Peptides and methods for use in the treatment of pain - Patents.com

Abstract

The present invention relates to novel peptides, compositions and kits comprising said peptides, for use as active ingredients for preventing or treating pain in a subject in need thereof. In vitro or ex vivo methods for modulating the expression of these peptides are also described herein.

Description

[Technical field]

[0001] FIELD OF THEINVENTION This application relates generally to the field of pain management. In particular, the present invention relates to a composition for preventing and / or treating pain, comprising a peptide fragment of TAFA-4 protein as an active ingredient, and a method for preventing and / or treating pain, in particular acute, subacute or chronic pain, of neuropathic, post-operative or inflammatory origin, or for preventing and / or treating hyperalgesia or allodynia resulting from injury-induced pain. [Background technology]

[0002] 2. Background of the Invention Pain is generally classified as acute or chronic. Acute pain is short-lived and essential for the maintenance of our physical integrity, whereas chronic pain persists beyond the normal time for healing and negatively impacts well-being. Chronic inflammatory, neuropathic or post-operative pain produces long-lasting sensory abnormalities, such as hyperalgesia (extreme pain induced by noxious stimuli) and mechanical allodynia (pain induced by non-noxious mechanical stimuli). These categories of pain differ in terms of etiology and clinical features, but share several mechanisms in common, including alterations to neuro-immune interactions and neuronal sensitization, both peripheral and central (Costigan et al., 2009). Growing evidence suggests 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). However, despite our extensive knowledge of the mechanisms and circuits underlying chronic pain in rodents, the translation of these findings into effective treatments for chronic pain in humans remains poor (Colloca et al., 2017). Indeed, nonsteroidal anti-inflammatory drugs (NSAIDs) have limited efficacy against chronic pain, and opioids have multiple adverse effects, including potentially fatal respiratory depression, nausea, constipation, hyperalgesia, tolerance, and physical and psychological dependence (Benyamin et al., 2008). Therefore, efforts to identify new targets with analgesic or analgesic potential for the treatment of chronic pain should be encouraged.

[0003] In the past few years, the inventors have discovered striking features of the secreted protein TAFA-4, which suggests that it may be an interesting drug for the treatment of chronic pain (WO2014180853). TAFA-4 belongs to a family of five highly conserved secreted neurokines (Sarver et al., 2021).

[0004] TAFA-4 contains a highly conserved core region with a signal peptide followed by 10 cysteine ​​residues that includes a CC-chemokine motif making it similar to cytokines (Tom Tang et al., 2004).

[0005] We have also previously shown that in mice lacking the TAFA-4 gene, nerve injury-induced mechanical hypersensitivity persists much longer than in wild-type (WT) mice (Delfini et al., 2013). These phenotypes are reversed by intrathecal injection of recombinant TAFA-4.

[0006] As described in WO2014180853, the present inventors have previously reported experimental evidence demonstrating that TAFA-4 possesses a potent 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. They also show that TAFA-4 can reverse nerve injury-induced neuronal sensitization of spinal cord lamina II interneurons, which have been reported to be responsible for mechanical threshold alteration.

[0007] However, since TAFA-4 is a cysteine-rich protein (10 cysteines in total), the production of recombinant mature TAFA-4 protein can be difficult. Indeed, refolding can result in obtaining non-native conformations or improper disulfide bridge patterns that can greatly affect the activity of the protein. Furthermore, several purification steps required to obtain recombinant TAFA-4 protein with a high degree of purity, along with possible aggregation problems, can limit the yield of properly folded protein. Furthermore, chemical synthesis of TAFA-4 protein has been performed, but this is also difficult due to the length of the protein, requiring the assembly of four or more (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, which results in low yields incompatible with industrial-scale production. Therefore, there is a substantial advantage in identifying novel compounds with activities similar to those of TAFA-4 protein that can be produced on an industrial scale and in a cost-effective manner. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2014180853 [Patent Document 2] PCT Patent Application Publication Number WO03042397 [Patent Document 3] U.S. Patent No. 6,632,670 [Patent Document 4] WO2017153424 [Non-patent literature]

[0009] [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, FM Ausubel et al., eds., 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, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [Non-Patent Document 13] Decosterd and Woolf, 2000; Pain, 87, 149-158 [Non-Patent Document 14] Bennett and Xie 1988 (A peripheral mononeuropathy in rats that produces disorders of pain sensation like those in man. Pain, 33:87-107) Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, there is a great need for alternative therapeutic agents and methods of use for effectively preventing or treating pain, particularly acute, subacute or chronic pain. [Means for solving the problem]

[0011] SUMMARY OF THE PRESENT APPLICATION The present invention is based, at least in part, on the identification of a novel peptide (also identified herein as "TT1") and variants thereof, such as the "TT6" peptide identified herein, having analgesic or analgesic activity, in particular analgesic activity. The peptide of interest is or is derived from a specific fragment of the human TAFA-4 protein. Advantageously, the peptide and variants thereof are easy to produce and highly effective against pain, in particular acute, subacute or chronic pain, typically injury-induced pain.

[0012] The present invention relates to an isolated, synthetic or recombinant peptide of sequence SEQ ID NO:1 or a peptide having at least 90% identity to SEQ ID NO:1 (also identified herein as a "peptide variant" or a "variant of a peptide of sequence SEQ ID NO:1"). In a particular embodiment, the peptide of sequence SEQ ID NO:1 or a variant thereof is a recombinant peptide. In a particular embodiment, the peptide variant is an isolated, synthetic or recombinant peptide of sequence SEQ ID NO:5.

[0013] Preferably, peptide variants having at least 90% identity to SEQ ID NO:1, such as the peptide of SEQ ID NO:5 ("TT6"), modulate the excitability of spinal interneurons (preferably spinal lamina IIi interneurons). In a preferred embodiment, the amino acid residues glutamine (Q) at position 13 and tyrosine (Y) at position 45 remain unchanged in peptide variants of the peptide of SEQ ID NO:1, with reference to the positions shown in SEQ ID NO:1.

[0014] The present invention also relates to nucleic acids encoding the peptides or variants thereof described herein; vectors allowing the expression of the peptides or peptide variants described herein above; and cells comprising nucleic acids encoding such peptides or variants thereof or cells modified using the vectors of the invention.

[0015] In another aspect, the present invention relates to a peptide or variant thereof as described herein for use as an active ingredient, e.g. as a drug or medicament. It also relates to a composition, typically a therapeutic, veterinary or dietary composition, comprising a peptide or variant thereof as described herein, a nucleic acid encoding such a peptide or variant thereof, and / or a vector or cell as described herein, and a pharma- ceutical and / or dietary acceptable support.

[0016] In this particular embodiment, the composition may further comprise at least one further (separate) active compound, preferably an active agent effective against pain (acute, subacute or chronic pain), even more preferably a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID) or an opioid drug.

[0017] A further object of the present invention relates to a peptide, a nucleic acid, a vector or a cell as described herein for use as an active ingredient for preventing or treating pain in a subject in need thereof. It also relates to the use of a peptide, a nucleic acid, a vector, a cell or a composition as described herein for the manufacture of a medicament for preventing or treating pain in a subject in need thereof. In particular, the present invention may be used to treat chronic pain, neuropathic pain, post-operative pain, inflammatory pain, hyperalgesia or allodynia.

[0018] 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 composition as described herein or a peptide of SEQ ID NO:1 or a variant thereof having at least 90% identity to SEQ ID NO:1, such as a peptide of SEQ ID NO:5.

[0019] The peptides or compositions described herein may be administered to a subject by any route, such as intramuscularly, intravenously, intraperitoneally, orally (per os), anally, cutaneously, subcutaneously, dermically, transdermically, or intrathecally. Preferably, the peptides or compositions described herein are administered to a subject subcutaneously or orally, and even more preferably orally.

[0020] The peptide or composition described herein can be part of a kit.Thus, the present invention also relates to a kit comprising i) the peptide, nucleic acid, vector, cell or composition described herein, ii) at least one further active compound, preferably separate from the peptide or variant thereof described herein, that is effective against pain, and optionally iii) instructions for using the kit.The kit described herein is typically used for the prevention or treatment of pain.

[0021] In certain embodiments, the products of the present invention may also be used in research. The nucleic acids encoding the peptides or variants described herein, the vectors enabling their expression, or the cells containing such nucleic acids or modified using such vectors may be used to express or modulate the expression in vitro or ex vivo of the peptide of SEQ ID NO: 1 or its functional variants having at least 90% identity to SEQ ID NO: 1, such as the peptide of SEQ ID NO: 5. In another particular embodiment, the present invention also relates to transgenic animals modified to express the nucleic acids described herein. BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0022] [Figure 1] Figure 1 shows the analgesic effect of peptide of SEQ ID NO:1 administered subcutaneously in vivo in the Spared Nerve Injury (SNI) model of neuropathic pain. The figure shows the mechanical force (expressed as its equivalent mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The peptide of SEQ ID NO:1 (referred to as "peptide") is compared to a negative control (vehicle) and a positive control (pregabalin). Statistical differences between peptide-treated mice compared to the vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). [Diagram 2]Figure 1 shows the analgesic effect of the peptide of SEQ ID NO: 1 orally administered in vivo in the SNI model of neuropathic pain. The figure shows the mechanical force (expressed as its equivalent mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The peptide of SEQ ID NO: 1 (referred to as "peptide") is compared with a negative control (vehicle) and a positive control (pregabalin). Statistical differences between peptide-treated mice compared to the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). [Diagram 3] Figure 1 shows the dose-dependent analgesic effect of the peptide of SEQ ID NO: 1 orally administered in vivo in the SNI model of neuropathic pain. Data are presented as percentage response to baseline levels. The peptide of SEQ ID NO: 1 (referred to as "peptide") is compared to a negative control (vehicle) and a positive control (pregabalin). Measurements were performed 1, 2, 4 and 24 hours after oral gavage. Statistical differences between peptide-treated mice compared to the vehicle group are shown in the table (two-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). [Figure 4] Figure 1 shows a comparison of the analgesic effect of TAFA-4 (full length protein) with the peptide of SEQ ID NO: 1 administered subcutaneously or orally in the SNI model of neuropathic pain. The figure shows the mechanical force (expressed as its equivalent in mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The peptide of SEQ ID NO: 1 (referred to as "peptide") is compared to a negative control (vehicle) and the TAFA-4 protein (full length) (referred to as TAFA4 sc). Statistical differences between peptide or TAFA4 treated mice compared to the vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). [Diagram 5]Figure 1 shows the analgesic effect of orally administered TAFA-4 (full length) protein in vivo in the SNI model of neuropathic pain. The figure shows the mechanical force (expressed as its equivalent in mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. TAFA-4 (full length) protein is compared to a negative control (vehicle) and a positive control (pregabalin). Statistical differences between pregabalin-treated mice compared to the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). No statistical differences are detected for oral administration of TAFA4. [Figure 6] Figure 1 shows the analgesic effect of the peptide of SEQ ID NO: 1 administered orally in vivo in an inflammatory pain model (carrageenan model). The figure shows the mechanical force (expressed as its equivalent mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The peptide of SEQ ID NO: 1 (referred to as "peptide") is compared with a negative control (vehicle) and a positive control (celecoxib). Statistical differences between peptide or celecoxib treated mice compared to the vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). [Figure 7] Figure 1 shows the analgesic effect of the peptide of SEQ ID NO:1 orally administered in vivo in a postoperative pain model (Brennan paw incision model of pain). The figure shows the mechanical force (expressed as its equivalent mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The peptide of SEQ ID NO:1 (referred to as "peptide") is compared to a negative control (vehicle) and a positive control (morphine). Statistical differences between peptide or morphine treated mice compared to the vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). [Figure 8]Figure 1 shows a comparison of the pain-relieving potency of recombinant TT1 (bio-produced) and TT1 obtained by chemical synthesis, administered orally in the SNI model of neuropathic pain. The figure shows the mechanical force (expressed as its equivalent mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The pain-relieving potency of the peptide of SEQ ID NO:1 produced by chemical synthesis ("TT1 synth") is compared to that of the peptide of SEQ ID NO:1 produced in bacteria (bio-) ("TT1 prod Bact"). Statistical differences between "TT1 synth" or "TT1 prod bact" treated mice compared to the vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). n=8; model=SNI; sex: male; administration: oral; concentration TT1 synth: 300 μg / kg, concentration TT1 prod bact: 300 μg / kg. [Figure 9] Figure 1 shows a comparison of pain-relieving efficacy between orally or subcutaneously administered bioproduced TT1 and TT6 in the SNI model of neuropathic pain. The figure shows the mechanical force (expressed as its equivalent mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. Peptide of SEQ ID NO:1 ("TT1") is compared to peptide of SEQ ID NO:5 ("TT6"), each peptide administered either subcutaneously (A) or orally (B). Statistical differences between TT1 or TT6 treated mice compared to the vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). n=8; model=SNI; sex: male; administration: oral or subcutaneous; concentration TT1: 300 μg / kg, concentration TT6: 300 μg / kg. [Figure 10]Figure 1 shows the preventive analgesic effect of the peptide of SEQ ID NO:1 administered subcutaneously in vivo in a postoperative pain model (Brennan paw incision model). A. Schematic of the protocol. TT1 or vehicle was administered subcutaneously twice a day at different time points: the day before surgery (D-1), 1 hour before and 1 hour after surgery (and awakening) on ​​day D, and on D+1 and D+2. Mechanical threshold response measurements on D+1 and D+2 were performed before administration of the peptide of SEQ ID NO:1 ("TT1"). B. The figure shows the mechanical force (expressed as its equivalent in mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The peptide of SEQ ID NO:1 (referred to as "TT1") is compared to a negative control (vehicle). Data are presented as percentage response relative to baseline levels. Statistical differences between peptide-treated mice compared to vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). n=23 for TT1, n=22 for vehicle; model=post-operative pain model; gender: 11 males and 12 females for TT1-treated mice, 11 males and 11 females for vehicle group; administration: subcutaneous; concentration TT1: 300 μg / kg. [Figure 11]Figure 1 shows the absence of tolerance after repeated oral administration of peptide of SEQ ID NO:1 in Chronic Constriction Injury (CCI) neuropathic pain model. After the establishment of CCI neuropathic pain model, mice were treated with peptide of SEQ ID NO:1 ("TT1") or vehicle for 14 consecutive days, starting 10 days after surgery. The analgesic effect of TT1 was determined every 2 days. The figure shows the mechanical force (expressed as its equivalent mass in grams) required to elicit a paw withdrawal response in 50% of mice as a function of time. The peptide of SEQ ID NO:1 (referred to as "TT1") is compared to the negative control (vehicle). Statistical differences between peptide-treated mice compared to the vehicle group are shown (2-way RM ANOVA followed by Bonferroni post-hoc test: *p<0.05, **p<0.01, ***p<0.001). Model=CCI neuropathic pain model; Gender: 10 males for TT1 treated mice, n=5 for vehicle group; Administration: Oral; Concentration TT1: 300 μg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention definition The terms used herein generally have their ordinary meaning in the art, within the context of the present invention and the specific context in which each term is used. Certain terms are discussed below or elsewhere herein to provide further guidance to those skilled in the art in describing the methods of the present invention and how to use them. It is further understood that the same item may be stated in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Synonyms are provided for certain terms. The recitation of one or more synonyms does not exclude the use of other synonyms.

