cow, etc.

[0060] In another specific embodiment, the subject has at least one mutant allele in the myo1A gene.

[0061] Peptide The inventors herein have identified a novel set of components that, when used in combination (either simultaneously or sequentially, preferably simultaneously), can prevent or treat pain, and in particular, can reverse mechanical hypersensitivity associated with a damaged or inflamed nervous system, in particular a set of peptides (the peptides are not linked to each other). Without being bound by any theory, the inventors believe that by modulating the excitability of the spinal cord network, these peptides exhibit either antalgic or analgesic activity, in particular antalgic activity, by specifically targeting mechanically and / or chemically induced pain signals.

[0062] The peptides described herein are specific fragments (SEQ ID NO: 5: SQHLRGHAGHHQIKQGTCEVVAVHRCCNKNRIEERSQTVKCSCFPGQVAGTTRAQPSCVEASIVIQKWWCHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR) derived from the human TAFA-4 mature protein of 105 amino acid residues, i.e., consisting of this specific fragment (as disclosed by Tang et al., 2004, resulting from the cleavage of the signal sequence and identified in the public database under accession number NP_0011005527 or Genbank accession AAP92409).

[0063] The inventors specifically describe, for the first time in this specification, a set of products / components, specifically, i) the first peptide of SEQ ID NO: 1 (CFPGQVAGTTRAQPSCVEASIVIQKWW, i.e., Cys-Phe-Pro-Gly-Gln-Val-Ala-Gly-Thr-Thr-Arg-Ala-Gln-Pro-Ser-Cys-Val-Glu-Ala-Ser-Ile-Val-Ile-Gln-Lys-Trp-Trp) or a peptide having at least 90% identity to SEQ ID NO: 1 (also identified herein as a "variant", "peptide variant", or "variant of the peptide" of the sequence of SEQ ID NO: 1), and ii) the second peptide of SEQ ID NO: 2 (CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR, i.e., Cys-His-Met-Asn-Pro-Cys-Leu-Glu-Gly-Glu-Asp-Cys-Lys-Val-Leu-Pro-Asp-Tyr-Ser-Gly-Trp-Ser-Cys-Ser-Ser-Gly-Asn-Lys-Val-Lys-Thr-Thr-Lys-Val-Thr-Arg) or a peptide having at least 90% identity to SEQ ID NO: 2 (also identified herein as a "variant", "peptide variant", or "variant of the peptide" of the sequence of SEQ ID NO: 2), and describe a set of isolated or synthetic peptides that comprises, consists of, or consists essentially of, and wherein the (at least two) peptides are not linked to each other.

[0064] A peptide having at least 90% identity to a particular amino acid / peptide sequence is identified herein as a "variant", "peptide variant", or "variant of the peptide" having said sequence. In a preferred embodiment, any variant present in the set or in a composition containing / expressing such a set is a functional variant further described below in this specification.

[0065] The term "consisting essentially of" has its generally assigned meaning. For example, this term permits elements not explicitly recited, but excludes elements found in the prior art or elements that affect the basic or novel characteristics of the invention. Accordingly, a set of peptides "consisting essentially of" the sequences refers to peptides that include or consist of these sequences (which are not linked to each other), and include other features that are not essential for the activity of these peptides.

[0066] The peptide of SEQ ID NO: 1 consists of 27 C-terminal amino acid residues of the human TAFA-4 mature protein and can be identified as amino acids 78-104 of UniProt accession number Q96LR4.

[0067] The peptide of SEQ ID NO: 2 consists of 36 amino acid residues of the human TAFA-4 mature protein and can be identified as amino acids 105-140 of UniProt accession number Q96LR4.

[0068] In certain embodiments, the peptide of SEQ ID NO: 1 and / or the peptide of SEQ ID NO: 2 are acetylated at the N-terminal position and / or amidated at the C-terminal position.

[0069]

Chemical formula

[0070] Q (Glu) represented in bold at position 13 within, and

[0071]

Chemical formula

[0072] The inventors believe that the two amino acid residues of Y (Tyr) represented in bold at position 18 within (based on the positions described in SEQ ID NO: 1 and SEQ ID NO: 2 respectively) imply the biological activity of these peptides.

[0073] Variants of the peptides of SEQ ID NO:1 and SEQ ID NO:2 are also for the first time disclosed herein by the inventors. A variant is intended to designate a peptide that is derived from the peptide of SEQ ID NO:1 or SEQ ID NO:2 and that has arisen by deletion or addition of one or more amino acids and / or substitution of one or more amino acids at one or more sites in the said peptide of SEQ ID NO:1 or SEQ ID NO:2. A variant is considered to be a "functional variant" if it substantially retains the functional activity of the peptide of SEQ ID NO:1 or SEQ ID NO:2, or even if it improves the said functional activity that enables prevention or treatment of pain. This activity can be measured using a functional assay such as a behavioral assay as carried out in the experimental part.

[0074] Variants of the peptides that can be used in connection with the present invention can have at least 90% sequence identity to the sequence encoding the said peptide (for example, SEQ ID NO:1 or SEQ ID NO:2), for example (at least) 90.5%, 91%, 92%, 92.1%, 93%, 93.7%, 94%, 95%, 95.2%, 96%, 96.8%, 97%, 98%, 98.4%, or 99% sequence identity.

[0075] In certain embodiments, a variant having at least 90% identity to SEQ ID NO:1 or SEQ ID NO:2 modulates the excitability of spinal cord interneurons (preferably, spinal cord layer IIi interneurons). In a preferred embodiment, this variant is a peptide comprising one or more point mutations (for example, 2, 3, 4, 5, or 6 point mutations) that add, delete, or substitute any of the amino acids present in the peptide sequence of the desired peptides described herein (for example, specifically SEQ ID NO:1 or SEQ ID NO:2).

[0076] In particular, with respect to peptide variants having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2, the amino acid residue Q at position 13 in SEQ ID NO: 1 remains unchanged in the amino acid sequence of a peptide having at least 90% identity to SEQ ID NO: 1, and the amino acid residue Y at position 18 in SEQ ID NO: 2 either remains unchanged in the amino acid sequence of a peptide having at least 90% identity to SEQ ID NO: 2 or is replaced by serine (S) (positions 13 and 18 are based on the positions described in SEQ ID NO: 1 and SEQ ID NO: 2, respectively), the previous paragraph applies. The peptide having the amino acid residue S at position 18 is SEQ ID NO: 6 (CHMNPCLEGEDCKVLPDSSGWSCSSGNKVKTTKVTR, i.e., Cys-His-Met-Asn-Pro-Cys-Leu-Glu-Gly-Glu-Asp-Cys-Lys-Val-Leu-Pro-Asp-Ser-Ser-Gly-Trp-Ser-Cys-Ser-Ser-Gly-Asn-Lys-Val-Lys-Thr-Thr-Lys-Val-Thr-Arg).

[0077] Therefore, in a preferred embodiment of the present invention, the amino acid Q (glutamine residue) at position 13 in the peptide of SEQ ID NO: 1 remains unchanged in the amino acid sequence of a variant of a peptide having at least 90% identity to SEQ ID NO: 1. Similarly, in another preferred embodiment, the amino acid Y (tyrosine residue) at position 18 in the peptide of SEQ ID NO: 2 either remains unchanged in the amino acid sequence of a variant of a peptide having at least 90% identity to SEQ ID NO: 2 or is replaced by S (serine).

[0078] In another preferred embodiment of the present invention, the amino acid Q at position 13 (based on SEQ ID NO: 1) remains unchanged in the amino acid sequence of a variant of a peptide having at least 90% identity to SEQ ID NO: 1, and the amino acid Y at position 18 (based on SEQ ID NO: 2) remains unchanged or is replaced by serine (S) in the amino acid sequence of a variant of a peptide having at least 90% identity to SEQ ID NO: 2.

[0079] In another specific embodiment, preferably, a variant of the peptide of SEQ ID NO: 1 that can be used in connection with the present invention can have at least 88.8%, 92.5%, 96.2% sequence identity to the peptide sequence of SEQ ID NO: 1, provided that the amino acid residue Q at position 13 based on SEQ ID NO: 1 is as described above. In another specific embodiment, preferably, a variant of the peptide of SEQ ID NO: 2 that can be used in connection with the present invention can have at least 91.6%, 94.4%, 97.2% sequence identity to the peptide sequence of SEQ ID NO: 2, provided that the amino acid residue Y at position 18 based on SEQ ID NO: 2 is as described above. Preferably, the variant modulates the excitability of spinal interneurons (preferably, spinal lamina IIi interneurons).

[0080] In a specific embodiment, the variant is a peptide containing one or more point mutations (e.g., 1, 2, or 3 point mutations) that add, delete, or substitute any of the amino acids present in the peptide sequence of the desired peptide described herein (e.g., specifically SEQ ID NO: 1 or SEQ ID NO: 2), provided that the amino acid residue Q at position 13 based on SEQ ID NO: 1 and the Y at position 18 based on SEQ ID NO: 2 are as described above.

[0081] In certain embodiments, one or more deletions are present at the N-terminus of the target sequence such as SEQ ID NO:1 or SEQ ID NO:2. In another embodiment, one or more deletions are present at the C-terminus or at any other position of the target sequence such as SEQ ID NO:1 or SEQ ID NO:2, provided that they are as described above with respect to amino acid residue Q at position 13 relative to SEQ ID NO:1 and Y at position 18 relative to SEQ ID NO:2. In yet another embodiment, two or more deletions are present at both the N-terminus and C-terminus of the target sequence such as SEQ ID NO:1 or SEQ ID NO:2. Such deletions at the N-terminus and / or C-terminus or in the core of the peptide of SEQ ID NO:1 or SEQ ID NO:2, for example, can result in truncated human peptide variants of SEQ ID NO:1 or SEQ ID NO:2.

[0082] In addition, any peptide of the set of peptides according to the invention can be fused to another peptide or protein to form a conjugate that can be used in the methods described herein, provided that said "another peptide or protein" is not one of the "at least two" peptides of the set of the invention. Indeed, the peptides of the set of peptides described herein (the active ingredients of this set) should not be linked to each other. The term "conjugate" in this context refers to an engineered fusion construct that combines the biological functions of two molecules within a single polypeptide (specifically, for example, a peptide of SEQ ID NO:1 or 2 or a variant thereof that can modulate the excitability of an afferent receptor or an intervening neuron), together with a polypeptide that specifically interacts with or binds to a target cell.