[0024] The terms "peptide", "peptide variant", "protein fragment", "protein segment" are used interchangeably to refer to a polymer of amino acid residues. Such a polymer of amino acid residues may contain natural or non-natural amino acid residues. These terms also include post-expression modifications of the peptide, such as glycosylation, sialylation, acetylation, phosphorylation, carbamethylation, etc. Furthermore, in the context of the present invention, "peptide" refers to a protein fragment or segment that contains modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native (wild-type) sequence, as long as the peptide maintains the desired activity, i.e., prevention or treatment of pain. These modifications are preferably obtained by deliberate mutations, for example, via site-directed mutagenesis.

[0025] The term "isolated peptide" refers to a peptide that has been removed from its original environment (i.e., the natural environment, if it occurs in nature). A peptide that naturally occurs in a natural system, e.g., in a living animal, is to be distinguished from the same peptide that has been isolated from all or a portion of the coexisting materials in said natural system. An isolated 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 occur in nature by itself.

[0026] The term "synthetic peptide" refers to a peptide obtained by chemical synthesis.

[0027] The term "recombinant peptide" refers to a peptide encoded by recombinant DNA cloned into a foreign expression system to support the expression of an exogenous gene. The recombinant DNA, usually the cDNA sequence of the target peptide, is typically designed to be placed under the control of a promoter that allows the expression of the target peptide in a selected host cell. Those skilled in the art can select the appropriate promoter to achieve high levels of protein expression.

[0028] The terms "sequence identity," "a sequence having at least X% identity," and "a sequence X% identical to" are used interchangeably to refer to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over a window of comparison, and the portions of the polynucleotide or polypeptide sequences in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where an identical nucleobase (e.g., A, T, C, G, U) or an identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where either an identical nucleobase or amino acid residue occurs in both sequences, or where the nucleobase or amino acid residue is aligned with a gap, to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, 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, herein SEQ ID NO: 1 ("CFPGQVAGTTRAQPSCVEASIVIQKWWCHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR"). Considering the specific example of SEQ ID NO: 5 ("SFPGQVAGTTRAQPSSVEASIVIQKWWSHMNPSLEGEDSKVLPDYSGWSSSSGNKVKTTKVTR"), said sequence is greater than 90% identical to SEQ ID NO: 1, specifically, it is 90.48% identical to SEQ ID NO: 1 using the BLAST algorithm.

[0029] Optimal alignment of sequences for comparison can be performed, 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 search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA), or by visual inspection (see generally Current Protocols in Molecular Biology, eds. FM Ausubel et al., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement)).

[0030] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are 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 via 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 that match or meet some positive threshold score "T" when aligned with words of the same length in database sequences. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using, for nucleotide sequences, the parameters "M" (reward score for a pair 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. The extension of the word hits in each direction is stopped when: the cumulative alignment score falls by an amount "X" from its maximum achieved value; when the cumulative score falls to zero or below due to the accumulation of one or more negatively scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation ("E") of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0031] The degree of percent amino acid sequence identity can also be obtained by ClustalW analysis (version W 1.8) by counting the number of identical matches in the alignment to achieve slow / exact pairwise optimal alignment, dividing such number of identical matches by the length of the reference sequence, and using the following default ClustalW parameters - gap opening penalty: 10; gap extension penalty: 0.10; protein mass matrix: Gonnet series; DNA mass matrix: IUB; Toggle Slow / Fast pairwise alignment = SLOW or FULL alignment.

[0032] The term "nucleic acid" or "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, these terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (similar to those typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may contain polymers of synthetic subunits, such as phosphoramidates, and thus may be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. Additionally, a double-stranded polynucleotide can be obtained from a single-stranded polynucleotide product of chemical synthesis by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase with an appropriate primer.

[0033] The term "vector" refers to a DNA or RNA molecule that contains a polynucleotide sequence that codes for a peptide, polypeptide or protein. A vector generally contains regulatory elements capable of directing the expression of the coding polynucleotide sequence, also called a transgene, in a cell into which the nucleic acid molecule is introduced. The term "transgene" refers to a polynucleotide that is introduced into a cell and that can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In certain aspects, it confers a desired property to the cell into which it is introduced or otherwise produces a desired technical effect, typically a therapeutic effect herein. A transgene may contain a sequence that codes for one or more proteins or one or more fragments of a protein.

[0034] The term "gene therapy" refers to treatment of a subject involving the delivery of genes / nucleic acids into the cells of an individual for the purpose of preventing or treating disease.

[0035] The term "transfection" refers to the uptake of an exogenous polynucleotide by a cell, e.g., a prokaryotic or eukaryotic cell. A cell is identified as "transfected" when an exogenous polynucleotide has been introduced into the cell. Several transfection 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). Such techniques can be used to introduce one or more exogenous nucleic acids into a suitable host cell.

[0036] The term "transduction" refers to the delivery of a nucleic acid molecule into a recipient host cell, for example, by a gene delivery vector, such as a recombinant viral vector, in particular a retrovirus, adenovirus, recombinant adeno-associated virus (AAV), herpes simplex virus, and lentivirus. For example, transduction of a target cell by a rAAV virion results in the transfer of the rAAV vector contained in the virion into the transduced cell.

[0037] Recombinant "adeno-associated virus (AAV)" is a small dependoparvovirus with a single-stranded linear DNA genome that is artificially produced using recombinant methods, lacks pathogenicity and exhibits low immunogenicity. Recombinant AAV (rAAV) preferably has tissue / cell-specific targeting capabilities, such that the transgene of the rAAV is specifically or preferentially delivered to one or more predetermined tissues / cells. The type of AAV capsid and regulatory region and the route of administration are important factors in determining these tissue-specific targeting capabilities.

[0038] The term "pain" refers in the context of the present invention to any pain or sensitivity associated with tissue damage.Preferably, the term pain, as used herein, is understood as abnormal sensitivity, i.e. typically as hypersensitivity, 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, somatoform pain, allodynia, hyperalgesia, or pain associated with nerve injury.

[0039] "Nociceptive" or "nociceptor-mediated" pain occurs in response to activation of a specific subset of peripheral sensory neurons (nociceptors) by intense or noxious stimuli. Nociceptive pain according to the present invention includes mechanical pain (crushing, laceration, etc.) and chemical pain (iodine on a cut, chili powder in the eye). Examples of nociceptive pain include, but are not limited to, traumatic or surgical pain, labor pain, sprains, fractures, burns, bumps, bruises, injections, dental procedures, skin biopsies, and occlusions. Nociceptive pain includes visceral pain and somatic pain, particularly deep somatic pain and superficial somatic pain.

[0040] Visceral pain is diffuse and difficult to locate, and often refers to distal, usually superficial structures. It may be accompanied by nausea and vomiting, which may be described as nauseating, deep, squeezing, and / or dull. Deep somatic pain is initiated by stimulation of nociceptors in ligaments, tendons, bones, blood vessels, fascia, and muscles, and is a dull, throbbing, less localized pain. Examples of deep somatic pain include sprains and broken bones. Superficial pain is initiated by activation of nociceptors in the skin or other superficial tissues, and is sharp, well-defined, and well-localized. Examples of injuries that produce superficial somatic pain include minor wounds and minor (first-degree) burns.

[0041] The term "injury-induced pain", within the context of the present invention, encompasses neuropathic pain, inflammatory pain and post-operative pain.

[0042] Inflammatory pain is pain that occurs in the presence of tissue injury or inflammation, including post-operative, post-traumatic pain, arthritic (rheumatoid or osteoarthritis) pain, pain associated with autoimmune diseases (e.g., psoriasis), and pain associated with damage to joints, muscles, and tendons, such as in axial low back pain. Inflammation is responsible for sensitizing peripheral sensory neurons, resulting in spontaneous pain and invalidating pain hypersensitivity. Acute or chronic pathological tissue inflammation strongly affects pain perception by sensitizing peripheral sensory neurons, resulting in localized, disabling pain hypersensitivity. Inflammatory mediators are known to enhance the excitability of nociceptive primary afferent fibers, in part, by modifying the expression and / or function of ion channels present in the nerve endings.