[0083] The present invention also relates to nucleic acids encoding the set of peptides of the invention (e.g., the peptide of the sequence of SEQ ID NO: 1, the peptide of the sequence of SEQ ID NO: 2, or variants thereof described herein). For example, any sequence encoding the peptide of SEQ ID NO: 1, the peptide of SEQ ID NO: 2, or variants thereof is encompassed by the present invention, as are similar sequences arising from the degeneracy of the genetic code. In certain embodiments, the nucleic acid encoding the peptides described herein comprises, consists of, or consists essentially of SEQ ID NO: 3: TGCTTCCCGGGACAGGTGGCGGGCACAACTCGGGCTCAACCTTCTTGTGTTGAAGCTTCCATTGTGATTCAGAAATGGTGG, or SEQ ID NO: 4: TGTCACATGAATCCGTGTTTGGAAGGAGAGGATTGTAAAGTGCTGCCAGATTACTCAGGTTGGTCCTGTAGCAGTGGCAATAAAGTCAAAACTACGAAGGTAACGCGG.

[0084] The inventors have specifically described herein a set of nucleic acids that enables the expression of (at least two) peptides that are not linked to each other, wherein the (first) nucleic acid comprises a sequence encoding the (first) peptide of interest (e.g., the peptide of SEQ ID NO: 1, or a peptide having at least 90% identity to SEQ ID NO: 1), and the (second) nucleic acid comprises a sequence encoding the (second) peptide of interest (e.g., the peptide of SEQ ID NO: 2, or a peptide having at least 90% identity to SEQ ID NO: 2). The inventors have also described herein a composition comprising such a set of nucleic acids, preferably together with an acceptable support, carrier, excipient, or vehicle described herein.

[0085] The above-described nucleic acid sequences encoding the peptides of SEQ ID NO: 1, SEQ ID NO: 2, or variants thereof can be adjacent to regulatory sequences for controlling their expression in a suitable host cell.

[0086] In another aspect, the present invention also relates to an expression cassette that, in the order from 5' to 3', - a promoter, - A nucleic acid sequence encoding a peptide of interest such as SEQ ID NO:1, SEQ ID NO:2, or a variant thereof described herein, and - A termination signal, such as a polyadenylation signal also relates to an expression cassette containing the same.

[0087] The above-described nucleic acid sequence encoding the peptide of interest, or the expression cassette disclosed herein, can be adjacent to a sequence suitable for packaging into a vector that optimizes transcription and / or translation in a cell.

[0088] In another aspect, the invention also relates to a vector that permits the expression of a peptide or set of peptides described herein. The invention also relates to a vector containing a set of nucleic acids that enables the expression of (at least two) peptides not linked to each other, or a set of expression cassettes defined herein.

[0089] In a further aspect, the invention relates to a set of vectors that enable the expression of (at least two peptides) not linked to each other, wherein the (first) vector contains a sequence encoding a (first) peptide of interest (e.g., the peptide of SEQ ID NO:1, or a peptide having at least 90% identity to SEQ ID NO:1), and the (second) vector contains a sequence encoding another (second) peptide of interest (e.g., the peptide of SEQ ID NO:2, or a peptide having at least 90% identity to SEQ ID NO:2). The inventors also describe herein a composition containing such a set of vectors, preferably together with an acceptable support, carrier, excipient, or vehicle described herein.

[0090] Method for producing a peptide To enable expression in a host cell, a nucleic acid encoding a peptide (or a variant thereof) described herein may be present in a vector, and after introduction of the vector into an appropriate host cell, this sequence can be expressed according to standard cloning techniques and expression techniques known in the art to produce the encoded peptide described herein (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Various expression vectors can be utilized to express the polynucleotide sequence encoding the peptide described herein. Expression vectors that can be used in the present invention include, but are not limited to, eukaryotic expression vectors, specifically mammalian expression vectors, virus-based expression vectors, baculovirus expression vectors, plant expression vectors, and any plasmid expression vectors for producing any of the peptides described herein in a host cell. Expression vectors may also potentially be vectors that enable expression of peptides in a bacterial system. When a single nucleic acid encodes several peptides or when a single vector enables expression of several peptides, the resulting peptides are not linked to each other.

[0091] The choice of expression vector depends on the intended host cell in which the vector can be expressed. This choice will be readily made by those skilled in the art. The present invention also relates to cells (specifically host cells) containing a nucleic acid sequence encoding a peptide described herein. Cells (host cells) modified using the vectors described herein are also for the first time disclosed herein by the inventors.

[0092] Cells that enable the expression of (at least two) peptides (or variants thereof) described herein that are not linked to each other are specifically disclosed herein. Such cells typically contain a (first) nucleic acid sequence or vector encoding a peptide of interest (e.g., the peptide of SEQ ID NO: 1, or a peptide having at least 90% identity to SEQ ID NO: 1) and a (second / different) nucleic acid sequence or vector encoding a (second) peptide of interest (e.g., the peptide of SEQ ID NO: 2, or a peptide having at least 90% identity to SEQ ID NO: 2). Preferably, compositions containing such cells are also disclosed, together with the acceptable supports, carriers, excipients, or vehicles described herein.

[0093] A set of cells that enable the expression of (at least two) peptides (or variants thereof) described herein that are not linked to each other is specifically disclosed herein. Each cell typically contains a nucleic acid sequence or vector encoding a peptide of interest (e.g., the peptide of SEQ ID NO: 1 or SEQ ID NO: 2, or a peptide having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2). Preferably, compositions containing such cells are also disclosed, together with the acceptable supports, carriers, excipients, or vehicles described herein.

[0094] The peptide of the present invention or its variant can be expressed in either a prokaryotic host cell or a eukaryotic host cell. Representative host cells include many Escherichia coli (E. coli) strains, mammalian cell lines such as CHO, CHO-K1, and HEK293; insect cells such as Sf9 cells; and yeast cells such as S. cerevisiae and P. pastoris.

[0095] The nucleic acids or vectors described herein can be introduced into host cells by standard techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells (e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc.). Alternatively, the nucleic acids or vectors described herein can be delivered into host cells by transduction using virus-based vectors.

[0096] The purity can be measured by any suitable standard method, for example, by column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis. An “isolated” or “synthetic” peptide also defines a degree of sterility that is safe for administration to a human subject (e.g., lacking infectious or toxic substances).

[0097] The peptides used in the compositions and methods described herein can also be produced by solid phase synthesis techniques. Direct chemical synthesis of peptides can be accomplished by methods well known to those skilled in the art such as native chemical ligation (NCL). This chemical approach consists of the coupling of unprotected peptide fragments where a peptide having an N-terminal cysteine reacts with a C-terminal thioester peptide. Immediately following this thioester exchange reaction, an intramolecular S,N-acyl shift occurs, thereby forming a native amide bond at the ligation site.

[0098] Gene therapy By cloning the nucleic acid sequences encoding the peptides described herein into suitable vectors, the inventors also provide herein novel gene delivery tools (specifically, gene constructs such as expression cassettes or vectors) for the gene therapy of pain (specifically, acute pain, subacute pain, or chronic pain, preferably chronic pain). Gene therapy can also enable the endogenous production of TAFA-4-peptide or its variants by specific cells of a subject. Gene therapy can be performed either in vivo or ex vivo. Ex vivo gene therapy requires at least the isolation and purification of a sample of the subject's cells, the introduction of at least one nucleic acid sequence (i.e., a transgene) encoding the set of peptides described herein into the isolated cells, and the reintroduction of the gene-altered / modified cells into the subject. In contrast, in in vivo gene therapy, the transgene is typically packaged for administration to the subject. The gene delivery construct can be either non-viral or viral. Preferably, the gene constructs described herein are prepared by viral elements, viral vectors, and / or any viral packaging system that can be used to express the transgene / coding sequence (i.e., the nucleic acid sequence encoding the set of peptides of interest or its variants) in the target tissue / cells. The viral vector can incorporate any suitable promoter and other transcriptional regulatory factors that enable or facilitate the expression of the transgene product in the target tissue / cells. The viral packaging system is preferably adapted to the target cells. Upon entry into the target cells, such a system facilitates delivery to the target tissue. The viral vectors that can be used in the methods described herein are preferably replication-deficient viruses such as, for example, adenoviral vectors or adeno-associated virus (AAV) vectors.

[0099] In some embodiments, the invention relates to a recombinant adeno-associated virus (AAV) that contains, in its genome, a nucleic acid sequence encoding at least one peptide (preferably at least two peptides, such as the peptide of SEQ ID NO: 1 and / or the peptide of SEQ ID NO: 2) of a set of interest or a variant thereof. The coding portion of this nucleic acid is typically operably linked to a promoter.

[0100] To date, at least several dozen different serotypes of AAV with various surface characteristics have been isolated and characterized from humans or non-human primates (NHPs). The term "serotype" enables those skilled in the art to distinguish AAVs having serologically distinct capsids. Serological characteristics are determined based on the lack of cross-reactivity between antibodies against a particular AAV serotype compared to other AAV serotypes. The rAAV (also referred to as rAAV vector or rAAV particle) described herein may have any one of the following known serotypes, i.e., it may be selected, for example, from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhIO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68. The rAAV vector may have enhanced tropism for specific cells, tissues, or organs. In relation to oral administration, the rAAV vector may have enhanced tropism for gastric, small intestine, or colon tissue, and more specifically, for the cells constituting these tissues, specifically, epithelial cells, such as enterocytes, goblet cells, enteroendocrine cells, Paneth cells, or tuft cells. In the case of target cells located in the intestine or target cells delivered to the intestine, any one of AAV4, AAV7, AAV8, AAV9, or AAV10 may be selected as particularly effective.

[0101] The gene therapy vector or cassette of the present invention can be produced by methods known to those skilled in the art and already described, for example, in WO 03 / 042397 and US Patent No. 6,632,670.

[0102] This specification also relates to a method for producing a recombinant vector (e.g., a viral vector such as AAV), comprising: a) culturing cells transfected / transduced with the recombinant vector described herein; and b) recovering the recombinant vector from the supernatant of the transfected / transduced cells. The present invention also relates to a method comprising the steps of:

[0103] The gene therapy vector of the present invention can be produced by transfecting 293 or 293T human embryonic kidney cell lines with two or three plasmids. In certain embodiments, the DNA encoding the therapeutic peptide is provided on one plasmid, and the capsid protein and replication genes derived from one or more serotypes of AAV, as well as helper functions derived from, for example, adenovirus, are all provided in trans by a second plasmid. In another specific embodiment, the DNA encoding the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 or variants thereof is provided on one plasmid, the capsid protein and replication genes derived from one or more serotypes of AAV are provided in trans by a second plasmid, and the helper functions derived from, for example, adenovirus are provided by a third plasmid. In certain embodiments, the first plasmid comprises an expression cassette comprising a nucleic acid sequence encoding the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2 or variants thereof operably linked to a promoter such as two adjacent inverted terminal repeats (ITRs). In another specific embodiment, the DNA encoding each of the therapeutic peptides is provided by the following two different plasmids: a first plasmid comprising a nucleic acid sequence encoding the peptide of SEQ ID NO: 1 or a variant thereof, and a second plasmid comprising a nucleic acid sequence encoding the peptide of SEQ ID NO: 2 or a variant thereof.