[0043] Neuropathic pain is a common type of chronic non-malignant pain that is the result of injury or dysfunction in the peripheral or central nervous system. Neuropathic pain can have different causes, for example, it can occur as a result of trauma, surgery, herniation of intervertebral discs, spinal cord injury, diabetes, infection with herpes zoster (shingles), HIV / AIDS, terminal cancer, amputation (including mastectomy), carpal tunnel syndrome, chronic alcohol use, exposure to radiation, and as an unintended side effect of neurotoxic treatments, for example, certain anti-HIV and chemotherapy drugs. A particular type of neuropathic pain is <<chemotherapy-induced peripheral neuropathic pain>> (CIPN) or <<chemotherapy-induced neuropathic pain>> (CINP). CINP or CIPN is the most severe side effect of anticancer drugs, for example, platinum-based drugs and taxane-based drugs (oxaliplatin, cisplatin, carboplatin and paclitaxel). CINP may indeed be a factor in the discontinuation of treatment, resulting in an increased risk of death. Neuropathic pain is often characterized by or responsible for the appearance of chronic allodynia (defined as pain resulting from stimuli that do not normally evoke a painful response, e.g., light touch) and / or hyperalgesia (defined as increased sensitivity to normally painful stimuli) and may persist for months or years, past any apparent healing of any damaged tissue. Pain may also occur in patients with cancer, which may result from multiple causes, e.g., inflammation, compression, invasion, metastatic spread into bone or other tissues. Pain also includes migraine headaches and headaches associated with activation of sensory fibers that innervate the meninges of the brain. The peptides according to the present invention, e.g., peptides of SEQ ID NO: 1 or SEQ ID NO: 5, are used to treat such above-mentioned types of pain.

[0044] The peptides of the invention may also be used to treat pain associated with Ehlers-Danlos syndrome, including chronic muscle and / or bone and / or joint pain.

[0045] Preferably, the peptide of the present invention is used to prevent or treat injury-induced pain. More preferably, the peptide of the present invention is used to prevent or treat neuropathic pain (e.g. chemotherapy-induced neuropathic pain or chemotherapy-induced peripheral neuropathic pain), postoperative pain and / or inflammatory pain. As shown in the experimental section, the inventors have also demonstrated that the peptide of the present invention is suitable for preventing subjects from developing postoperative mechanical allodynia. Advantageously, the peptide of the present invention can be used without inducing any tolerance in subjects.

[0046] Typically, the peptides of the present invention are used to prevent or treat chronic injury-induced pain. Typically, the peptides of the present invention are used to prevent or treat chronic neuropathic pain, such as chronic chemotherapy-induced peripheral neuropathic pain (CIPN), chronic chemotherapy-induced neuropathic pain (CINP) or chronic nerve injury-induced pain; chronic post-operative pain; and / or chronic inflammatory pain.

[0047] Within the context of the present invention, the term "treatment" or "treating" pain in a subject refers to delaying, stabilizing, healing, curing, mitigating, relieving, altering, ameliorating, improving, correcting or affecting any form of pain in a subject as described herein, or any disease or condition associated with pain, in particular acute, subacute or chronic pain (in particular any neuropathic condition associated with chronic pain typically resulting from neuropathic pain, post-operative pain or inflammatory pain), or any symptom of such disease or condition, following application or administration of a peptide of sequence SEQ ID NO: 1 or a variant thereof as appropriate as defined herein, or a composition according to the present invention.

[0048] The term "treatment" or "treating" also refers to any indicator of success in treating pain (which may be associated with any injury, pathology, or condition), including any objective or subjective parameter, such as remission, relief, slowing of progression or severity, stabilization, attenuation of the symptoms of pain, or making it tolerable or more tolerable to the subject. The term "treating" pain also includes increasing pain tolerance and / or decreasing perceived pain. In certain embodiments, the methods, compounds, and compositions of the present invention are for increasing pain tolerance and / or decreasing perceived pain. As used herein, the term "pain tolerance" refers to the amount of pain a subject can perceive and tolerate before becoming emotionally and / or physically overwhelmed. Pain tolerance is distinct from pain threshold (the minimum mechanical stimulation required to cause pain). As used herein, "increasing pain tolerance" generally refers to a situation in which a subject can develop a higher pain tolerance (i.e., less perceived pain) compared to a previous state following administration to the subject of, for example, an appropriate peptide of sequence SEQ ID NO: 1 or a variant thereof, or a composition comprising said peptide or variant.

[0049] Within the context of the present invention, "preventing" or "prevention" in relation to pain in a subject refers to at least a reduction in the likelihood of the risk (or susceptibility) of the subject acquiring any type of pain following application or administration of the appropriate peptide of sequence SEQ ID NO: 1 or a variant thereof or a composition according to the present invention. For example, "preventing" includes not developing at least one of the clinical symptoms of pain in a subject who may be exposed to or predisposed to pain, but who has not yet experienced or exhibited symptoms of pain.

[0050] In the context of the present invention, "subject" or "patient" refers to an animal, particularly a mammal, that is in need of treatment for a disease or disorder or symptoms thereof. The subject may be a subject that has been diagnosed with a disease or disorder or has been determined to be at risk of developing a disease or disorder, which disease or disorder is known to cause the subject to feel pain. In certain examples, the subject has been diagnosed with or is suffering from pain, including neuropathic pain, post-operative pain, inflammatory pain, hyperalgesia and / or allodynia, for example, acute pain and / or subacute pain or chronic pain.

[0051] In certain embodiments, the subject is a human-being.

[0052] In another particular embodiment, the subject is an animal, particularly a livestock or breeding animal, particularly a horse, dog, cat, cow, and the like.

[0053] In another specific embodiment, the subject has at least one mutated allele in the myolA gene.

[0054] peptide The present inventors have identified novel peptides that can prevent or treat pain, in particular reverse mechanical hypersensitivity in the context of injured or inflamed nervous systems. The present inventors believe that these peptides exhibit either analgesic or analgesic activity, in particular analgesic activity, by specifically targeting mechanically and / or chemically induced nociceptive signals, by modulating the excitability of spinal cord networks.

[0055] The peptides described herein are / consist of a specific fragment from the 105 amino acid residue human TAFA-4 mature protein (resulting from cleavage of the signal sequence and identified in public databases under accession number NP_0011005527 or under Genbank accession AAP92409, as disclosed by Tang et al., 2004).

[0056] In a first aspect, the present invention relates to an isolated, synthetic or recombinant peptide of the sequence SEQ ID NO:1, or a peptide having at least 90% identity to SEQ ID NO:1, such as a variant peptide of SEQ ID NO:5.

[0057] In a particular embodiment, the peptide of sequence SEQ ID NO: 1 or a variant thereof, such as the variant peptide of SEQ ID NO: 5, is a recombinant peptide (ie, a biologically produced peptide).

[0058] In a particular embodiment, the peptide according to the invention has the amino acid sequence CFPGQVAGTTRAQPSCVEASIVIQKWWCHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR (SEQ ID NO: 1), i.e. Cys-Phe-Pro-Gly-Gln-Val-Ala-Gly-Thr-Thr-Arg-Ala-Gln-Pro-Ser-Cys-Val-Glu- The peptide contains: Ala-Ser-Ile-Val-Ile-Gln-Lys-Trp-Trp-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.

[0059] In another particular embodiment, the peptide according to the invention is a peptide consisting or consisting essentially of the amino acid sequence shown in SEQ ID NO:1.

[0060] In a further particular embodiment, the peptide according to the invention is a peptide consisting or consisting essentially of the amino acid sequence shown in SEQ ID NO:5.

[0061] The terms "consisting essentially of" have the meanings commonly ascribed to them, e.g., they allow for elements not explicitly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention. Thus, a peptide "consisting essentially of" a specified sequence refers to a peptide that includes or consists of that sequence, including other features that are not essential to the activity of the peptide.

[0062] The peptide of SEQ ID NO:1 consists of or consists essentially of the C-terminal 63 amino acid residues of the human TAFA-4 mature protein, and can be identified as amino acids 78 to 140 of UniProt Accession No. Q96LR4.

[0063] With reference to the position shown in SEQ ID NO:1, [ka] Two amino acid residues appearing in bold within: Q (Glu) at position 13 and Y (Tyr) at position 45, are believed by the inventors to be implicated in the biological activity of the peptide, in particular in the modulation of excitability of spinal interneurons, preferably spinal lamina IIi interneurons.

[0064] Variants of the peptide of SEQ ID NO: 1 are also disclosed herein for the first time by the inventors. Variants are intended to denote peptides derived from the peptide of SEQ ID NO: 1 and resulting from the deletion or addition of one or more amino acids and / or the substitution of one or more amino acids at one or more sites in the peptide of SEQ ID NO: 1. A variant is considered as a functional variant if it substantially retains the functional activity of the peptide of SEQ ID NO: 1, or even if it improves said functional activity and allows the prevention or treatment of pain. Activity can be measured using functional assays, for example behavioral assays as carried out in the experimental part.

[0065] Peptide variants that can be used in the context of the present invention may have at least 90%, 90.5%, 91%, 92%, 92.1%, 93%, 93.7%, 94%, 95%, 95.2%, 96%, 96.8%, 97%, 98%, 98.4% or 99% sequence identity to SEQ ID NO:1.

[0066] A particular preferred variant of the peptide of SEQ ID NO:1 ("TT1") has a molecular weight of 6820 g / mol, SEQ ID NO:5 ("TT6"):SFPGQVAGTTRAQPSSVEASIVIQKWWSHMNPSLEGEDSKVLPDYSGWSSSSGNKVKTTKVTR, i.e., Ser-Phe-Pro-Gly-Gln-Val-Ala-Gly-Thr-Thr-Arg-Ala-Gln-Pro-Ser-Ser-V al-Glu-Ala-Ser-Ile-Val-Ile-Gln-Lys-Trp-Trp-Ser-His-Met-Asn-Pro-Ser-Leu-Glu-Gly-Glu-Asp-Ser-Lys-Va The peptide is l-Leu-Pro-Asp-Tyr-Ser-Gly-Trp-Ser-Ser-Ser-Gly-Asn-Lys-Val-Lys-Thr-Thr-Lys-Val-Thr-Arg.

[0067] In certain embodiments, variants having at least 90% identity to SEQ ID NO: 1 modulate the excitability of spinal interneurons (preferably spinal lamina IIi interneurons). In a preferred embodiment, the variant is a peptide that includes one or more point mutations (e.g., 2, 3, 4, 5 or 6 point mutations) that add, delete or substitute any of the amino acids present in SEQ ID NO: 1, with the proviso that amino acid residues Q at position 13 and Y at position 45 in SEQ ID NO: 1 (positions 13 and 45 with reference to the positions shown in SEQ ID NO: 1) remain unchanged.

[0068] Thus, in a preferred embodiment of the invention, two amino acids (Q at position 13 and Y at position 45 of SEQ ID NO:1) remain unchanged in the amino acid sequence of a peptide variant having at least 90% identity to SEQ ID NO:1, for example with respect to the peptide of SEQ ID NO:5.

[0069] In another particular embodiment, variants of the peptide usable in the context of the present invention may have at least 84.1%, 85%, 85.7%, 86%, 87%, 87.3%, 88%, 88.9% or 89% sequence identity to SEQ ID NO: 1. Preferably, variants having at least 85% identity to SEQ ID NO: 1 modulate the excitability of spinal interneurons (preferably spinal lamina IIi interneurons).

[0070] In a particular embodiment, the variant is a peptide that comprises seven or more point mutations (e.g., eight, nine or ten point mutations) that add, delete or substitute any of the amino acids present in SEQ ID NO:1, with the proviso that amino acid residues Q at position 13 and Y at position 45 in SEQ ID NO:1 (positions 13 and 45 with reference to the positions shown in SEQ ID NO:1) remain unchanged.

[0071] In a particular embodiment, the deletion or deletions are at the N-terminus of SEQ ID NO: 1. In another embodiment, the deletion or deletions are at the C-terminus of SEQ ID NO: 1, or in any other position, with the proviso that the two amino acids (Q and Y) at positions 13 and 45 of SEQ ID NO: 1 remain unchanged. In yet another embodiment, two or more deletions are at both the N-terminus and C-terminus of SEQ ID NO: 1. Such deletions at the N-terminus and / or C-terminus of the peptide of SEQ ID NO: 1, or in its core, may result in truncated human peptide variants of SEQ ID NO: 1.

[0072] Furthermore, the 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. The term "conjugate" in this context refers to an engineered fusion construct that combines the biological functions of two molecules in a single polypeptide, i.e., a peptide of SEQ ID NO: 1 or a variant thereof can be combined with a polypeptide that specifically interacts or binds to a target cell, for example to modulate the excitability of a nociceptor or interneuron.

[0073] The present invention also relates to nucleic acids (of SEQ ID NO: 2 or 9) that encode the peptides of SEQ ID NO: 1 or variants thereof described herein. Any sequence that encodes the peptides of SEQ ID NO: 1 or any of its variants is encompassed by the present invention, as well as similar sequences resulting from degeneration of the genetic code. In a particular embodiment, the nucleic acid that encodes the peptides described herein comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 2: TGCTTCCGGGACAGGTGGCGGGCACAACTCGGGCTCAACCTTCTTGTGTTGAAGCTTCCATTGTGATTCAGAAATGGTGGTGTCACATGAATCCGTGTTTGGAAGGAGAGGATTGTAAAGTGCTGCCAGATTACTCAGGTTGGTCCTGTAGCAGTGGCAATAAAGTCAAAACTACGAAGGTAACGCGG.

[0074] In another particular embodiment, a nucleic acid encoding a peptide described herein comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 9: TGCTTTCCAGGTCAAGTTGCGGGAACAACTCGTGCACAACCATCGTGCGTAGAGGCCTCAATTGTTATCCAAAAGTGGTGGTGTCACATGAACCCCTGCCTCGAAGGAGAGGACTGTAAGGTACTGCCTGACTACAGCGGGTGGTCATGTTCATCAGGCAATAAGGTGAAGACGACCAAAGTTACCCGT. The nucleic acid of SEQ ID NO: 9 is particularly adapted for production of the peptide of SEQ ID NO: 1 in bacteria.

[0075] The present invention also relates to a nucleic acid (of SEQ ID NO: 6) that encodes the peptide of SEQ ID NO: 5 or a variant thereof described herein. Any sequence that encodes the peptide of SEQ ID NO: 5 or any of its variants, as well as similar sequences resulting from the degeneration of the genetic code, are encompassed by the present invention. In a particular embodiment, the nucleic acid that encodes the peptide described herein comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 6: TCGTTTCCAGGTCAAGTTGCGGGAACAACTCGTGCACAACCATCGTCGGTAGAGGCCTCAATTGTTATCCAAAAGTGGTGGTCGCACATGAACCCCTCGCTCGAAGGAGAGGACTCGAAGGTACTGCCTGACTACAGCGGGTGGTCATCGTCATCAGGCAATAAGGTGAAGACGACCAAAGTTACCCGT.

[0076] The above-mentioned nucleic acid sequences encoding the peptide of SEQ ID NO: 1 or a variant thereof may be flanked by regulatory sequences for controlling its expression in a suitable host cell.

[0077] In another aspect, the present invention also relates to an expression cassette comprising, in this order from 5' to 3': - Promoters, - a nucleic acid sequence encoding the peptide of SEQ ID NO: 1 or a variant thereof as described herein; and - Termination signals, such as, for example, polyadenylation signals.

[0078] The above-mentioned nucleic acid sequences encoding the peptide of SEQ ID NO:1 or a variant thereof, or expression cassettes disclosed herein, may be flanked by suitable sequences for their packaging into vectors that optimize their transcription and / or translation in cells.

[0079] In another aspect, the present invention also relates to vectors allowing the expression of the peptides described herein.

[0080] Methods for Producing Peptides To allow expression in a host cell, the nucleic acid encoding the peptides described herein (or one of its variants) can be present in a vector, following standard cloning and expression techniques well known in the art (e.g., as described in Sambrook, J., Fritsh, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and after introduction of the vector into a suitable host cell, the sequence can be expressed to produce the encoded peptides described herein. A variety of expression vectors can be used to express polynucleotide sequences encoding the peptides described herein. Expression vectors that can be used in the present invention non-inclusively include eukaryotic expression vectors, particularly mammalian expression vectors, virus-based expression vectors, baculovirus expression vectors, plant expression vectors and any plasmid expression vectors for producing any one of the peptides described herein in a host cell. The expression vector can also be a vector that allows expression of the peptide in a bacterial system.

[0081] The choice of expression vector depends on the intended host cell in which the vector is expressed. This choice can be easily made by those skilled in the art. The present invention also relates to cells, particularly host cells, that contain the nucleic acid sequence encoding the peptides described herein. The (host) cells modified using the vectors described herein are also disclosed herein for the first time by the inventors.

[0082] The peptides of the present invention or variants thereof can be expressed in either prokaryotic or eukaryotic host cells. Representative host cells include many strains of E. coli, mammalian cell lines such as CHO, CH0-K1 and HEK293, insect cells such as Sf9 cells, and yeast cells such as S. cerevisiae and P. pastoris.

[0083] The nucleic acids or vectors described herein can be transfected into host cells by standard techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as 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 viral-based vectors.

[0084] Purity can be measured by any appropriate 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 renders it safe for administration to a human subject, e.g., lacking infectious or toxic agents.

[0085] 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 achieved by methods well known to those skilled in the art, for example, native chemical ligation (NCL). This chemical approach consists of coupling of unprotected peptide fragments: a peptide with an N-terminal cysteine ​​reacts with a C-terminal thioester peptide. This transthioesterification is quickly followed by an intramolecular S,N-acyl shift that results in the formation of a native amide bond at the ligation site. When two peptides are ligated in a one-step ligation followed by one-step purification, the expected yield is very high and therefore compatible with large-scale production for therapeutic use.

[0086] Gene Therapy By cloning the nucleic acid sequence encoding the peptides described herein into a suitable vector, the inventors also provide herein a novel gene delivery tool, in particular a gene construct (such as an expression cassette or vector) for gene therapy of pain, in particular acute, subacute or chronic pain, preferably chronic pain. Gene therapy can be used to enable endogenous production of TAFA-4-peptide, TT1 peptide or its variants, such as TT6 peptide, by specific cells in a subject. Gene therapy can be performed either in vivo or ex vivo. Ex vivo gene therapy requires the isolation and purification of at least one sample of cells of a subject, the introduction of a nucleic acid sequence encoding a peptide described herein (i.e., a transgene) into the isolated cells, and the introduction of the genetically altered / modified cells back into the subject. In contrast, in in vivo gene therapy, the transgene is typically packaged for administration to a subject. The gene delivery construct can be either non-viral or viral. Preferably, the genetic constructs described herein are prepared using 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 peptide of SEQ ID NO: 1 or a variant thereof) in the targeted tissue / cell. The viral vector may incorporate any suitable promoter and other transcriptional regulators that allow or facilitate the expression of the transgene product in the targeted tissue / cell. The viral packaging system is preferably adapted to the targeted cell. Once inside the target cell, such a system facilitates delivery to the targeted tissue. The viral vectors that can be used in the methods described herein are preferably replication-defective viruses, such as adenovirus or adeno-associated virus (AAV) vectors.

[0087] In some aspects, the present invention relates to a recombinant adeno-associated virus (AAV) comprising in its genome a nucleic acid sequence encoding a peptide of SEQ ID NO:1 or a variant thereof, e.g., a peptide of SEQ ID NO:5, typically operably linked to a promoter.

[0088] To date, at least 12 different serotypes of AAV, with variations in their surface properties, have been isolated and characterized from humans or non-human primates (NHPs). The term "serotype" allows the skilled artisan to distinguish between AAVs with serologically distinct capsids. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies against one AAV serotype compared to other AAV serotypes. The rAAV described herein, also referred to as rAAV vectors or rAAV particles, can have any one of the following known serotypes, i.e., 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.Anc80, AAV.Rh ...8, AAV.rhM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80, AAV.Rh8, AAV.rhIO, AAV.rh20, AAV.rh39, AAV.Rh8, AAV.rhM4-1, AAV.hu37, AAV.Anc80, AAV.Anc , 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 the context of administration by the oral route, the rAAV vector may have enhanced tropism for stomach, small intestine or colon tissue, more specifically for cells that compose these tissues, particularly epithelial cells, such as enterocytes, goblet cells, enteroendrocine cells, Paneth cells or Tuft cells, etc. For targeted cells located in the intestine, or to target cells for delivery to the intestine, any one of AAV4, AAV7, AAV8, AAV9 or AAV10 may be selected as being particularly effective.

[0089] The gene therapy vectors or cassettes of the present invention can be produced by methods well known to those of skill in the art and previously described, for example, in PCT Patent Application Publication No. WO03042397 and US Pat. No. 6,632,670.

[0090] Described herein is a method for producing a recombinant vector, e.g., a viral vector, e.g., AAV, comprising: a) culturing cells transfected / transduced with a 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:

[0091] The gene therapy vector of the present invention can be produced by transfection of two or three plasmids into 293 or 293T human embryonic kidney cell lines. In some embodiments, DNA encoding a therapeutic gene is provided by one plasmid; capsid proteins and replication genes from one or more serotypes of AAV, and helper functions, for example, from adenovirus, are all provided in trans by a second plasmid. In some embodiments, DNA encoding a peptide of SEQ ID NO: 1 or a variant thereof is provided by one plasmid; capsid proteins and replication genes from one or more serotypes of AAV are provided in trans by a second plasmid, and helper functions, for example, from adenovirus, are provided by a third plasmid. In certain embodiments, the first plasmid comprises an expression cassette comprising a nucleic acid sequence encoding a peptide as described herein operably linked to a promoter comprising two flanking inverted terminal repeats (ITRs).

[0092] 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 one of skill in the art, such as, for example, by using an iodixanol step gradient followed by ion exchange chromatography on a Hi-Trap QHP column. The resulting gene therapy vector can then be concentrated by spin column, and the purified vector can be stored frozen (at -60°C or below), for example, in phosphate buffered saline.

[0093] Related aspects of the invention include host cells transfected or transduced with a recombinant vector described herein, e.g., transduced with an AAV vector. Further related aspects of the invention include any nucleic acid molecule comprising or consisting (essentially) of the genome of a recombinant vector described herein, e.g., an AAV.

[0094] composition One or more of the peptides described herein may be formulated for separate or combined use, either individually or in the form of a composition. The composition may be a dietary or pharmaceutical composition and may be used in the therapeutic or prophylactic methods described herein.

[0095] In a further aspect, the invention relates to a composition comprising a peptide, a nucleic acid, a vector or a cell as described herein and a dietary or pharma- ceutically acceptable support.

[0096] The term "dietarily acceptable support" relates to a carrier that allows the subject to ingest and digest the composition comprising the peptide, nucleic acid sequence, vector or cell described herein without risk, and is capable of protecting said peptide from any attack associated with food digestion that may modify it before it exerts its therapeutic action at the exact site and at the exact moment depending in particular on the nature and localization of the pain.

[0097] In the context of oral administration, the composition may further comprise at least one gastrointestinal protectant, preferably an acid inhibitor, present in an amount effective to raise the subject's intragastric pH 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 gastric acid secretion and increases intragastric pH. Acid inhibitors may include, but are not limited to, H2 blockers, including cimetidine, ranitidine, ebrotidine, pabutidine, lafutidine, loxtidine, famotidine; proton pump inhibitors, including omeprazole, esomeprazole, pantoprazole, lansoprazole, dexlansoprazole, rabeprazole, pariprazole, leminoprazole, and tenatoprazole; or any combination thereof.