[0104] After cell culture, the gene therapy vector can be released from the cells, for example, by freeze-thaw cycles and purified by any method known to those skilled in the art. For example, it can be purified by using an iodixanol step gradient and subsequent ion exchange chromatography on a Hi-Trap QHP column. The obtained gene therapy vector can then be concentrated by a spin column, and the purified vector can be cryopreserved (at -60°C or below), for example, in phosphate-buffered saline.

[0105] Related aspects of the present invention include host cells that have been transfected or transduced with at least one recombinant vector described herein (e.g., transduced with at least one AAV vector); or a set of cells that have been transfected with a vector or set of vectors containing DNA encoding a therapeutic peptide or variant thereof as defined herein. Further related aspects of the present invention include any nucleic acid molecule that contains or (essentially) consists of the genome of a recombinant vector (e.g., AAV) described herein.

[0106] Composition A composition described herein that contains a set of (at least two) peptides that are not linked to each other, or a product (e.g., nucleic acid, cell, set of nucleic acids, or set of cells) described herein that enables the expression of the (at least two) peptides can be a pharmaceutical composition, a therapeutic composition, a prophylactic composition, or a veterinary composition. This composition can also be a nutritional / dietary composition (optionally, simultaneously).

[0107] The specific compositions described herein include a peptide of the sequence of SEQ ID NO: 1 or a (typically functional) variant thereof (i.e., a peptide having at least 90% identity to SEQ ID NO: 1), and a peptide of the sequence of SEQ ID NO: 2 or a (typically functional) variant thereof (i.e., a peptide having at least 90% identity to SEQ ID NO: 2), wherein said (at least two) peptides are not linked to each other.

[0108] A set of two or more peptides described herein is advantageously formulated for use in combining these peptides separately or simultaneously. Thus, each peptide of the set can be prepared in a separate form, or in the form of a composition comprising a mixture of at least two peptides of the set. This composition can be a dietary / nutritional composition and / or a pharmaceutical composition, and can be used in the treatment methods or prevention methods described herein.

[0109] In certain aspects, the invention relates to a composition comprising at least two peptides or variants thereof described herein that are not linked to each other, typically a set of (at least two) peptides that are not linked to each other, a nucleic acid or set of nucleic acids encoding (at least two) peptides that are not linked to each other, an expression cassette or set of expression cassettes enabling the expression of (at least two) peptides that are not linked to each other, a vector or set of vectors enabling the expression of (at least two) peptides that are not linked to each other, or a cell or set of cells enabling the expression of (at least two) peptides that are not linked to each other, and preferably a permitted support, carrier, excipient, or vehicle described herein, specifically a dietary and / or pharmaceutically permitted support, carrier, excipient, or vehicle.

[0110] The term "dietarily acceptable carrier" relates to a carrier that allows a composition containing a peptide or set of peptides, nucleic acid sequence or set of nucleic acid sequences, expression cassette or set of expression cassettes, vector or set of vectors, or cell or set of cells, as described herein, to be ingested or digested by a subject without risk, and protects the peptide from any attack (specifically, any attack related to food digestion) that may alter the peptide before the peptide exerts its therapeutic effect at the appropriate site and at the appropriate time, depending on the nature and location of the pain.

[0111] In certain embodiments where the composition comprises two peptides, the first and second peptides may be present in the composition of the invention in equal or different ratios. In certain compositions, the first and second peptides are present in the composition in equal (equimolar) ratios, i.e., each peptide occupies 50% of the peptide component of the composition, which means that the composition contains the same number of moles of each of the two peptides.

[0112] Another particular peptide composition may contain a particular peptide in a higher proportion compared to another peptide or other peptides. For example, a particular peptide may occupy at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the peptide component of the composition.

[0113] In relation to oral administration, the composition may further comprise at least one gastroprotective agent (preferably, an acid inhibitor) present in an amount effective to raise the pH of the subject's stomach to at least 2, at least 3, at least 4, more preferably at least 5 or 6. The term "acid inhibitor" refers to an agent that inhibits the secretion of gastric acid and raises the pH of the stomach. Examples of acid inhibitors include, but are not limited to: H2 blockers, such as cimetidine, ranitidine, ebrotidine, pabutidine, raf tidine, roxatidine, famotidine; proton pump inhibitors, such as omeprazole, esomeprazole, pantoprazole, lansoprazole, dexlansoprazole, rabeprazole, pariprazole, reminiprazole, and tenatoprazole; or any combination thereof.

[0114] A "pharmaceutically acceptable support / vehicle / carrier" can be a diluent, adjuvant, or excipient administered together with the active agent (i.e., at least two peptides of the invention or variants thereof, and optionally any additional different active agent). Such pharmaceutical carriers can be sterile liquids such as water or oil, of petroleum, animal, vegetable, or synthetic origin, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline, as well as aqueous dextrose and glycerol solutions, can also be used particularly as liquid carriers for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0115] The pharmaceutically acceptable support / vehicle / carrier can consist of or include ascorbic acid and / or an ascorbate. Examples of ascorbates are magnesium ascorbate, sodium ascorbate, calcium ascorbate, and ascorbyl palmitate.

[0116] In one embodiment, the composition of the present invention contains ascorbic acid or an ascorbate.

[0117] When the composition (for example, a pharmaceutical composition) is suitable for oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients, and examples of such excipients include: binders (for example, pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (for example, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc, or silica), disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets can be coated by any method known in the art. Liquid formulations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or can be presented as dry products for reconstitution with water or another suitable vehicle before use. Such liquid formulations can be prepared by conventional means with pharmaceutically acceptable additives, and examples of such additives include: suspending agents (for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (for example, methyl or propyl - p - hydroxybenzoate or sorbic acid). The formulation can also optionally contain buffering salts, flavorings, colorants, and sweeteners.

[0118] The composition of the present invention (for example, a pharmaceutical composition, a prophylactic composition, or a therapeutic composition) can further contain at least one additional (different) active compound. Preferably, this additional active compound is an active agent effective against pain. "Effective against pain" means an active agent having antalgic or analgesic properties (measurable and felt by the subject). More preferably, this additional active compound is a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID), or an opioid drug.

[0119] Examples of said may include, but are not limited to: hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone, fludrocortisone, or any combination thereof.

[0120] Examples of NSAID may include, but are not limited to: celecoxib, rofecoxib, lumiracoxib, valdecoxib, parecoxib, etoricoxib, CS-502, JTE-522, L-745,337, NS398, aspirin, acetaminophen (considered an NSAID for the purposes of this disclosure), ibuprofen, flurbiprofen, ketoprofen, naproxen, oxaprozin, etodolac, indomethacin, ketorolac, lornoxicam, meloxicam, piroxicam, droxicam, tenoxicam, nabumetone, diclofenac, meclofenamate, mefenamic acid, diflunisal, sulindac, tolmetin, fenoprofen, suprofen, benoxaprofen, aceclofenac, tolfenamic acid, oxyphenbutazone, azapropazone, phenylbutazone, or any combination thereof.

[0121] Examples of opioid drugs include, but are not limited to: (dextro)propoxyphene, A-methylfentanyl, alfentanil, allylprodine, bezitramide, buprenorphine, butorphanol, carfentanil, desmethylprodine, dextromoramide, dezocine, diacetylmorphine, dihydrocodeinone, dihydroetorphine, dimorphon, diphenoxylate, dipipanone, etorphine, fentanyl, ketobemidone, levetamine, levacetylmethadol, levomethorphan, levorphanol, loperamide, meperidine, meptazinol, methadone, methylmorphine, morphine, nalbufine, nicomorphine, orimiphentanyl, olipavine, oxycodone, oxymorphone, PEPAP, paramorphine, pentazocine, phenazocine, pirtramide, prodine, remifentanil, sufentanil, tapentadol, tilidine, tramadol, or an opioid antagonist, for example, nalmeffene, naloxone, naltrexone, or any combination thereof.

[0122] Therapeutic use and prophylactic use As used herein, for use as an active prophylactic or therapeutic ingredient, preferably for use as a pharmaceutical composition, drug, or medicament, the peptides, nucleic acids, expression cassettes, vectors, cells, and sets thereof, or any combination thereof, described above, and compositions containing such peptides, nucleic acids, expression cassettes, vectors, cells, and / or sets thereof are disclosed.

[0123] In certain embodiments, the peptides described herein, specifically, the isolated or synthetic peptide of SEQ ID NO: 1 and / or the isolated or synthetic peptide of SEQ ID NO: 2, preferably, a set of said peptides, any set enabling the expression of such peptides, or any composition containing such peptides, nucleic acids, expression cassettes, vectors, cells, and / or sets are for use as a pharmaceutical composition, drug, or medicament, specifically, for use for the purpose of preventing or treating pain.

[0124] In another aspect, the present specification relates to the use of the peptides, nucleic acids, expression cassettes, vectors, or cells described herein, and / or the sets described herein, as active ingredients / agents for preventing or treating pain in a subject in need thereof.

[0125] The present specification specifically relates to the use [as an active ingredient] for preventing or treating pain in a subject in need thereof of a product disclosed herein, or any set of products disclosed herein, for example, a set of cells described herein, typically a set of peptides, or cells that contain or express such a set of peptides.

[0126] In certain aspects, the inventors herein describe the use of any of the products of the invention described herein for the manufacture of a pharmaceutical composition, drug, or medicament for the prevention and treatment of pain in a subject in need thereof, for example, the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof described herein, or any combination thereof described herein. The inventors also herein describe the corresponding method for preventing or treating pain, typically comprising administering to a subject in need thereof the peptides, nucleic acids, expression cassettes, vectors, cells, sets, and / or compositions described herein.

[0127] Specifically, provided herein is a method for preventing or treating pain in a subject in need thereof, the method comprising administering a (therapeutically) effective amount of a product (e.g., externally, a set of peptides and / or a composition described herein, preferably a set of peptides comprising the peptide of SEQ ID NO: 1 or a variant thereof having at least 90% identity to SEQ ID NO: 1, and the peptide of SEQ ID NO: 2 or a variant thereof having at least 90% identity to SEQ ID NO: 2, or a composition comprising such a set).