[0098] A "pharmaceutical acceptable support / vehicle / carrier" may be a diluent, adjuvant or excipient with which an active agent (i.e., the peptide of the present invention or its variant and, optionally, any further separate active agents) is administered. Such pharmaceutical carriers may be sterile liquids, such as water or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0099] When the pharmaceutical composition is adapted for oral administration, tablets or capsules can be prepared by conventional means using pharma- ceutically acceptable excipients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by any method well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or can be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may be prepared by conventional means using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents, as appropriate.

[0100] The composition of the present invention may further comprise at least one further active compound.Preferably, the further active compound is an active agent that is effective against pain. "Effective against pain" means an active agent that has analgesic or analgesic properties (measurably felt by the subject).More preferably, the further active compound is a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID) or an opioid drug.

[0101] SAIDs may include, but are not limited to, hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone, fludrocortisone, or any combination thereof.

[0102] NSAIDs may include, but are not limited to, celecoxib, rofecoxib, lumiracoxib, valdecoxib, parecoxib, etoricoxib, CS-502, JTE-522, L-745,337, NS398, aspirin, acetaminophen (which will be considered an NSAID for 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.

[0103] Opioid drugs include (dextro)propoxyphene, A-methylfentanyl, alfentanil, allylprozine, bezitramide, buprenorphine, butorphanol, carfentanyl, desmethylprozine, dextromoramide, dezocine, diacetylmorphine, dihydrocodeinone, dihydroetorphine, dimorphone, diphenoxylate, dipipanone, etorphine, fentanyl, ketobemidone, lefetamine, levacetylmethadol, levomethorphan, and levorphanol. The therapeutic agent may include, but is not limited to, morphine, loperamide, meperidine, meptazinol, methadone, methylmorphine, morphine, nalbuphine, nicomorphine, omefentanil, oripavine, oxycodone, oxymorphone, PEPAP, paramorphine, pentazocine, phenazocine, piritramide, prodine, remifentanil, sufentanil, tapentadol, tilidine, tramadol, or an opioid antagonist, such as nalmefene, naloxone, naltrexone, or any combination thereof.

[0104] Therapeutic and Prophylactic Uses The peptides described herein are typically used to prevent or treat pain.

[0105] The present invention also relates to the peptides described herein, in particular isolated, recombinant or synthetic peptides of the sequence SEQ ID NO:1 or peptides having at least 90% identity to SEQ ID NO:1, for use as a medicament.

[0106] In another particular and preferred aspect, the present invention relates to an isolated, synthetic or recombinant peptide of sequence SEQ ID NO: 5 as defined herein, or a variant thereof, for use as a medicament.

[0107] In another aspect, we herein describe a peptide, a nucleic acid, a vector or a cell as described herein for use as an active ingredient / medicament for preventing or treating pain in a subject in need thereof.

[0108] In a particular aspect, the inventors herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein for the manufacture of a medicament for the treatment of pain in a subject in need thereof. They also herein describe a peptide, nucleic acid, vector, cell or composition described herein for use in the prevention or treatment of pain in a subject in need thereof, and a corresponding method for preventing or treating pain comprising administering to a subject in need thereof said peptide, nucleic acid, vector, cell and / or composition described herein.

[0109] In a particular aspect, we herein describe the use of a peptide, a nucleic acid, a vector, a cell or a composition as described herein for preventing or treating pain, in particular acute, subacute or chronic pain, preferably chronic pain.

[0110] Preferably, the pain is neuropathic pain (eg chemotherapy-induced peripheral neuropathic pain or chemotherapy-induced neuropathic pain), post-operative pain, inflammatory pain, hyperalgesia or allodynia.

[0111] In certain aspects, the inventors herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein to prevent or treat neuropathic pain (e.g. chemotherapy-induced peripheral neuropathic pain or chemotherapy-induced neuropathic pain), post-operative pain or inflammatory pain.

[0112] In certain aspects, we describe herein the use of a peptide, nucleic acid, vector, cell or composition described herein to prevent or treat chronic neuropathic pain (e.g. chronic chemotherapy-induced peripheral neuropathic pain or chronic chemotherapy-induced neuropathic pain), chronic post-operative pain or chronic inflammatory pain.

[0113] In a particular aspect, we describe herein the use of a peptide, nucleic acid, vector, cell or composition as described herein for preventing or treating hyperalgesia, in particular thermal (e.g. heat or cold hyperalgesia, preferably cold hyperalgesia) or mechanical hyperalgesia, preferably mechanical hyperalgesia, even more preferably injury-induced mechanical hyperalgesia.

[0114] In another particular aspect, the inventors herein describe the use of a peptide, a nucleic acid, a vector, a cell or a composition described herein for preventing or treating allodynia, in particular mechanical allodynia, preferably nerve injury-induced mechanical allodynia or static type mechanical allodynia.

[0115] In a further particular aspect, the inventors herein describe the use of a peptide, nucleic acid, vector, cell or composition as described herein for preventing or treating allodynia, in particular thermal allodynia, e.g. heat allodynia (i.e. perceived pain in response to a normally non-painful warm stimulus) or cold allodynia (i.e. perceived pain in response to a normally non-painful cold stimulus), preferably cold allodynia.

[0116] In a particular aspect, we describe herein the use of a peptide, a nucleic acid, a vector, a cell or a composition described herein to prevent or treat mechanical hypersensitivity (also called hypersensitivity to mechanical stimuli, or simply hyperalgesia), preferably injury-induced mechanical hypersensitivity, in a subject in need thereof.

[0117] Treatment may result in improvement in one or more of the following sensations: touch, burning or cold, "pins and needles", numbness, itching, excruciating pain, and difficulty in accurately sensing temperature. In certain embodiments, treatment relieves pain. In other particular embodiments, treatment reduces symptoms of pain, particularly those of neuropathic pain (allodynia and / or hyperalgesia). The methods of the present invention make neuropathic pain more manageable (i.e., improve quality of life), even if they do not relieve it.

[0118] Standard tests well known to those skilled in the art are available in the art to assess whether pain, especially (chronic) neuropathic pain, has been treated using a particular peptide of the invention. For example, the assessment of pain sensitivity in a subject is standardized using quantitative sensory tests (pinprick, pressure algometer, von Frey filament, touch, pinching, or light pressure with a finger) or using a pain rating scale.

[0119] subject In the context of the present invention, the subject or patient is an animal, preferably a mammal. In certain embodiments, the subject is a domestic animal, such as a horse, dog, cat, cow, etc. In another particular preferred embodiment, the subject is a human being.

[0120] Subjects with chronic pain of neuropathic origin may suffer from disorders classically associated with such neuropathic pain, such as fibromyalgia, complex regional pain syndrome, postherpetic neuralgia, Ehlers-Danlos syndrome, and erythromelalgia.

[0121] 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 neural circuits involving the perception, transmission and processing of afferent nociceptive stimuli, with widespread manifestations of pain at the level of the motor system. The main symptoms of the disease are muscle stiffness, joint stiffness, insomnia, fatigue, mood disorders, cognitive dysfunction, anxiety, depression, general sensitivity, and the inability to perform normal daily activities. FM may also be associated with certain diseases, such as infectious diseases, diabetes, rheumatic diseases, and / or psychiatric or neurological disorders.

[0122] In a particular aspect, we herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein for preventing or treating fibromyalgia (FM).

[0123] In another aspect, the subject suffers from Complex Regional Pain Syndrome (CRPS). Complex regional pain syndrome is a chronic neurological condition involving the limbs, characterized by severe pain, along with sensory, autonomic, motor and nutritional disorders (Goh et al., 2017). The condition can be induced by surgery, trauma or minor injury, and has a variable course ranging from mild and self-limiting to chronic disease that impairs activities of daily living and health-related quality of life. CRPS can be classified into two types: Type I and Type II CRPS, characterized by the absence or presence of identifiable nerve injury. Type I CRPS is a syndrome that usually develops after an initiating noxious event, is not limited to the distribution of a single peripheral nerve, and is disproportionate to the inciting event. It is associated with edema, changes in cutaneous blood flow, abnormal sudomotor activity in areas of pain, allodynia and hyperalgesia, and generally involves the distal aspect of the affected limb or a distal to proximal gradient. Type II CRPS can be defined as burning pain, allodynia and hyperpathy occurring in an area of ​​the limb following partial injury of the nerve or one of its major branches that innervates that area.

[0124] In a particular aspect, we herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein for preventing or treating complex regional pain syndrome (CRPS).

[0125] In yet another embodiment, the subject has postherpetic neuralgia (PHN). Postherpetic neuralgia is the most common complication of varicella zoster (HZ), an infection caused by reactivation of dormant varicella zoster virus in sensory ganglia (Ngo et al., 2020). It is characterized by a localized blistering rash and pain along the associated dermatome. PHN is defined as persistent pain for at least 90 days after the initial onset of the HZ rash and significantly reduces the quality of life of affected patients. PHN is subcategorized into hypersensitive nociceptors and deafferentation models. During VZV reactivation, the virus replicates and spreads from the dorsal root ganglia to its respective periphery. This proliferation initiates immune responses and inflammation that damage peripheral nerves. This damage reduces neuronal inhibition of pain and lowers the threshold for depolarization of pain signals. This results in painful perception in response to non-painful stimuli, a process called peripheral sensitization. Repetitive activation of subtype C-nociceptors also causes a state of heightened excitability in the dorsal horn. Direct viral damage from HZ weakens descending pain inhibitory pathways, resulting in chronic activation of second-order neurons in the dorsal horn. Furthermore, loss of inhibitory gamma-aminobutyric acid (GABA)-producing interneurons in the dorsal horn has been reported in HZ patients with PHN compared to HZ patients without PHN. These factors amplify all subsequent responses from afferent inputs in a process called central sensitization. In PHN, this process is accompanied by anatomical reorganization of low-threshold mechanoreceptive afferents called Aβ-fibers that normally relay innocuous tactile stimuli to the central nervous system. When viral damage results in loss of C-nociceptors in the periphery, these fibers connect in a compensatory manner with second-order neurons originally connected to C-nociceptor afferents. This process is called deafferentation, and patients suffering from allodynia exhibit a severe loss of sensory function.

[0126] In a particular aspect, we herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein for preventing or treating post-herpetic neuralgia (PHN).

[0127] In yet another aspect, the subject suffers from erythromelalgia (EM). Erythromelalgia is a rare, episodic acrosyndrome that affects mainly both lower limbs bilaterally and symmetrically or unilaterally, with the classic triad of redness, warmth and burning pain (Maria Bibiana Leroux, 2018). EM is classified together with chronic painful syndromes. Primary EM is an autosomal dominant genetic disorder coded by OMIN (Online Mendelian Inheritance in Man) as number 133020. It is associated with alterations to the alpha subunit protein of sodium channel type 9 (SCN9A), affecting the Nav1.7 channel, which is mainly expressed in dorsal root ganglion and sympathetic ganglion neurons. Secondary EM is associated with myeloproliferative disorders, paraneoplasia, autoimmune diseases, contact with toxins and infections.

[0128] In a particular aspect, we herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein for preventing or treating erythromelalgia (EM).

[0129] In a particular aspect, the inventors herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein to prevent or treat any pain-related condition in which signals transmitted by nociceptors or interneurons are impaired.

[0130] The present inventors have previously demonstrated that Myo1a expresses functional Myo1a + / + It has been established that the absence, reduced or insufficient expression, or non-functional expression of Myosin IA (Myo1a) in a subject compared to the expression observed in a reference subject predisposes the subject to developing injury-induced chronic mechanical pain and / or inflammation-induced chronic temperature pain (WO2017153424).

[0131] In yet another embodiment, the subject has one or two mutated alleles in the myo1A gene. A "mutated" allele refers to a substitution, deletion, or insertion in the nucleic acid of the myo1A gene (in a coding or non-coding region) that alters the expression or level of expression of Myo1a. The mutation can affect one or more nucleobases. The substitution can be a single nucleotide polymorphism (SNP).

[0132] In a particular aspect, we herein describe the use of a peptide, nucleic acid, vector, cell or composition described herein to prevent or treat injury-induced chronic mechanical pain and / or inflammation-induced chronic temperature pain in such a subject.

[0133] dose The compositions according to the present invention are preferably administered directly to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of the peptide of SEQ ID NO: 1 or any variant thereof required to treat, ameliorate or prevent pain in a subject. The therapeutically effective amount can be initially estimated either 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. Such information can then be used to determine useful doses and routes for administration in humans.

[0134] The dosage of the peptides used in the methods described herein may vary depending on the subject's general health, age, sex and weight, the nature and severity / intensity of the pain, the time, frequency and duration of administration, the particular peptide being used, the drug combination, reaction sensitivity and tolerance / response to treatment. The appropriate effective dosage can be determined by routine experimentation and is under the judgment of the clinician.

[0135] To obtain an adequate analgesic or analgesic effect, the effective dose of the peptides or variants thereof described herein by the inventors per kg of body weight per 24 hours is between 1 μg / kg / day and 100 mg / kg / day, preferably between 5 μg / kg / day and 80 mg / kg / day, more preferably between 10 μg / kg / day and 50 mg / kg / day in animals, typically mammals.

[0136] When the subject is a human, the effective dose of the peptide or variant thereof described herein by the inventors is preferably between 2.5 μg / kg / day and 0.6 mg / kg / day, preferably between 5 μg / kg / day or 10 μg / kg / day and 0.5 mg / kg / day, more preferably between 50 μg / kg / day or 75 μg / kg / day and 0.3 mg / kg / day in a human subject.

[0137] The dose may be administered in bolus form or divided into several portions administered separately over the course of the day. In other words, the treatment may be a single dose schedule or a multiple dose schedule. An effective dose may be administered over a period of days, weeks, months or years.

[0138] Delivery of the peptides or compositions described herein to a subject can be accomplished by several routes.

[0139] In certain aspects, the peptides or compositions described herein are administered to a subject intramuscularly, intravenously, intraperitoneally, orally (per os), anally, cutaneously, subcutaneously, cutaneously, transdermally or intrathecally, preferably subcutaneously or orally, and even more preferably orally.

[0140] As shown in the experimental section, the inventors have demonstrated that the peptide of SEQ ID NO: 1 and its variant of SEQ ID NO: 5 have analgesic effect when administered subcutaneously to treat neuropathic pain, postoperative pain and / or inflammatory pain. Surprisingly, the same analgesic effect is also observed when the peptide of SEQ ID NO: 1 or 5 is administered orally (per os). Even more surprising and advantageously, the peptide of the present invention shows better analgesic effect when administered orally compared to TAFA-4 (full length) protein, which makes it more convenient and suitable to administer to patients than said full length protein.

[0141] The inventors demonstrate herein that a variant of SEQ ID NO:5, which contains approximately 90% identity to the peptide of SEQ ID NO:1, achieves similar results in terms of potency when compared to the peptide of SEQ ID NO:1.

[0142] kit The present invention also relates to a kit comprising i) a peptide, a nucleic acid, a vector, a cell and / or a composition disclosed herein, and ii) preferably at least one additional separate active compound effective against pain, separate from the peptide or variant thereof described herein. In certain embodiments, the kit further comprises iii) instructions for using the kit.

[0143] The kit is preferably a kit-of-parts comprising at least two parts, e.g. two separate containers, a first part comprising a peptide, a nucleic acid, a vector, a cell or a composition disclosed herein and a second part comprising at least one further separate 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 disclosed herein.

[0144] In certain embodiments, the kit comprises i) a peptide of SEQ ID NO:1, ii) at least one additional separate active compound effective against pain, and iii) optionally instructions for using the kit.

[0145] In another particular embodiment, the kit comprises i) a peptide having at least 90% sequence identity to SEQ ID NO:1, e.g., the peptide of SEQ ID NO:5, ii) at least one additional separate active compound effective against pain, and iii) optionally instructions for using the kit.

[0146] In one embodiment, the peptide, nucleic acid, vector, cell or composition of the kit is in a form adapted for intramuscular, intravenous, intraperitoneal, oral (per os), anal, cutaneous, subcutaneous, dermical, transdermical or intrathecal route, preferably the subcutaneous or oral route, even more preferably the oral route.

[0147] In another embodiment, the at least one additional separate active compound effective against pain is in a form adapted for intramuscular, intravenous, intraperitoneal, oral (per os), anal, cutaneous, subcutaneous, transdermal or intrathecal route.

[0148] Depending on the nature, origin, intensity of the pain to be treated, and also depending on the nature of the contents of the peptide, nucleic acid, vector, cell or composition and the nature of the at least one further separate active compound effective against pain, the products are or are not co-administered, simultaneously / concomitantly or sequentially.

[0149] 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 pain as described herein above, such as chronic pain, neuropathic pain, post-operative pain, inflammatory pain, hyperalgesia or allodynia. The kit of the present invention may also be used in the prevention or treatment of acute or subacute pain.

[0150] Also disclosed herein is the use of the kit of the present invention for the manufacture of a medicament for preventing or treating pain as described herein above, such as chronic pain, neuropathic pain, post-operative pain, inflammatory pain, hyperalgesia or allodynia, in a subject in need thereof. The kit of the present invention may also be used for the manufacture of a medicament for preventing or treating acute or subacute pain in a subject in need thereof.

[0151] Research Tools The present invention also includes the use of the peptides or any of the products described herein above as research tools for studying pain.

[0152] The peptides described herein can be used to modulate neuronal excitability in biological tissues or in cell cultures, for example to study mechanically and / or chemically induced pain nociceptive signals.

[0153] The present invention also relates to the use of a nucleic acid encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, such as the peptide of SEQ ID NO: 5, or a vector enabling its expression, for expressing or modulating (the level of) expression of a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, such as the peptide of SEQ ID NO: 5, in biological tissue or cell cultures.

[0154] The nucleic acid molecules described herein can also be used to create transgenic animals. This can be done locally, by somatic cell modification, or via germline therapy for germ cells to incorporate heritable modifications. Thus, the present invention also relates to transgenic organisms (e.g., animals) that comprise (i.e., contain) a nucleic acid of SEQ ID NO: 2, 6, or 9, or a variant thereof; a vector that comprises a nucleic acid of SEQ ID NO: 2, SEQ ID NO: 6, or 9, or a variant thereof; or a peptide of SEQ ID NO: 1, or a peptide having at least 90% identity to SEQ ID NO: 1, such as the peptide of SEQ ID NO: 5. Thus, the present invention also relates to host cells or transgenic organisms that contain a nucleic acid sequence encoding any of the peptides described herein, or a vector that allows its expression.

[0155] The following examples are provided to demonstrate and further illustrate certain preferred embodiments of the present invention and are not to be construed as limiting its scope. EXAMPLES

[0156] Example 1 Chemical synthesis Materials and Methods Peptide synthesis The peptide of SEQ ID NO:1 is produced using the native chemical ligation (NCL) method. Briefly, two short peptides are produced. A first 27 amino acid N-terminal peptide with the amino acid sequence CFPGQVAGTTRAQPSCVEASIVIQKWW (SEQ ID NO:3) is synthesized. A second 36 amino acid C-terminal peptide (containing a cysteine ​​residue at its N-terminal portion) with the amino acid sequence CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR (SEQ ID NO:4) is synthesized. The two peptides are ligated using a one-step ligation followed by a one-step purification.

[0157] mouse C57 / B16J mice (8-12 weeks old) were purchased from Charles River Laboratories. Mice of both sexes were used in all experiments. No differences were noticed between males and females, so the data for the two sexes were combined. Mice were kept under standard housing conditions (22°C, 40% humidity, 12-h light cycle, and free access to food and water). Particular efforts were made to minimize the number of mice used in this study and the stress and distress to which they were subjected. All experiments were performed in accordance with the European guidelines for the care and use of laboratory animals (Council Directive 86 / 609 / EEC). All experimental procedures were approved by an independent ethical committee for animal experiments (APAFIS) as required by French law and in accordance with the relevant institutional regulations of the French law for animal experimentation. All experiments were performed in accordance with the ARRIVE guidelines.

[0158] Pain model 1: Spatial 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 as a neuropathic pain model. The SNI model consists of the transection of the tibial branch of the sciatic nerve and the common peroneal nerve; the sural nerve remains intact. The latter then develops signs of neuropathic pain accompanied by substantial mechanical allodynia. The SNI model has a number of advantages: - Neuropathic pain is persistent, which makes it possible to understand the phenomenon of habituation upon repeated injections of the peptide. - The pain experienced is robust. - This model is highly repeatable.

[0159] Mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP) and the left sciatic nerve was exposed under aseptic conditions. The distal trifurcation of the sciatic nerve was identified and the tibial and common peroneal branches were ligated with polypropylene nonabsorbable 6-0 suture (Ethicon); 1 mm was clipped and the sural branch was left intact. The wound was closed with sutures and the animals were allowed to recover and returned to their cages.

[0160] Pain Model 2: Foot incision – post-operative pain. Paw incision surgery was performed as described by Brennan and coworkers (1999) (Brennan, 1999). Mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP) and a longitudinal incision was made through the skin and fascia of the right hind paw. Using forceps, the flexor digitorum brevis muscle was elevated longitudinally and an incision was made with a scalpel through the muscle to cut it into two halves. The wound was closed with sutures and the animals were allowed to recover and returned to their cages. Paw incision was used as a postoperative pain model.

[0161] Pain model 3: Carrageenan injection – inflammatory pain. We 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 mice using a Hamilton syringe. Carrageenan injection was used as an inflammatory pain model.

[0162] Von Frey Test Von Frey uses Von Frey (VF) hairs or fibers, which are small pieces of nylon rods approximately 50 mm in length, to test rodent sensitivity to mechanical stimuli. In this test, the animal stands on an elevated mesh platform and a Von Frey hair is inserted through the mesh to impale the animal's hind paw. Normal responses for the animal include paw withdrawal or licking or shaking. An "up-down" Von Frey method is used to determine the mechanical force required to elicit a paw withdrawal response in 50% of the animals. Herein, mice were placed in a plastic chamber on a wire mesh grid and stimulated with von Frey filaments (Bioseb) by the "up-down" method (45), starting with a 1 g filament and using 0.04 and 4 g filaments as cutoffs.

[0163] statistical analysis Results are expressed as mean ± SEM. Statistical analysis was performed using Prism 7 (Graphpad Software, La Jolla, Calif., USA).

[0164] result Pain model 1: Spatial nerve injury (SNI) model - neuropathic pain. 1.1 Analgesic effect of peptide of SEQ ID NO: 1 administered subcutaneously The purpose of these experiments was to evaluate the analgesic effect of a peptide of sequence number 1 according to the present invention (identified herein as "TT1") by subcutaneous injection in a spared nerve injury (SNI) model resulting in neuropathic pain.

[0165] The experiment was carried out on 8-week-old male WT C57B16 mice. Three groups of 8 mice were used. The peptide of SEQ ID NO: 1 was resuspended in 0.9% NaCl at [0.6 mg / mL]. The peptide was used in this study at a concentration of 0.3 mg / kg.

[0166] 0.9% NaCl solution is used as a negative control (vehicle) and pregabalin 5 mg / kg, a well-known drug used to treat neuropathic pain, is used as a positive control.

[0167] After measuring the basal threshold of the mouse using the Von Frey (VF) filament with the up-down method, the establishment of the SNI model begins. The mouse is anesthetized, and the ligation of the tibial and peroneal nerves is performed, and then these two nerves are cut. The sural nerve, which remains intact, develops neuropathy very quickly. The occurrence of neuropathy is confirmed 3 days after the operation. A decrease in the response threshold to the Von Frey filament of the ipsilateral paw is thereby observed.