[0128] In certain embodiments, the inventors describe herein the use of any of the products of the invention described herein for preventing or treating pain (specifically, acute pain, subacute pain, or chronic pain, preferably chronic pain), e.g., the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets described herein, or any combination or composition thereof described herein.

[0129] Preferably, the pain is neuropathic pain (e.g., chemotherapy-induced peripheral neuropathic pain, or chemotherapy-induced neuropathic pain), postoperative pain, inflammatory pain, hyperalgesia, or allodynia.

[0130] In certain embodiments, the inventors describe herein the use of any of the products of the invention described herein for preventing or treating neuropathic pain (e.g., chemotherapy-induced peripheral neuropathic pain, or chemotherapy-induced neuropathic pain), postoperative pain, or inflammatory pain, e.g., the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and / or sets described herein, or any combination or composition thereof described herein.

[0131] In certain embodiments, the inventors herein describe the use of any of the products of the invention described herein for preventing or treating chronic neuropathic pain (e.g., chronic chemotherapy-induced peripheral neuropathic pain, or chronic chemotherapy-induced neuropathic pain), chronic postoperative pain, or chronic inflammatory pain, e.g., the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and their corresponding sets described herein, or any of the compositions thereof described herein.

[0132] In more specific embodiments, the inventors herein describe the use of any of the products of the invention described herein for preventing or treating osteoarthritis pain, e.g., the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and their corresponding sets described herein, or any of the compositions thereof described herein.

[0133] In certain embodiments, the inventors herein describe the use of any of the products of the invention described herein for preventing or treating hyperalgesia, specifically, thermal hyperalgesia (e.g., heat or cold hyperalgesia, preferably cold hyperalgesia) or mechanical hyperalgesia, preferably mechanical hyperalgesia, and even more preferably injury-induced hyperalgesia, e.g., the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and their corresponding sets described herein, or any of the compositions thereof described herein.

[0134] In another specific aspect, the inventors herein describe the use of any of the products of the invention described herein for preventing or treating allodynia (specifically, mechanical allodynia), for example, the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof, or any of the compositions thereof described herein. Preferably, this mechanical allodynia is nerve injury-induced mechanical allodynia or mechanical allodynia of the static type.

[0135] In a more specific aspect, the inventors herein describe the use of any of the products of the invention described herein for preventing or treating allodynia, specifically, thermal allodynia, such as heat allodynia (i.e., pain perceived in response to a warm stimulus that is not normally painful) or cold allodynia (i.e., pain perceived in response to a cold stimulus that is not normally painful), preferably cold allodynia, for example, the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof, or any of the compositions thereof described herein.

[0136] In a specific aspect, the inventors herein describe the use of any of the products of the invention described herein for preventing or treating mechanical hypersensitivity (hypersensitivity to mechanical stimuli, also simply referred to as hyperalgesia) in a subject in need thereof, preferably injury-induced mechanical hypersensitivity, for example, the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof, or any of the compositions thereof described herein.

[0137] In certain aspects, the inventors herein describe the use of any of the products of the invention described herein for preventing or treating pain due to sunburn or pain due to frostbite, for example, the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof described herein, or any of the compositions thereof described herein.

[0138] Treatment can improve one or more of touch, heat or cold sensation, "pins and needles", numbness, itching, sharp pain, and difficulty accurately perceiving temperature. In certain aspects, this treatment eliminates pain. In another specific aspect, this treatment reduces the symptoms of pain, specifically, the symptoms of neuropathic pain (allodynia and / or hyperalgesia). By the methods of the invention, even if neuropathic pain is not eliminated, neuropathic pain becomes more manageable (i.e., quality of life is improved).

[0139] Standard tests known to those of skill in the art are available in the art to evaluate whether pain (specifically, (chronic) neuropathic pain) is being treated using a particular peptide of the invention. For example, assessment of a subject's pain sensitivity is standardized using quantitative sensory testing (pinprick, dolorimetry, von Frey filaments, touch, pinch, or light pressure by finger) or a pain assessment scale.

[0140] Subject In connection with the present invention, a subject or patient is an animal, preferably a mammal. In certain aspects, the subject is a domestic animal, such as a horse, dog, cat, cow, etc. In another specific preferred aspect, the subject is a human.

[0141] Subjects having chronic pain due to neuropathic pain may be suffering from diseases typically associated with such neuropathic pain (e.g., fibromyalgia, complex regional pain syndrome, postherpetic neuralgia, Ehlers-Danlos syndrome, and erythromelalgia).

[0142] In certain embodiments, the subject has fibromyalgia (FM). Fibromyalgia is a syndrome characterized by chronic musculoskeletal pain (Siracusa et al., 2021). FM is caused by a central sensitization phenomenon characterized by dysfunction of the neural circuits involved in the perception, transmission, and processing of nociceptive stimuli, and pain is widespread at the level of the motor system. The main symptoms of this disease are muscle stiffness, joint stiffness, insomnia, fatigue, mood disorders, cognitive impairment, anxiety, depression, general hypersensitivity, and the inability to perform normal daily activities. FM may also be associated with certain diseases such as infections, diabetes, rheumatic diseases, and / or mental or neurological disorders.

[0143] In certain embodiments, the inventors herein describe the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof (i.e., sets of such peptides, nucleic acids, expression cassettes, vectors, or cells), or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells, or sets described herein for preventing or treating fibromyalgia (FM).

[0144] In another aspect, the subject has complex regional pain syndrome (CRPS). Complex regional pain syndrome is a chronic neurological condition affecting the extremities, characterized by severe pain accompanied by sensory, autonomic, motor, and trophic disturbances (Goh et al., 2017). This condition can be induced by surgery, trauma, or minor injury, and can range from mild and self-limiting to a chronic disease that impairs activities of daily living and health-related quality of life. CRPS can be classified into two types: CRPS type I, characterized by the absence of identifiable nerve injury, and CRPS type II, characterized by the presence of identifiable nerve injury. CRPS type I usually develops after an inciting harmful event, is not limited to the distribution of a single peripheral nerve, and is a disproportionate syndrome relative to the inciting event. It is accompanied by edema, changes in skin blood flow, abnormal sweating activity in the pain region, allodynia, and hyperalgesia, and usually affects the distal part of the affected limb or has a distal-to-proximal gradient. CRPS type II can be defined as a burning pain, allodynia, and hyperalgesia that occur in the region of the limb after partial injury of the nerve or its main branches that are distributed in this region.

[0145] In certain aspects, the inventors herein describe the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof (i.e., sets of such peptides, nucleic acids, expression cassettes, vectors, or cells) described herein, or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells, or sets described herein, for preventing or treating complex regional pain syndrome (CRPS).

[0146] In yet another aspect, the subject has postherpetic neuralgia (PHN). Postherpetic neuralgia is the most common complication of herpes zoster (HZ), an infectious disease caused by the reactivation of varicella-zoster virus that is dormant in the sensory ganglia (Ngo et al., 2020). PHN is characterized by a localized vesicular rash and pain along the associated dermatome. PHN is defined as pain that persists for at least 90 days after the onset of the initial symptoms of HZ rash and significantly reduces the quality of life of affected patients. PHN is reclassified into irritable nociceptors and afferent blockade models. During the reactivation of VZV, the virus replicates and spreads from the dorsal root ganglia of the spinal cord to each periphery. This spread induces an immune response and inflammation, which damages the peripheral nerves. This damage reduces the pain inhibition of neurons and lowers the depolarization threshold of pain signals. As a result, a process called peripheral sensitization occurs, in which pain sensation to non-painful stimuli occurs. Repeated activation of subtype C nociceptors also causes an increase in excitatory states in the posterior horn. Direct viral damage by HZ weakens the descending inhibitory pain pathway and causes chronic activation of secondary neurons in the posterior horn. Furthermore, in HZ patients with PHN, a loss of inhibitory gamma-aminobutyric acid (GABA)-producing interneurons in the posterior horn has been reported compared to HZ patients without PHN. These factors amplify all subsequent responses from afferent input in a process called central sensitization. In PHN, this process involves the anatomical reorganization of low-threshold mechanoreceptive afferent fibers (called Aβ fibers) that normally transmit harmless tactile stimuli to the central nervous system. When viral infection-induced damage leads to the loss of peripheral C nociceptors, this fiber compensatorily connects to the secondary neurons that were originally wired to the C nociceptor afferent fibers. This process is called afferent blockade, and patients suffering from allodynia exhibit severe loss of sensory function.

[0147] In certain embodiments, the inventors herein describe the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof (i.e., sets of such peptides, nucleic acids, expression cassettes, vectors, or cells), or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells, or sets described herein, for preventing or treating postherpetic neuralgia (PHN).

[0148] In yet another embodiment, the subject has erythromelalgia (EM). Erythromelalgia is a paroxysmal acral syndrome that mainly occurs symmetrically or unilaterally in both lower extremities or rarely, and is accompanied by the typical triad of erythema, heat sensation, and burning pain (Maria Bibiana Leroux, 2018). EM is classified as a chronic pain syndrome. Primary EM is an autosomal dominant genetic disease encoded by OMIM (Online Mendelian Inheritance in Man) as #133020. EM is mainly associated with modifications in the α subunit protein of sodium channel type 9 (SCN9A) expressed mainly in dorsal root ganglia and sympathetic ganglion neurons (affecting the Nav1.7 channel). Secondary EM is associated with myeloproliferative disorders, tumor-associated conditions, autoimmune diseases, toxin exposure, and infections.

[0149] In certain embodiments, the inventors herein describe the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof (i.e., sets of such peptides, nucleic acids, expression cassettes, vectors, or cells), or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells, or sets described herein, for preventing or treating erythromelalgia (EM).

[0150] In certain aspects, the inventors herein describe the use of the peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof (i.e., sets of such peptides, nucleic acids, expression cassettes, vectors, or cells), or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells, or sets described herein, for preventing or treating any pain-related condition in which signals transmitted by nociceptors or interneurons are impaired.

[0151] The present inventors have demonstrated that Myo1a expresses functional Myo1a + / + It has previously been established that absence, reduced or insufficient expression, or non-functional expression of Myosin IA (Myo1a) in a subject, when compared to expression observed in a reference subject, predisposes the subject to developing injury-induced chronic mechanical pain and / or inflammation-induced chronic heat pain (WO2017153424).