[0168] Seven days after surgery, the response thresholds are measured again, followed by subcutaneous injections of 100 μl / 10 g of each of the peptide solution, vehicle and pregabalin solution in a blinded experimenter manner.

[0169] Response thresholds are measured 1, 2, 4 and then 24 hours after injection.

[0170] At 7 days after SNI, all mice show a dramatic decrease in mechanical threshold (Figure 1). Subcutaneous injection of the peptide of SEQ ID NO: 1 (referred to as "peptide" or "TT1") induced a strong increase in response (i.e., paw withdrawal) threshold as early as 1 hour after administration, reaching a maximum increase at 2 hours, with values ​​comparable to pregabalin. Statistical analysis shows a strong significant result (with a p-value < 0.001) at 2 hours compared to the negative control.

[0171] 1.2 Analgesic effect of orally administered peptide of SEQ ID NO:1 The administration of peptide, vehicle and pregabalin used the same protocol as described in 1.1, except that for peptide and vehicle, oral administration was used and was performed 14 days after surgery.

[0172] For oral administration, the peptide of SEQ ID NO:1 was diluted at a concentration of 30 μg / mL in a solution of 1% w / v hydroxypropylmethylcellulose (Sigma-Aldrich, no. 423238, batch MKCD3665), 0.5% v / v Tween 80 (Euromedex, no. 2002-A, batch 100412 / 16S407), hereafter referred to as HPMC solution (vehicle).

[0173] Vehicle and peptide solutions were administered orally using two gavage needles. Pregabalin (5 mg / kg) was injected subcutaneously by a different experimenter, so the experimenter performing the VF measurements was blinded to the treatment (n=8 for each treatment). Administration was performed 14 days after surgery.

[0174] At 14 days after SNI, all mice show a dramatic decrease in mechanical threshold (Figure 2). Oral administration of vehicle had no effect. Oral administration of peptide of SEQ ID NO: 1 also induced a strong increase in response (i.e. paw withdrawal) threshold as early as 1 hour after administration, which slowly decreased up to 24 hours. This was significantly different compared to vehicle-treated mice up to 4 hours (p-values ​​< 0.001 at 1 and 2 hours and p-values ​​< 0.05 at 4 hours).

[0175] 1.3 Dose-dependent analgesic effect of orally administered peptide of SEQ ID NO:1 The peptide of SEQ ID NO:1 is resuspended in HMPC solution at five different concentrations (1, 5, 30, 90 and 180 μg / mL).

[0176] Von Frey filament measurements using the up-down method are performed to determine the baseline, as previously described. The installation of the SNI model is then started. Response thresholds are measured 14 days after surgery (day 14 - D14) to check for the development of neuropathic pain. Blind oral administration of 100 μl / 10 g peptide solution in five different concentrations (n=8 for 10 μg / kg; n=8 for 50 μg / kg; n=13 for 300 μg / kg, n=8 for 900 μg / kg, n=8 for 1,8 mg / kg) is then performed. Response thresholds are measured 1 h, 2 h, 4 h and then 24 h after oral administration. Pregabalin (5 mg / kg, n=6) and vehicle (n=9) were also administered by oral gavage.

[0177] At 14 days after SNI, all mice show a dramatic decrease in mechanical thresholds, indicating strong mechanical hypersensitivity, compared to baseline thresholds (Figure 3). Oral administration of vehicle had no effect. The analgesic effect of peptide SEQ ID NO: 1 was dose-dependent, with the maximum reversal effect on mechanical thresholds observed at 2 hours with a dose of 300 μg / kg. Interestingly, the two highest doses (0.9 and 1.8 mg / kg) produced the same effect at 2 hours, but also persisted for a longer period, with still significant effects at 4 hours after administration. The maximum reversal effect of pregabalin on mechanical thresholds is observed 2 hours after administration; this effect disappears completely at 4 hours after administration.

[0178] 1.4 Comparison of the analgesic effect of TAFA-4 (full-length protein) with the peptide of SEQ ID NO:1 administered subcutaneously or orally The inventors compared the analgesic effect of the peptide of the present invention with that of the prior art TAFA-4 (full length) protein. The same protocol as described in 1.1 and 1.2 was used, except that the administration of the peptide and TAFA-4 (full length) protein was performed 14 days after surgery.

[0179] Surprisingly, the peptides of the present invention maintain the same analgesic effect as the full-length TAFA-4 protein when administered subcutaneously (FIG. 4).

[0180] The present inventors also performed a comparison between TAFA-4 full-length protein and the peptide of the present invention when administered orally. The same experiment was performed using TAFA-4 full-length protein (TAFA-4 protein administration was performed 7 days after surgery). TAFA-4 full-length protein did not show significant analgesic effect when administered orally (i.e., per os) (Figure 5).

[0181] From the results shown in Figures 4 and 5, it can be concluded that the peptide of the present invention has a much better analgesic effect than TAFA-4 (full length) protein when orally administered.

[0182] In summary, Example 1 shows that the peptide of the present invention induces analgesic effect by subcutaneous and oral administration in SNI model (neuropathic pain model). Advantageously, the peptide of the present invention maintains its activity when administered subcutaneously, compared to TAFA-4 (full length) protein. Even more surprisingly, the peptide of the present invention shows better analgesic effect than TAFA-4 (full length) protein when administered orally.

[0183] Pain model 2: Carrageenan injection – inflammatory pain. The experiment was carried out on 8-week-old male WT C57B16 mice. Three groups of 8 mice were used. The peptide of SEQ ID NO: 1 is resuspended in HMPC solution at a concentration of 30 μg / mL, for an injection of 10 μl per gram.

[0184] Measurements were performed using Von Frey filaments with the up-down method to determine the baseline as described previously. Then, an intraplantar injection of 20 μl of carrageenan (1%) was performed in the hind paw. Response thresholds were measured 24 hours after injection (D1) and then blinded by the experimenter, oral administration of 0.3 mg / kg peptide solution (n=8) or vehicle solution (n=8) or subcutaneous injection of 20 mg / kg celecoxib solution (n=8). Celecoxib is a well-known drug used as a positive control. It is a COX-2 inhibitor and a nonsteroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation in several diseases. Response thresholds were measured 1, 2, 4 and 24 hours after administration of peptide or celecoxib.

[0185] After injection of carrageenan, the mice developed mechanical allodynia (see D1) (Figure 6). Subcutaneous administration of a positive control (celecoxib) causes an increase in the response threshold. Similarly, oral (per os) administration of the peptide of the invention also induced a statistically significant increase in the response threshold. Interestingly, the maximum analgesic effect of the peptide is obtained after 1 hour, whereas for celecoxib this takes 2 hours.

[0186] These results show that the peptides of the present invention induced analgesic effects via oral administration in the carrageenan model (inflammatory pain model). Advantageously, the peptides of the present invention had a rapid onset of analgesic effect.

[0187] Pain Model 3: Foot incision – post-operative pain. The experiment was carried out on 8-week-old male WT C57B16 mice. Three groups of 8 mice were used. The peptide of SEQ ID NO: 1 is resuspended in HMPC solution NaCl at a concentration of 30 μg / mL, for an injection of 10 μl per gram.

[0188] Measurements were performed using Von Frey filaments with the up-down method to determine the baseline, as previously performed. Paw incision surgery was then performed according to the protocol described above. Response thresholds were measured 24 hours after injection (D1), followed by blinded oral administration of 0.3 mg / kg peptide solution (n=8), vehicle solution (n=8) or 1 mg / kg morphine solution (positive control) (n=8). Response thresholds were measured 1, 2, 4 and 24 hours after administration.

[0189] After paw incision, mice developed mechanical allodynia (see D1 in FIG. 7). Oral administration of a positive control (morphine) caused an increase in response threshold. Similarly, oral administration of the peptide of the present invention also induced a statistically significant increase in response threshold 1 hour after administration, with the maximum effect occurring 2 hours after administration.

[0190] These results demonstrate that the peptide of the present invention caused an analgesic effect by oral administration in a paw incision model (postoperative pain model).

[0191] Conclusion: Taken together, our results show the analgesic effect of the peptide of SEQ ID NO: 1 in neuropathic, inflammatory and postoperative pain models. The mechanical allodynia (reduction in response threshold) induced by these models can be inhibited by subcutaneous injection or oral (per os) administration of the peptide. We also demonstrated that the analgesic effect of the peptide of SEQ ID NO: 1 is dose-dependent.

[0192] Advantageously, the peptides of the present invention are much easier to produce and obtain (and therefore cheaper) than the full-length TAFA-4 protein, yet maintain their analgesic activity when administered subcutaneously compared to the latter. Even more surprisingly and advantageously, when administered orally (per os), the peptides of the present invention show better analgesic effects compared to the TAFA-4 (full-length) protein, making them more convenient and suitable for administration to patients than the TAFA-4 full-length protein.

[0193] Example 2 Biological production of TT1 and its variant TT6 The inventors have produced the peptide of SEQ ID NO:1 ("TT1") and the peptide of SEQ ID NO:5 ("TT6") by recombinant pathways ("TT1 bioproduct" and "TT6 bioproduct") using the protocols described herein below.

[0194] The amino acid sequence of the peptide of SEQ ID NO:5 is 90.48% identical to the amino acid sequence of the peptide of SEQ ID NO:1.

[0195] Using the behavioral assay (pain model 1: SNI) described in Example 1, the inventors obtained confirmation that synthetically obtained TT1 and thus obtained biologically produced TT1 peptide have the same pain-relieving potency (see FIG. 8). They also obtained confirmation that both thus obtained biologically produced TT1 and TT6 peptides have the same pain-relieving potency as observed with synthetically obtained TT1 peptide (see FIG. 9).

[0196] material Plasmid pEt28A+, kanamycin resistance E. coli BL21(DE3)pLysS strain, competent, chloramphenicol resistant Recombinant 6xHis-TEV protease produced and purified in the AFMB lab (Academic MTA) NZY Auto-induction LB medium (powder): NZYTECH (MB17903) 2xYT broth (powder): MP Biomedicals (3012032) Sonicator, centrifuge, Petri dish (d 10cm) MF-Millipore membrane, cellulose ester, hydrophilic, 0.22 μm, 47 mm, white: MERCK HisTrap excel 5mL cartridge: Cytiva Imidazole buffer substance ACS. CAS 288-32-4: Millipore (MERCK) Dialysis tubing Zellu / Trans / ROTH T2: MWO 6000-8000, 50mm, 30m: CARL ROTH Affinity Chromatography Media, Chelating Sepharose™ Fast Flow: Cytiva AMICON Ultra-15 Centrifugal Filter Unit, 3K: Millipore Superdex S200 increase 10 / 300 GL column: Cytiva SDS-PAGE gel with 20% reticulation

[0197] method 1. Production of 6xHis-(TEV site)-TT1 (or 6xHis-(TEV site)-TT6) fusion proteins (identified herein as SEQ ID NOs: 7 and 8, respectively) a. Transformation of E. coli BL21(DE3)pLysS strain - Thaw the competent bacteria BL21(DE3)pLysS, which is resistant to chloramphenicol. - Add 1 μL of plasmid (1000 ng / μL) per 50 μL of competent bacteria - Keep on ice for 5 minutes - Perform heat shock: 37°C for 1 min - Keep on ice for 5 minutes - Add 500 μL of NZY autoinducing LB medium or 2YT broth medium (in sterile conditions) - Incubate at 37°C with agitation (200 rpm) for at least 30 minutes. - Heat the LB agar until it is completely dissolved. Mix and pour the following into two Petri dishes: 40mL melted LB agar 40 μL Kanamycin for plasmids 40 μL of chloramphenicol for strains Once solidified, spread the transformed bacteria at 50-100μL per dish.

[0198] b. Pre-incubation For a 500 mL culture: - 30mL of NZY autoinducing LB medium or 2YT broth medium - For plasmids, 30 μL Kanamycin - 30 μL of chloramphenicol for strains - 1 colony Incubate overnight at 37°C with agitation (200 rpm).

[0199] c. Culture - In a 2L Erlenmeyer flask: NZY autoinducing LB medium - 500mL of medium - 500 μL Kanamycin for plasmids - 500 μL chloramphenicol for stocks - Volume of pre-culture to reach an OD600nm of approximately 0.1 - Incubate at 37°C with agitation (200 rpm) until OD600nm reaches approximately 0.6-0.8. Incubate overnight at 17°C with agitation (200 rpm). Using 2YT broth medium - 500mL of medium - 500 μL Kanamycin for plasmids - 500 μL chloramphenicol for stocks - Volume of pre-culture to reach an OD600nm of approximately 0.1 - Incubate at 37°C with agitation (200 rpm) until OD600nm reaches approximately 0.6-0.8. - Add 500 μL of IPTG (1M stock solution) - Incubate overnight at 17°C with agitation (200 rpm) - Centrifuge at 5000 rpm for 20 minutes at room temperature - Resuspend the pellet in 50 mL of lysis buffer: Tris 50 mM pH 8.0, NaCl 300 mM, Imidazole 10 mM pH 8.0, Lysozyme 0.5 mg / mL - Flash freeze in liquid nitrogen - Store at -80℃

[0200] 2. Purification and cleavage of the fusion protein a. Cell lysis: total lysis - Thaw the stored pellet (resuspended in lysis buffer) at 37°C for 10 min. - Add DNAse 1 / 100 (v / v) (2 mg / mL stock solution) and MgSO4 1 / 200 (v / v) (2 M stock solution) - Shake / incubate for 20 minutes at 4°C - Sonicate for 6 minutes, e.g., 30 seconds on, 30 seconds off, 40% amplitude (beaker containing sample should be kept on ice) - Centrifuge at 20000 rpm for 40 minutes at 4°C - Filter the supernatant through a 0.22 μm filter membrane

[0201] b. Standard Immobilized Metal Chelation Chromatography (IMAC) (AKTA Purifier) - Equilibrate a HisTrap excel 5mL column with Buffer A: Tris 50mM pH8, NaCl 300mM, Imidazole 10mM pH8) at a flow rate of 3mL / min. - Load the supernatant and then wash the column with 20 CV, i.e. 100 mL of buffer A - The fusion protein is eluted with 10 CV, i.e. 50 mL of buffer B: Tris 50 mM pH 8, NaCl 300 mM, Imidazole 250 mM pH 8) - Measure the volume and OD280nm of the pooled elution fractions - Calculate the concentration and amount using A1%o=1.1UDO / mg at 280nm (if no disulfide bonds are present) - Using dialysis tubing with a cut-off of 6-8 kDa: Dialyze the elution pool overnight at 4°C against 2 L of buffer A: Tris 50 mM pH 8, NaCl 1 M, Imidazole 10 mM pH 8

[0202] c. Cutting - Measure the OD280nm of the dialyzed eluate - To ensure that cleavage occurs correctly, the eluate should be diluted to have a concentration between 0.5-0.7 mg / mL. - Add recombinant TEV (1 / 10 w / w) during elution - Incubate at room temperature for 4 hours - Incubate overnight at 4 °C. - Add recombinant TEV (1 / 20 w / w) during elution - Incubate at room temperature for 4 hours - Incubate overnight at 4 °C. - Centrifuge at 7500 rpm for 15 minutes at 4°C to remove any precipitates

[0203] 3. Purification of TT1 or TT6 a. Resin preparation For samples derived from 1 L bacterial culture: - 4mL of resin - 800 μL NiSO4 0.2M, and incubate for 15-30 minutes - Wash with 15 mL of H2O and centrifuge at 5000 rpm for 5 min at 4°C - Discard the supernatant - Wash with 15 mL of H2O and centrifuge at 5000 rpm for 5 min at 4°C - Discard the supernatant - Wash with 15mL of Tris 50mM pH8.0, NaCl 1M and centrifuge at 5000rpm for 5 minutes at 4°C - Discard the supernatant - Repeat the process until the liquid becomes clear. - Wash with 15 mL of Tris 50 mM pH 8.0, NaCl 1 M, Imidazole 10 mM pH 8.0, incubate for 10 min and centrifuge at 5000 rpm for 5 min at 4 °C - Discard the supernatant - Wash with 15 mL of Tris 50 mM pH 8.0, NaCl 1 M, Imidazole 10 mM pH 8.0, incubate for 10 min and centrifuge at 5000 rpm for 5 min at 4 °C - Discard the supernatant - Add 15mL of Tris 50mM pH 8.0, NaCl 1M, Imidazole 10mM pH 8.0 and incubate overnight at 4°C

[0204] b. "Flow-through" immobilized metal (Ni 2+ ) Immunochemical Chelation Chromatography (IMAC) (batch) - The cleaved samples are incubated with the pre-equilibrated resin for 2 hours at 4°C. - Transfer the resin / cleaved elution mixture to an empty column - Collect the flow-through containing only TT1 or TT6 (the remaining fusion protein - if present - and TEV remain on the column)

[0205] c. Concentration - Concentrate the flow-through using an AMICON 3K concentrator (3900 rpm, 4 °C) until a volume of 200-500 µL is obtained (smaller is better). - Centrifuge at 10,000 rpm for 5 minutes to remove any precipitate and transfer the supernatant to a clean tube.

[0206] d. Size-exclusion chromatography (SEC) (AKTA Purifier) - Equilibrate the Superdex S200 increase 10 / 300 GL column with the buffer Tris 50 mM pH 8.0, NaCl 1 M, Imidazole 10 mM pH 8.0 using a flow rate of 0.5 mL / min - Load and elute concentrated flow-through (TT1 and TT6 start to elute at approximately 19 mL) - Measure the volume and OD280nm of the pooled TT1 or TT6 fractions - Calculate the concentration and amount using A1%o=2,6UDO / mg at 280nm (if no disulfide bonds are present)

[0207] e. SDS-PAGE (20% reticulated gel, reducing conditions) - For each purification step and each elution peak: take 1-4 μg of protein, denature (SDS), reduce (DTT or 2 / beta-mercaptoethanol) the sample and store at 4°C or -20°C until final use. - Load and migrate onto SDS-PAGE gel (200V)

[0208] Example 3 Preventive analgesic effect of TT1 The purpose of this experiment was to evaluate whether the peptide of SEQ ID NO:1 ("TT1") also has a prophylactic analgesic effect in the paw incision model (as described above) by subcutaneous injection of TT1.

[0209] Materials and Methods TT1 or vehicle was administered subcutaneously twice a day at different time points: the day before surgery (D-1), 1 hour before and 1 hour after surgery (and awakening) on ​​day D, and on D+1 and D+2 (FIG. 10A). Mechanical threshold response measurements were performed on D+1 (before TT1 injection on D+1), D+2 (before TT1 injection on D+2) and D+3.

[0210] result After paw incision, mice treated with vehicle developed mechanical allodynia (see D1, D2 and D3 in FIG. 10B). In contrast, mice treated with TT1 did not develop mechanical allodynia. Compared with mice treated with vehicle, mice treated with TT1 showed statistically significant higher response thresholds at D1, D2 and D3.

[0211] These results indicate that the peptide TT1 of the present invention can be used prophylactically in a paw incision model (postoperative pain model).

[0212] Example 4 Absence of tolerance following repeated oral administration of TT1 in the CCI neuropathic pain model The purpose of this experiment was to evaluate whether subjects treated with the peptide of SEQ ID NO:1 ("TT1") were able to induce tolerance to this peptide.

[0213] Materials and Methods CCI neuropathic pain model: Chronic constriction injury (CCI) was performed as previously described by Bennett and Xie 1988 (A peripheral mononeuropathy in rats that produces disorders of pain sensation like those in man. Pain, vol. 33:87-107). Briefly, unilateral peripheral mononeuropathy was induced in mice anesthetized with Ketamine / Xylasine (100 mg / kg and 10 mg / kg ip, respectively) with two loose ligatures (6-0 Monocryl, Ethicon) tied around the common sciatic nerve (approximately 1 mm apart). The nerve was constricted just enough to be barely noticeable so that circulation via the epineural vasculature was not interrupted.

[0214] After the establishment of the above CCI neuropathic pain model, mice were treated daily with peptide of SEQ ID NO:1 ("TT1") or vehicle for 14 consecutive days, starting 10 days after surgery (D10). The analgesic effect of TT1 was determined every 2 days until 24 days after surgery (D24).

[0215] result Subcutaneous injection of peptide of TT1 induces a strong increase in response (i.e. paw withdrawal) threshold, demonstrating the analgesic effect of TT1.Interestingly, the analgesic effect of TT1 remains constant throughout the entire duration of treatment (14 days), without any decrease in TT1 potency, even at D24.These results show that peptide TT1 of the present invention can be used repeatedly for a long period of time without inducing tolerance in subjects.

[0216] References [Table 1] [Table 2]

Claims

1. An isolated, synthetic or recombinant peptide of the sequence SEQ ID NO:1, or a peptide having at least 90% identity to SEQ ID NO:

1.

2. 2. The peptide of claim 1, wherein the peptide having at least 90% identity to SEQ ID NO: 1 is capable of modulating the excitability of spinal interneurons (preferably spinal cord layer IIi interneurons).

3. array 【Chemistry 1】 3. The peptide of claim 1 or 2, wherein the amino acids Q and Y appearing in bold in (SEQ ID NO: 1) remain unchanged in the amino acid sequence of said peptide having at least 90% identity to SEQ ID NO:

1.

4. 3. The peptide of claim 1 or 2, wherein the peptide is a recombinant peptide.

5. 3. The peptide of claim 1 or 2, wherein the peptide is a peptide of SEQ ID NO:

5.

6. 6. A composition comprising a peptide according to any one of claims 1 to 5 for use as a medicament.

7. A nucleic acid sequence encoding the peptide of any one of claims 1 to 5.

8. A vector allowing the expression of a peptide according to any one of claims 1 to 5.

9. 9. A cell comprising the nucleic acid sequence of claim 7 or modified using the vector of claim 8.

10. 10. A composition comprising a peptide according to any one of claims 1 to 5, a nucleic acid according to claim 7, a vector according to claim 8 or a cell according to claim 9, and a dietarily or pharmaceutically acceptable support.

11. 11. The composition of claim 10, further comprising at least one further active compound, preferably an active agent effective against pain, even more preferably an SAID, NSAID or opioid drug.

12. 12. A composition according to claim 6 or a composition according to claim 10 or 11 for use as a medicament, wherein the peptide or composition is administered to a subject intramuscularly, intravenously, intraperitoneally, orally (per os), anally, cutaneously, subcutaneously, cutaneously, transdermally or intrathecally, preferably subcutaneously or orally, even more preferably orally.

13. 13. The composition of claim 6, 10, 11 or 12 for use as a medicament, wherein the dose of the peptide is between 1 μg / kg / day and 100 mg / kg / day in mammals, preferably between 2.5 μg / kg / day and 0.6 mg / kg / day in human subjects.

14. 10. A composition comprising a peptide according to any one of claims 1 to 5, a nucleic acid according to claim 7, a vector according to claim 8 or a cell according to claim 9 for use as an active ingredient for preventing or treating pain in a subject in need thereof.

15. 15. The composition of claim 14, wherein the pain is chronic pain, neuropathic pain, post-operative pain, inflammatory pain, hyperalgesia, or allodynia.

16. The composition of claim 14 or 15, wherein the subject is a mammal, in particular a human.

17. The composition of claim 16, wherein the subject has at least one mutated allele in the myo1A gene.

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