[0152] In yet another embodiment, the subject has one or two mutant alleles in the myo1A gene. A "mutated" allele refers to a substitution, deletion, or insertion in the nucleic acid (coding or non-coding region) of the myo1A gene that alters the expression or expression level of Myo1a. The mutation can affect one or more nucleic acid bases. The substitution can be a single nucleotide polymorphism (SNP).

[0153] In certain aspects, the inventors herein describe the use of peptides, nucleic acids, expression cassettes, vectors, cells, and corresponding sets thereof (i.e., sets of such peptides, nucleic acids, expression cassettes, vectors, or cells), or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells, or sets described herein, for preventing or treating injury-induced chronic mechanical pain and / or inflammation-induced chronic heat pain in such subjects.

[0154] dose The product according to the present invention (specifically, a peptide, a set of peptides, or a composition comprising or enabling the expression of such a set) is preferably administered directly to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of the peptide of interest (specifically, the peptides of SEQ ID NO: 1 and SEQ ID NO: 2, or functional variants thereof) necessary to treat, alleviate, or prevent pain in the subject. A therapeutically effective amount can first be estimated in cell culture assays or animal models. Animal models can also be used to determine the appropriate concentration range and route of administration of the peptide (set). Such information can then be used to determine useful dosages and routes of administration in humans.

[0155] The dosage of the peptide used in the methods described herein can vary depending on the general health status, age, sex, and weight of the subject, the nature and severity / intensity of the pain, the time, frequency, and duration of administration, the particular peptide used, the combination of drugs, the sensitivity of the response, and the tolerance / reactivity to the treatment. This appropriate and effective dosage can be determined by routine experimentation and is left to the discretion of the clinician.

[0156] The dosage can be administered in bolus form or divided into several portions / units and administered separately throughout the day. In other words, the treatment can be on a single-dose schedule or a multiple-dose schedule. The effective dosage can be administered over several days, weeks, months, or years.

[0157] To obtain an appropriate antalgic or analgesic effect, the effective total dosage of the peptide or its variant present in the set of peptides described herein by the inventors or present in a composition comprising such a set of peptides (typically, the effective total dosage of both the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2) is 1 μg to 10 g per unit dosage, preferably 1 μg to 100 mg, more preferably 25 μg to 10 mg, and even more preferably 2 mg to 10 mg.

[0158] In certain embodiments of the invention, a set of peptides, or a composition comprising such a set of peptides, is administered to a mammal at a dose of 1 μg (i.e., 1 μg / kg / day) to 100 mg / kg / day per kg of body weight per 24 hours, preferably at a dose of 2.5 μg / kg / day to 0.6 mg / kg / day.

[0159] When the subject is a human, the effective dose of the peptide or a variant thereof described herein to be administered to the subject is preferably 2.5 μg / kg / day to 0.6 mg / kg / day, preferably 5 μg or 10 μg / kg / day to 0.5 mg / kg / day, and more preferably 50 μg or 75 μg / kg / day to 0.3 mg / kg / day when the subject is a human subject.

[0160] In certain embodiments of the invention, a set of peptides, or a composition comprising such a set of peptides, is administered at a dose in which the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2 are at equimolar concentrations.

[0161] In a more specific embodiment of the invention, a set of peptides, or a composition comprising such a set of peptides, is administered at a dose comprising 50 μg / kg to 150 μg / kg of the peptide of SEQ ID NO: 1 and 100 μg / kg to 250 μg / kg of the peptide of SEQ ID NO: 2.

[0162] Delivery of the products described herein (specifically, the peptides or compositions described herein) to a subject in need thereof can be accomplished by any route.

[0163] The products described herein can be administered, for example, intramuscularly, intravenously, intraperitoneally, orally (per os), anal, cutaneous, subcutaneous, topical, dermically, transdermically, or intrathecally. Preferably, the products described herein are administered subcutaneously, orally, or intravenously, and even more preferably, subcutaneously or orally.

[0164] As shown in the experimental part, the inventors have demonstrated that a set of peptides comprising the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2 has an analgesic effect when administered subcutaneously for treating pain (specifically, neuropathic pain). Surprisingly, this analgesic effect is also observed when the peptide set comprising the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2 is administered orally (per os) or intravenously. Even more surprisingly and advantageously, the set of peptides of the present invention shows an excellent analgesic effect compared to the TAFA-4 (full-length) protein when administered orally or subcutaneously, whereby this set is convenient and suitable for administration to patients compared to the full-length protein.

[0165] Advantageously, the set of two peptides of the present invention, or a composition comprising such a set, is much easier (and as a result, less expensive) to manufacture compared to the full-length TAFA-4 protein, but when administered subcutaneously, induces a higher analgesic activity / effect compared to the latter. Even more surprisingly, when administered orally (per os), the set of peptides of the present invention shows an excellent analgesic effect compared to the TAFA-4 (full-length) protein, and thus is convenient and suitable for administration to patients compared to the TAFA-4 full-length protein. Intravenous injection of this set of two peptides advantageously provides a third route of administration for treating neuropathic pain.

[0166] Kit Each of the products described herein (e.g., each of the products described herein, preferably each peptide, each set of peptides, or each composition described herein) can be part of a kit.

[0167] A typical kit of the present invention comprises at least two products as described herein selected from a peptide or a variant thereof, a nucleic acid, an expression cassette, a vector, a cell, a set of peptides, a set of nucleic acids, a set of expression cassettes, a set of vectors, a set of cells, and a composition comprising such a peptide, nucleic acid, expression cassette, vector, cell, or set, and optionally at least one additional different active compound effective against pain. In certain embodiments, the kit further comprises written instructions for using the kit.

[0168] This kit is preferably a kit of parts comprising at least two parts (e.g., two different containers), wherein the first part comprises a peptide, nucleic acid, expression cassette, vector, cell, set of peptides, set of nucleic acids, set of expression cassettes, set of vectors, or set of cells, or a composition as described herein, and the second part comprises at least one additional different active compound effective against pain. Preferably, the active compound effective against pain is a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID), or an opioid drug as disclosed herein.

[0169] In certain embodiments, the kit comprises i) the first peptide of SEQ ID NO: 1, a peptide having at least 90% identity to SEQ ID NO: 1, or a nucleic acid sequence encoding such a peptide, ii) the second peptide of SEQ ID NO: 2, a peptide having at least 90% identity to SEQ ID NO: 2, or a nucleic acid sequence encoding such a peptide, and optionally iii) at least one additional different active compound effective against pain, and / or iv) written instructions for using the kit, wherein the first and second peptides, or the nucleic acids encoding said peptides, are in separate containers.

[0170] In one aspect, the peptide, nucleic acid, expression cassette, vector, cell, set, or composition of this kit is in a form suitable for intramuscular, intravenous, intraperitoneal, oral (per os), anal, dermal, subcutaneous, topical, transdermal, or intrathecal routes, preferably in a form suitable for subcutaneous or oral routes, and even more preferably in a form suitable for oral routes.

[0171] In another aspect, at least one additional different active compound effective against pain is in a form suitable for intramuscular, intravenous, intraperitoneal, oral (per os), anal, dermal, subcutaneous, topical, transdermal, or intrathecal routes.

[0172] Depending on the nature, cause, and intensity of the pain to be treated, as well as the nature of the contents of the peptide, nucleic acid, expression cassette, vector, cell, set, or composition, and the nature of at least one additional different active compound effective against pain, the product is co-administered simultaneously / concomitantly or sequentially, or not co-administered.

[0173] The present invention also relates to the in vivo, ex vivo, or in vitro use of the kit of the present invention for preventing or treating the pain (e.g., chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia, or allodynia) described above. The kit of the present invention can also be used for the prevention or treatment of acute pain or subacute pain.

[0174] The use of the kit of the present invention for the manufacture of a pharmaceutical composition, drug, or medicament for preventing or treating the pain (e.g., chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia, or allodynia) described above in a subject in need thereof is also disclosed herein. The kit of the present invention can also be used for the manufacture of a pharmaceutical composition, drug, or medicament for the prevention or treatment of acute pain or subacute pain in a subject in need thereof.

[0175] Research tool In certain embodiments, the products of the invention may also be used in connection with research.

[0176] The invention also encompasses the use of the peptides or variants thereof (specifically, sets of peptides) described herein as research tools, or the use of any other product described above.

[0177] Typically, nucleic acids encoding the peptides or variants thereof described herein, expression cassettes or vectors that permit their expression, or cells modified using such nucleic acids or such expression cassettes or vectors are used to express in vitro or ex vivo a peptide of interest (specifically, the peptide of SEQ ID NO: 1 or a functional variant thereof having at least 90% identity to SEQ ID NO: 1, and / or the peptide of SEQ ID NO: 2 or a functional variant thereof having at least 90% identity to SEQ ID NO: 2), or to regulate the expression of this peptide in vitro or ex vivo.

[0178] This tool can be used to study pain or to modulate the excitability of neurons in biological tissues or cell cultures, for example, to study mechanically and / or chemically induced pain nociceptive signals.

[0179] The invention also relates to the use of nucleic acids encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1 and a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2, or the use of an expression cassette or vector that permits this expression, to express or to regulate the expression (level) of such peptides in a culture of biological tissue or cells.

[0180] Transgenic animals can also be created using the nucleic acid molecules described herein. This can be done locally by modifying somatic cells or by germline therapy to incorporate genetic modifications into germ cells. Accordingly, the invention also relates to a transgenic organism (e.g., an animal) that contains (i.e., comprises) the nucleic acid of SEQ ID NO: 3 and / or SEQ ID NO: 4 or variants thereof; an expression cassette or vector that contains the nucleic acid of SEQ ID NO: 3 and / or SEQ ID NO: 4 or variants thereof; or a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, and / or a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2. Accordingly, the invention also relates to a host cell or transgenic organism (specifically, a transgenic animal) that contains a nucleic acid sequence encoding any set of (at least two) peptides described herein, or an expression cassette or vector that permits the expression of any such set.

[0181] The following examples are provided to demonstrate and further illustrate certain preferred embodiments of the invention and should not be construed as limiting the scope of the invention.

Examples

[0182] Materials and Methods Peptide Synthesis All peptides were synthesized on a Symphony X instrument (Protein Technologies, USA) using Fmoc-SPPS at a 200 μmol scale. Synthetic grade Fmoc-AA reagents were purchased from Iris Biotech. Specific L-pseudo proline peptides were used to facilitate peptide synthesis. HATU, 2-chlorotrityl chloride resin (200 - 400 mesh, 1.2 mmol / g), and RINK amide resin (200 - 400 mesh, 0.42 mmol / g) were purchased from IRIS Biotech. DMF, DCM, methanol, diethyl ether, and HPLC grade acetonitrile were purchased from Aldrich. The standard deprotection-coupling cycle for each residue consisted of the following six steps: 1. Wash with 5 mL of DMF (3 times × 30 s) 2. Deprotect the Fmoc group with 5 mL of 20% piperidine in DMF (3 times × 3 min) 3. Wash with 5 mL of DMF (3 times × 30 s) 4. Couple Fmoc-AA (2 times × 60 min) a. Dissolve 5 mL of Fmoc-AA in DMF at 200 mM b. Dissolve 2 mL of HATU in NMP at 500 mM c. Dissolve 2 mL of iPr2NEt in NMP at 1 M 5. Capping with 5 mL of 10% Ac2O in DMF (7 min) 6. Wash with 5 mL of DMF (3 times × 30 s)

[0183] Upon completion of this synthesis, the peptide resin was washed three times with DMF and three times with DCM. The peptide resin was then cleaved with one of the following three TFA cleavage cocktails (per 100 μmol scale synthesis): · 35 mL of TFA, 1000 μL of TIS, 2000 μL of water, 1000 mg of DTT

[0184] After 3 hours of TFA cleavage, the cleavage solution was filtered from the resin and added to 40 mL of ice-cold ether to precipitate the peptide. After more than 1 hour at -20 °C, the ether solution was centrifuged at 3500 RCF and the supernatant was decanted. The pellet was washed more than 2 times with ether and then dried in a vacuum desiccator for more than 3 hours, dissolved, subjected to analytical characterization, and purified.

[0185] Schematic preparation method of peptide resin hydrazide 0.35 mmol of Fmoc-NH-NH2 was dissolved in 30 mL of DCM, and then 1.5 mmol of DIPEA was added to this solution. Next, this solution was added to 1 g of resin and mixed at room temperature for 2 hours. Unreacted groups were capped with 17:2:1 DCM:MeOH:DIPEA for 30 minutes. Then, the Fmoc-AA resin was washed 3 times with DMF, 3 times with DCM, and dried in vacuo for more than 20 minutes. The loading amount was determined by titration (UV, 301 nm) of Fmoc group deprotection.

[0186] Analysis and purification The peptide was analyzed by UPLC and ESI-MS mass spectrometry. The instrument was equipped with BEH C18 (WATERS), 150×2.1 mm (150×2.1 mm) (flow rate: 0.6 mL / min). Solvents A and B were 0.1% TFA in water and 0.1% TFA in acetonitrile.

[0187] Purification of the crude peptide was carried out using a preparative reversed-phase HPLC (Waters Delta Prep 4000) system with a reversed-phase column (Vydac Denali prep C-18, 10 μm, 120 Å, 50×300 mm) and an appropriate gradient of increasing concentration of buffer B in buffer A (flow rate 80 mL / min). Fractions containing the purified target peptide were identified by UV measurement at 214 nm (Waters 2489 UV / Visible detector), and then the selected fractions were pooled and lyophilized.

[0188] Synthesis of Ac-CFPGQVAGTTRAQPSCVEASIVIQKWW-NHNH2 (Peptide F1 - SEQ ID NO: 1): Peptide F1 (Ac-CFPGQVAGTTRAQPSCVEASIVIQKWW-NHNH2) was prepared by standard Fmoc-SPPS as described above using hydrazide resin, and standard peptide cleavage conditions (described above) were employed as follows.

[0189] After the standard cleavage procedure, the crude F1 peptide was dissolved in water and purified by RP-HPLC. Fractions containing the purified target peptide were identified and then the selected fractions were pooled and lyophilized.

[0190] Synthesis of CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR-NH2 (Peptide F2 - SEQ ID NO: 2): Peptide F2 (CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR-NH2) was prepared by standard Fmoc-SPPS as described above using RINK amide resin.

[0191] After the standard cleavage procedure, the crude F2 peptide was dissolved in water and purified by RP-HPLC. Fractions containing the purified target peptide were identified and then the selected fractions were pooled and lyophilized.

[0192] Mouse C57 / Bl6J mice (8 - 12 weeks old) were purchased from Charles River Laboratories. Both male and female mice were used in all experiments. Since no differences were observed between males and females, data for the two sexes were combined. The mice were maintained under standard housing conditions (22 °C, 40% humidity, 12 - hour light cycle, and free access to food and water). Particular efforts were made to minimize the number of mice used in this study and to minimize the stress and pain suffered by the mice. All experiments were carried out in accordance with the European guidelines for the care and use of experimental animals (Council Directive 86 / 609 / EEC). All experimental procedures were approved by the independent ethics committee (APAFIS) as required by French law and in accordance with the relevant regulations of French law regarding animal experiments. All experiments were conducted in accordance with the ARRIVE guidelines.

[0193] Pain model 1: Spared Nerve Injury (SNI) model - Neuropathic pain. The Spared Nerve Injury (SNI) model developed by Decosterd and Woolf, 2000; Pain, Vol. 87, pp. 149 - 158 was used. This model was used as a neuropathic pain model. The SNI model consists of the transection of the tibial and common peroneal branches of the sciatic nerve, with the sural nerve remaining intact. The latter then exhibits signs of neuropathic pain with significant mechanical allodynia. The SNI model has many advantages as follows: - Neuropathic pain is persistent. This allows the habituation phenomenon during repeated peptide injections to be grasped. - The resulting pain is robust. - This model is highly reproducible.

[0194] The mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP), and the left sciatic nerve was exposed under sterile conditions. The distal trifurcation of the sciatic nerve was identified, and the tibial branch and the common peroneal branch were ligated with polypropylene non-absorbable 6-0 suture (Ethicon) and transected by 1 mm, leaving the peroneal branch intact. The wound was sutured, and the animal was allowed to recover and returned to the cage.

[0195] Pain model 2: Plantar incision - postoperative pain. As described by Brennan and co-workers (1999) (Brennan, 1999), a plantar incision surgery was performed. The mice were anesthetized with ketamine (100 mg / kg, IP) and xylazine (10 mg / kg, IP), and the skin and fascia of the right hind paw were longitudinally incised. Forceps were used to lift the flexor digitorum brevis muscle longitudinally, and the muscle was incised with a scalpel and transected into two parts. The wound was sutured, and the animal was allowed to recover and returned to the cage. The plantar incision was used as a postoperative pain model.

[0196] Pain model 3: Carrageenan injection - inflammatory pain. The inventors injected 20 μl of 1% λ-carrageenan (Sigma-Aldrich, 22049-5G-F) in 1×PBS into the plantar surface of the left hind paw of the mice using a Hamilton syringe. This carrageenan injection was used as an inflammatory pain model.

[0197] Pain model 4: Chronic constriction injury (CCI) model - neuropathic CCI Neuropathic Pain Model: As already described by Bennett and Xie 1988 (A peripheral mononeuropathy in rats that produces disorders of pain sensation like those in man. Pain Vol. 33: pp. 87 - 107), chronic constriction injury (CCI) was performed. Briefly, in mice anesthetized with ketamine / xylazine (100 mg / kg and 10 mg / kg ip, respectively), two ligatures (6 - 0 Monocryl, Ethicon) were loosely tied (at approximately 1 mm intervals) around the entire sciatic nerve to induce unilateral peripheral mononeuropathy. The nerve was constricted to a barely recognizable degree so that the circulation through the vasa nervorum was not interrupted.

[0198] Pain Model 5: Capsaicin - induced Inflammatory Pain. A stock solution of capsaicin (Sigma) (dissolved in absolute ethanol, 16.7 mg / mL) was diluted to 0.25 mg / mL with 0.9% NaCl and then 10 μL was injected onto the plantar surface of the left hind paw of the mouse using a Hamilton syringe and a 26G needle.

[0199] Pain Model 6: Osteoarthritis Pain Model (Monoiodoacetate Model). The MIA model is a standard model of joint destruction in rodent osteoarthritis. Briefly, mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP), and then, as described by Pitcher et al. (2016), 1 mg / 10 μL of MIA (monoiodoacetate, Sigma) in 0.9% NaCl was injected into the synovial fluid of the knee joint of the left hind paw of the mouse using a Hamilton syringe and a 26G needle.

[0200] Pain Model 7: Ultraviolet Burn Pain Model The mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP), and completely covered except for the plantar side of one hind paw. This paw was irradiated with a UV lamp (VL-215.MC Vilmer) with a wavelength of 312 nm at a distance of 20 cm for 60 minutes. When they woke up, the animals were returned to their cages.

[0201] Pain model 8: Cryogenic burn pain model The mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP). The hind paw was exposed to cold air through a copper tube filled with dry ice and contacted with a piece of filter paper soaked in NaCl for 40 seconds. When they woke up, the animals were returned to their cages.

[0202] von Frey test In the von Frey test, the sensitivity of rodents to mechanical stimuli is tested using von Frey (VF) hairs or fibers, which are small pieces of nylon rod approximately 50 mm in length. In this test, the animal is placed on a high-mesh platform, and the von Frey hair is inserted through the mesh to poke the animal's hind paw. Normal responses of the animal include withdrawing the paw, licking it, or vibrating it. The "up-down" von Frey method is used to determine the mechanical force required to produce a paw withdrawal response in 50% of the animals. Here, the mice were placed in a plastic chamber on a wire mesh grid, and stimulated with von Frey fibers (Vivotech) by the "up-down" method (45), starting with 1 g fibers and using 0.04 g and 4 g fibers as cutoffs.

[0203] Statistical analysis The results are expressed as mean ± SEM. Statistical analysis was performed using Prism 7 (Graphpad Software, La Jolla, CA, USA).

[0204] Results (Example 1: Pain model 1: Spared nerve injury (SNI) model - Neuropathic pain.) 1.1 Comparison of the analgesic effects of TAFA-4 full-length protein (TAFA-4 FL) and a set of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (peptide 1+2) in subcutaneous administration. The purpose of these experiments was to evaluate the antalgic effect of a set of two peptides according to the present invention (this set is identified herein as "peptide 1+2" or "peptide F1+F2") by subcutaneous injection in a nerve partial injury (SNI) model of neuropathic pain, and to compare this effect with the effect obtained with TAFA-4 full-length protein (TAFA-4 FL).

[0205] This experiment was performed on 8-week-old male WT C57Bl6 mice. Four groups consisting of 6 - 12 mice were used. The peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were resuspended in 0.9% NaCl at [0.6 mg / mL]. In this study, the two peptides were diluted to obtain equimolar concentrations (12.8 μg / ml for the peptide of SEQ ID NO: 1 and 17.2 μg / ml for the peptide of SEQ ID NO: 2) for use at a final total concentration of 0.3 mg / kg of "peptide 1+2". Similarly, TAFA-4 FL was resuspended in 0.9% NaCl, diluted, and used at a final concentration of 0.3 mg / kg.

[0206] A 0.9% NaCl solution was used as a negative control (vehicle), and 3 mg / kg of pregabalin, a known drug used to treat neuropathic pain, was used as a positive control.

[0207] Using von Frey (VF) fibers, the basic threshold (“baseline”) of mice was measured by the up / down method [showing the mechanical force (expressed in gram equivalents) required to produce a foot withdrawal response in 50% of mice as a function of time under various conditions], and then the SNI model was set up. The mice were anesthetized, ligations of the tibial nerve and the peroneal nerve were performed, and then these two nerves were transected. The intact peroneal nerve develops neuropathy very rapidly. The onset of neuropathy is confirmed 3 days after surgery. As a result, a decrease in the response threshold to von Frey fibers in the ipsilateral foot is observed.

[0208] Fourteen days after surgery, the response threshold is measured again (see “D14” in Figure 1). Then, 100 μl / 10 g of each solution containing a set of two peptides, the TAFA-4 full-length protein, the vehicle, or pregabalin is blindly subcutaneously injected into the mice by the experimenter.

[0209] One hour, two hours, and then four hours after administration / injection, the response threshold is measured.

[0210] On the 14th day after SNI, all mice showed a significant decrease in mechanical threshold and strong mechanical hypersensitivity compared to the baseline threshold (see Figure 1). Subcutaneous administration of the vehicle had no effect. Surprisingly, at 1 hour after administration, the analgesic effect of the set / composition of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (“peptide 1+2”) was maximal, significantly higher (p value <0.01) compared to the analgesic effect of the TAFA-4 full-length protein (“TAFA4 FL”), and also higher compared to the analgesic effect of pregabalin. Statistical analysis showed strong significant results (p value <0.001) at 1 hour and 2 hours for the set / composition of two peptides compared to the negative control (vehicle).

[0211] 1.2 Comparison of the analgesic effects of the TAFA-4 full-length protein (“TAFA-4 FL”) and the set of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (“peptide 1+2”) in oral administration. The protocol described in 1.1 was used, except that administration of the set of two peptides, TAFA-4 FL, vehicle, and pregabalin was performed 14 days after surgery using oral administration.

[0212] For oral administration, the two peptides of SEQ ID NO:1 and SEQ ID NO:2 were diluted to a total concentration of 30 μg / mL in a solution of 1% w / v hydroxypropyl methylcellulose (Sigma-aldrich #423238, batch MKCD3665), 0.5% v / v Tween 80 (Euromedex #2002-A, batch 100412 / 16S407) (hereinafter referred to as HPMC solution or "vehicle"). The same protocol was applied for TAFA-4 FL.

[0213] The vehicle, TAFA-4 FL solution, and the solution containing the set of two peptides were orally administered using two forced oral needles. Pregabalin (3 mg / kg) was orally administered ( "orally") by an experimenter different from the experimenter who performed the VF measurement, so the latter worked blindly with respect to the treatment (n = 8 for each treatment). The administration was performed 14 days after surgery.

[0214] On the 14th day after SNI, all mice showed a significant decrease in mechanical threshold (Figure 2). Oral administration of the vehicle had no effect. The full-length TAFA-4 protein did not show a significant analgesic effect when administered orally (i.e., per os). Surprisingly, oral administration of the set / composition of the two peptides of the present invention induced a statistically significant reversal effect on the mechanical threshold immediately 1 hour after administration (p-value < 0.01), and the maximum effect appeared 2 hours after administration (p-value < 0.001). The analgesic effect of the set / composition of the two peptides of the present invention was similar to the analgesic effect of pregabalin at 1 hour and 2 hours after administration, but was significantly higher than the analgesic effect of the full-length TAFA-4 protein ( "TAFA-4 FL") at 2 hours after administration. From Figure 2, it can be concluded that when administered orally, the set / composition of the two peptides of the present invention has a much better analgesic effect than the full-length TAFA-4 protein.

[0215] 1.3 Analgesic effect of the set of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 administered intravenously ( "Peptide 1+2") The protocol described in 1.1 was used, except that the administration of the set of two peptides or the vehicle was performed 14 days after surgery using the intravenous route.

[0216] The peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were resuspended in 0.9% NaCl at a total concentration of 150 μg / mL.

[0217] As previously described, von Frey fiber measurements were performed by the up / down method to determine the baseline. Then, the SNI model was set up. The response threshold was measured 14 days after surgery (day 14 - D14) to confirm the occurrence of neuropathic pain. Then, a blinded injection of a 20 μl / 10 g solution containing the set of two peptides at a total concentration of 300 μg / kg was gently performed via the tail vein. The response threshold was measured 1 hour, 2 hours, and then 4 hours after intravenous administration. The vehicle (n = 11) was also administered intravenously.

[0218] On the 14th day after SNI, all mice showed a significant decrease in mechanical threshold and exhibited strong mechanical hypersensitivity compared to the baseline threshold (Figure 3). Intravenous administration of the vehicle had no effect. Surprisingly, a strong and significant analgesic effect of the set of two peptides of SEQ ID NO:1 and SEQ ID NO:2 (“Peptide 1+2”) was observed, and the maximum reversal effect on the mechanical threshold was observed at 2 hours (p<0.001).

[0219] Overall, this experiment has shown that the set of two peptides of the present invention induced an analgesic effect by subcutaneous administration, oral administration, and intravenous administration in the SNI model (neuropathic pain model). Advantageously, the very early (i.e., at 1 hour after administration) analgesic effect of the set of two peptides of the present invention is remarkable compared to the analgesic effect observed with the TAFA-4 (full-length) protein when administered subcutaneously. More surprisingly, the set of two peptides of the present invention shows an excellent analgesic effect compared to the TAFA-4 (full-length) protein when administered orally at 2 hours after administration.

[0220] From the results of the present inventors, the analgesic effect of the set of two peptides of SEQ ID NO:1 and SEQ ID NO:2 in the neuropathic pain model was demonstrated. The mechanical allodynia (decrease in pain sensitivity threshold) induced by this model can be reduced by subcutaneous administration, intravenous administration, or oral (per os) administration of the set of peptides of the present invention.

[0221] Advantageously, a set of two peptides of the present invention, or a composition comprising such a set, is much easier (and as a result, less expensive) to produce compared to the full-length TAFA-4 protein, but when administered subcutaneously, induces a higher analgesic activity / effect compared to the latter. Even more surprisingly, when administered orally (per os), the set of peptides of the present invention exhibits an excellent analgesic effect compared to the TAFA-4 (full-length) protein, and thus is convenient and suitable for administration to patients compared to the said TAFA-4 full-length protein. Intravenous injection of this set of two peptides advantageously provides a third route of administration for treating neuropathic pain.

[0222] (Example 2: Plantar incision - postoperative pain -) The purpose of these experiments was to evaluate the antalgic effect of a set of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 according to the present invention (this set is identified herein as "peptide 1+2") by subcutaneous injection in a postoperative pain model (Brennan plantar incision model).

[0223] This experiment was performed on 8-week-old male and female WT C57Bl6 mice. For the "peptide 1+2" treatment group, 12 males and 12 females were used, and for the vehicle group, 11 males and 11 females were used. An equimolar combination of the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2 was resuspended in ultrapure water at [4 mg / mL]. In this study, these two peptides were diluted to obtain a final total concentration of "peptide 1+2" of 0.3 mg / kg.

[0224] "Peptide 1+2" or vehicle was administered subcutaneously twice a day at the following various time points: the day before surgery (D-1), the day of surgery (D0), and D+1 and D+2 after surgery. The administration at D0 was performed 1 hour before and 1 hour after (and after waking up) the surgery, respectively. The measurement of the mechanical threshold response was performed at D+1 (before injection at D+1), D+2 (before injection at D+2), and D+3 (Figure 4A).

[0225] After plantar incision, mice treated with vehicle developed mechanical allodynia (see D+1, D+2, and D+3 in FIG. 4B). In contrast, mice treated with "Peptide 1+2" did not develop mechanical allodynia. Compared to mice treated with vehicle, mice treated with "Peptide 1+2" showed statistically significantly higher response thresholds at D+1, D+2, and D+3 (FIG. 4B).

[0226] These results indicate that the set of peptides of the present invention can be used as an analgesic in a plantar incision model (postoperative pain model).

[0227] (Example 3: Lack of tolerance in a chronic constriction injury (CCI) neuropathic pain model) The purpose of this experiment was to evaluate whether subjects treated with the set of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("Peptide 1+2") could induce tolerance to this set of peptides after repeated subcutaneous administration of the set of peptides.

[0228] The CCI model was introduced according to the protocol described above. Starting from D7 after surgery, mice were subcutaneously injected with "Peptide 1+2" (0.3 mg / kg) twice a day for 14 consecutive days. Before surgery and 7 days after surgery, von Frey filaments were used to test mechanical sensitivity by the up / down method to verify CCI-induced mechanical allodynia. After the first administration, a time-course response was performed. Then, the analgesic effect of "Peptide 1+2" was tested every other day. Then, a complete time-course response was performed after the last administration (D20) (FIG. 5A). The last measurement was performed at D+30 (10 days after the end of treatment) to verify the proper recovery of the mice.

[0229] The first subcutaneous injection of "Peptide 1+2" caused a significant increase in the response (i.e., foot withdrawal) threshold, thereby demonstrating the analgesic effect of "Peptide 1+2" (Figure 5B - comparison of the response measured in the "before drug" state and the response measured 2 hours after administration at the first injection). Interestingly, the analgesic effect of "Peptide 1+2" remained constant throughout the treatment period (14 days) without any decrease in efficacy even at the last injection corresponding to D20 (Figure 5B, no significant difference in the response measured at the first injection compared to the response measured at the last injection). These results indicate that the set of peptides of the present invention can be repeatedly used over a long period without inducing tolerance in the subject.

[0230] (Example 4: Comparison of various doses of treatment in a postoperative pain model - preventive effect) The purpose of this experiment was to test various posologies for treating subjects suffering from postoperative pain with a set of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("Peptide 1+2") when administered subcutaneously in vivo in a postoperative pain model.

[0231] "Peptide 1+2" or vehicle was administered subcutaneously twice a day according to three different protocols. "D-1 / D0" protocol: administration was carried out on the day before surgery (D-1) and the day of surgery (D0); "D0" protocol: administration was carried out on the day of surgery (D0); "D0 / D+1 / D+2" protocol: administration was carried out on the day before surgery (D-1), the day of surgery (D0), and on days D+1 and D+2 after surgery. The administration at D0 was carried out 1 hour before surgery and 1 hour after surgery (and after waking up) respectively. The measurement of the mechanical threshold response at D+1 and D+2 was carried out before the administration of the set of two peptides or vehicle (Figure 6A).

[0232] After plantar incision, mice treated with vehicle developed mechanical allodynia (see D+1, D+2, and D+3 in Figure 6B). In contrast, mice treated with "Peptide 1+2" did not develop mechanical allodynia regardless of the protocol used. Interestingly, mice treated with the "D-1 / D0" and "D0" protocols that were not treated after surgery did not develop mechanical allodynia, thereby demonstrating that the set of peptides of the present invention can be used prophylactically in a plantar incision model (postoperative pain model).

[0233] (Example 5: Analgesic effect in an inflammatory pain model) The purpose of this experiment was to evaluate the antalgic effect of a set of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ( "Peptide 1+2") administered subcutaneously in vivo in an inflammatory pain model (capsaicin inflammatory pain model).

[0234] This experiment was performed on 8-week-old male WT C57Bl6 mice. Two groups of 8 mice were used. "Peptide 1+2" was resuspended in an HMPC solution at a concentration of 30 μg / mL for injection at 10 μL per gram.

[0235] A capsaicin (Sigma) stock solution (dissolved in absolute ethanol, 16.7 mg / mL) was diluted to 0.25 mg / mL with 0.9% NaCl and then 10 μL was injected onto the plantar surface of the left hind paw of the mice using a Hamilton syringe and a 26G needle. As previously described, von Frey fiber measurements were performed by the up / down method to determine the baseline. Twenty-four hours after capsaicin injection (D+1), the two groups of mice were treated with "Peptide 1+2" or vehicle, respectively. The reaction threshold was measured at 1 hour (+1h), 2 hours (+2h), and then 4 hours (+4h) after subcutaneous administration of the "Peptide 1+2" solution or vehicle solution (n = 8) at 0.3 mg / kg (n = 8).

[0236] Twenty-four hours after injection of capsaicin, the mice developed mechanical allodynia (see D+1 in FIG. 7), whereas subcutaneous administration of the set of peptides of the present invention induced a statistically significant increase in the response threshold 2 hours after administration.

[0237] These results indicate that the set of peptides of the present invention caused an analgesic effect by subcutaneous administration in the capsaicin model (inflammatory pain model).

[0238] (Example 6: Analgesic effect in osteoarthritis pain model) The purpose of this experiment was to evaluate the analgesic effect of the set of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ( "peptide 1+2") administered subcutaneously in vivo in an osteoarthritis pain model (monoiodoacetate model).

[0239] Two groups of 8 mice were used. In each group, mechanical sensitivity was tested using von Frey fibers by the up / down method before (baseline) and 14 days after injection of monoiodoacetate (MIA). Fourteen days after injection of MIA, the mice in the two groups were treated with "peptide 1+2" or vehicle (NaCl solution), respectively. Time-course responses were performed to follow the analgesic effect of "peptide 1+2". The response threshold was measured at 1 hour (+1h), 2 hours (+2h), and then 4 hours (+4h) after administration of "peptide 1+2" or vehicle.

[0240] On day 14 after MIA injection, all mice showed a significant decrease in mechanical threshold and strong mechanical hyperalgesia compared to the baseline threshold (FIG. 8). Subcutaneous administration of the vehicle had no analgesic effect. In contrast, the analgesic effect of "peptide 1+2" was statistically significant compared to the negative control (vehicle) at 1 hour and 2 hours after administration (FIG. 8).

[0241] These results indicate that the set of peptides of the present invention caused an analgesic effect by subcutaneous administration in the arthritis pain model.

[0242] (Example 7: Analgesic effect in the frostbite pain model) The purpose of this experiment was to evaluate the antalgic effect of the set of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (''peptide 1+2'') administered subcutaneously in vivo in the frostbite pain model.

[0243] Two groups consisting of 16 mice were used. Each group consisted of 8 males and 8 females. Mechanical sensitivity was tested using von Frey fibers by the up / down method before cold exposure (baseline) and 24 hours later (D+1). After administration of ''peptide 1+2'' or vehicle (NaCl solution) 24 hours after cold exposure, time-course responses were performed to follow the analgesic effect of ''peptide 1+2'' at 1 hour (D1+1), 2 hours (D1+2h), and 4 hours (D1+4h) after administration.

[0244] Twenty-four hours after cold exposure, all mice showed a significant decrease in mechanical threshold and strong mechanical hyperalgesia compared to the baseline threshold (Figure 9). Subcutaneous administration of vehicle had no analgesic effect. In contrast, the analgesic effect of ''peptide 1+2'' was statistically significant compared to the negative control (vehicle) at 1 hour and 2 hours after administration (Figure 9).

[0245] These results indicate that the set of peptides of the present invention caused an analgesic effect by subcutaneous administration in the frostbite (or ice burn) pain model. Such a model is also considered a type of acute pain model.

[0246] (Example 8: Analgesic effect in the ultraviolet burn pain model) The purpose of this experiment was to evaluate the antalgic effect of the set of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (''peptide 1+2'') administered subcutaneously in vivo in the ultraviolet burn pain model (also called the sunburn pain model).

[0247] Two groups consisting of 16 mice were used. Each group consisted of 8 males and 8 females. Mechanical sensitivity was tested using von Frey filaments by the up / down method before ultraviolet irradiation (baseline) and 24 hours later (D+1). After administration of "Peptide 1+2" or vehicle (NaCl solution), time-course reactions were performed to follow the analgesic effect of "Peptide 1+2" at 1 hour (D1+1h), 2 hours (D1+2h), and 4 hours (D1+4h) after administration.

[0248] Twenty-four hours after ultraviolet irradiation, all mice showed a significant decrease in mechanical threshold, indicating strong mechanical hypersensitivity compared to the baseline threshold (Figure 10). Subcutaneous administration of the vehicle had no analgesic effect. In contrast, at 1 hour and 2 hours after administration, the analgesic effect of the "Peptide 1+2" set was statistically significant compared to the negative control (vehicle) (Figure 10).

[0249] These results indicate that the set of peptides of the present invention caused an analgesic effect by subcutaneous administration in a UV burn (or sunburn) pain model. Such a model is also considered a type of acute pain model.

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Claims

1. A composition comprising a first peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1 and a second peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2, wherein the two peptides are not linked to each other.

2. When the composition comprises a peptide having at least 90% identity to SEQ ID NO: 1 and / or a peptide having at least 90% identity to SEQ ID NO: 2, the glutamine (Q) residue at position 13 in the peptide of SEQ ID NO: 1 remains unchanged in the amino acid sequence of the peptide having at least 90% identity to SEQ ID NO: 1, and the tyrosine (Y) residue at position 18 in the peptide of SEQ ID NO: 2 remains unchanged or is replaced by serine (S) in the amino acid sequence of the peptide having at least 90% identity to SEQ ID NO:

2. The composition according to Claim 1.

3. A set or composition of components comprising a nucleic acid enabling the expression of two non-linked peptides, wherein the first nucleic acid comprises a sequence encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, and the second nucleic acid comprises a sequence encoding a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO:

2. A set or composition of components.

4. A set or composition of components comprising a vector enabling the expression of two non-linked peptides, wherein the first vector comprises a sequence encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, and the second vector comprises a sequence encoding a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO:

2. A set or composition of components.

5. A cell or a composition comprising the cell and an acceptable support, wherein the cell enables the expression of two non-linked peptides, and the cell comprises a first nucleic acid sequence or vector encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1 and a second nucleic acid sequence or vector encoding a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO:

2. A cell or composition.

6. The composition according to any one of claims 1 to 5, further comprising at least one additional active compound, preferably an active compound effective against pain, and even more preferably a SAID, NSAID, or opioid drug.

7. The composition according to any one of claims 1 to 6, comprising a pharmaceutically and / or nutritionally acceptable carrier.

8. The composition according to any one of claims 1 to 7, further comprising ascorbic acid or an ascorbate.

9. A set of a first peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1 and a second peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2, wherein the two peptides are not linked to each other, and the set is for use as a medicament in a subject in need thereof, the set according to claim 3 or 4, which is for use as a medicament in a subject in need thereof, the composition according to any one of claims 1 to 8, which is for use as a medicament in a subject in need thereof, or the cell according to claim 5, which is for use as a medicament in a subject in need thereof.

10. A set of a first peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1 and a second peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2, wherein the two peptides are not linked to each other, and the set is for use in the prevention or treatment of pain in a subject in need thereof, the set according to claim 3 or 4, which is for use in the prevention or treatment of pain in a subject in need thereof, the composition according to any one of claims 1 to 8, which is for use in the prevention or treatment of pain in a subject in need thereof, or the cell according to claim 5, which is for use in the prevention or treatment of pain in a subject in need thereof.

11. The pain is chronic pain, neuropathic pain, postoperative pain, inflammatory pain, arthritic pain, pain associated with joint injury, pain associated with Ehlers-Danlos syndrome, pain due to sunburn, pain due to frostbite, hyperalgesia, allodynia, or acute pain, the set, composition, or cell according to claim 10.

12. The subject is a mammal, particularly a human, the set, composition, or cell according to any one of claims 9 to 11.

13. The composition is administered to the subject intramuscularly, intravenously, intraperitoneally, orally (per os), rectally, dermally, subcutaneously, topically, dermically, transdermally, or intrathecally, preferably subcutaneously or orally, the set or composition according to any one of claims 9 to 12.

14. The set or composition contains both the first and second peptides at 1 μg to 100 mg per single dose, preferably 25 μg to 10 mg, the set or composition according to any one of claims 9 to 13.

15. The set or composition is administered to a mammal at a dose of 1 μg / kg / day to 100 mg / kg / day, preferably 2.5 μg / kg / day to 0.6 mg / kg / day, the set or composition according to any one of claims 9 to 13.

16. A kit comprising: i) the first peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, or a nucleic acid sequence encoding said peptide; ii) the second peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2, or a nucleic acid sequence encoding said peptide; and optionally iii) written instructions for using the kit, wherein the first and second peptides or the nucleic acids encoding said peptides are in separate containers.

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