Novel CD47-activating proteins

Abstract

The present invention relates to novel CD47-activating proteins derived from TSP-1 and to the use thereof for treating inflammatory disorders such as age-related macular degeneration.

Description

[0001] DESCRIPTION

[0002] TITLE: Novel CD47 Activating Proteins

[0003] The present invention relates to CD47 activating proteins and their use for the treatment of inflammatory disorders such as age-related macular degeneration.

[0004] Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. The disease presents with an early form characterized by accumulations of lipoprotein debris called drusen and pseudodrusen below and above the retinal pigment epithelium (RPE). The disease can subsequently progress to an early intermediate form and then to one of two clinical forms of debilitating late-onset AMD:

[0005] - the exudative form (wet or neovascular AMD) which develops rapidly if left untreated, defined by choroidal neovascularization (CNV) or intraretinal neovascularization (Retinal angiomatous proliferation) and

[0006] - the slower-developing atrophic form, characterized by photoreceptor degeneration and RPE atrophy. It is also known as the geographic form (or geographic atrophy, GA, or "dry" AMD) (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002).

[0007] Although AMD is often classified into "atrophic" and "wet" forms, mixed forms exist. An "atrophic" form can also develop into a "wet" form and vice versa. Both forms develop with common manifestations such as increased activation of innate immunity and are associated with the same polymorphisms such as those of complementary factor H, and a haplotype of chromosome 10q26 defined by SNPs near the Serine Protease High Temperature Requirement Al (HTAR1) and Age-Related Maculopathy Susceptibility 2 (ARMSD2) genes (Fritsche et al., 2016, 10.1038 / ng.3448).

[0008] Regardless of the stage and form of the disease, it is characterized by the accumulation of mononuclear phagocytes (PMs) in the photoreceptor layer, the subretinal space (SRS), and the choroid. PMs constitute a group of cells that includes monocytes (Mos), resident macrophages (rM4>), such as microglial cells (MCs) for the central nervous system (CNS), and monocyte-derived inflammatory macrophages (MdMs). The choroid of patients with age-related macular degeneration (AMD) and surgically removed neovascular membranes are also infiltrated by lymphocytes, including IL17+ T cells. Patients with atrophy are also characterized by an increased number of degranulated (activated) choroidal mast cells (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002).

[0009] The subretinal space, located between the RPE and the outer segments of the photoreceptors, is an area of ​​immune privilege mediated by RPE immunosuppressive signals, notably FasL (CD95L), known for its ability to induce leukocyte death. However, microparticles (MPs) accumulate in the subretinal space in the intermediate and two advanced forms of age-related macular degeneration (AMD) that threaten vision (Klein et al., 2004, 10.1016 / j.ajo.2003.11.069). They are then in close contact with the RPE during choroidal neovascularization and near RPE lesions in geographic atrophy (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002). In animal models, MPs contribute to CCN and photoreceptor degeneration in AG (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002).Subretinal microparticles (MPs) have also been shown to be present in and around soft drusen, which are a significant risk factor for developing late-onset AMD (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002). However, the reasons for the impaired subretinal immunosuppression and MP accumulation in AMD remain poorly understood.

[0010] AMD is associated with chronic (also referred to as "non-resolving") and low-grade inflammation which mainly involves the innate immune system and more specifically the accumulation of MP (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002).

[0011] The major role of inflammation is the body's response to tissue injury and microbial invasion. Ideally, it rapidly and efficiently eliminates pathogens and repairs tissue damage through either regeneration or scarring. If the inflammatory response is not rapidly controlled, it can become pathogenic and contribute to disease progression, as occurs in chronic inflammatory diseases. Chronic low-grade inflammation is observed in contexts such as metabolic diseases (obesity, atherosclerosis), neurodegenerative diseases, and cancers, and contributes significantly to pathogenesis (Nathan et al., 2010, 10.1016 / j.cell.2010.02.029).Indeed, while chronic low-grade inflammation is not the triggering cause of these diseases, it contributes significantly to their pathogenesis because the microbicidal mediators produced by neutrophils and macrophages (reactive oxygen species, proteases and inflammatory cytokines...) can cause considerable collateral damage to host cells, a phenomenon which itself maintains inflammation.

[0012] Therefore, there is a need to identify active ingredients to prevent and / or treat chronic low-grade inflammation, particularly inflammation associated with the accumulation of mononuclear phagocytes, and especially age-related macular degeneration (AMD). As part of their work on AMD, the inventors specifically studied the role of thrombospondin-1 (TSP-1).

[0013] Thrombospondin-1 (TSP-1) is a multimeric glycoprotein located on the cell surface and in the extracellular matrix (ECM). It belongs to the thrombospondin family, which consists of five members, subdivided into two groups based on their oligomerization domain and size. TSP-1 and TSP-2 form homotrimeric molecules composed of three 145 kDa protein chains. TSP-3 and TSP-5 are comparatively shorter and associate into homo- or heteropentameric molecules (Lawler et al., 1986 J Cell Biol 103(5): 1635-1648; Bornstein, 2001, 10.1172 / JCI12749).

[0014] As illustrated in Figure IA, TSP-1 (SEQ. ID. No. 1) is composed of the following domains:

[0015] - the heparin-binding domain (HBD) in the N-terminal region which interacts with cell surface heparan sulfate proteoglycans and low-density lipoprotein receptor-like protein (LRP1, CD91);

[0016] - a spiral oligomerization sequence, known as "coiled-coil", comprising two highly conserved cysteine ​​residues that form disulfide bonds between the monomers of TSP-1 in its trimerized structure (Tan et al., 2006, 10.1016 / j.str.2005.09.017);

[0017] - a von Willebrand factor (vWC) type C domain; - three "type 1" repeats including regions capable of binding to the CD36 receptor and the latent TGF|3-bound LAP peptide (LTBP) allowing TSP-1 to release an active form of TGF|3;

[0018] - three repeats of "type 2" including a region involved in the activation of phospholipase Cy (PLCy), it has been shown that a more active mutant of this protein was associated with auto-inflammatory pathologies (Koss et al., 2014, 10.1016 / j.tibs.2014.09.004);

[0019] - the "type 3" repeats comprising 7 DxDxDGxxDxxD motifs (Kvansakul et al., 2004, 10.1038 / sj.emboj.7600166) and including several calcium binding sites;

[0020] - The cell-binding domain (CBD) in the C-terminal region of TSP-1 contains two valine-valine-methionine (WM) sequences, each of which can interact with a CD47 receptor. Efficient activation of CD47 by TSP-1 requires the presence of both WM sites (McDonald et al., 2003, 10.1021 / bi0341408). CD47 is known to play a key role in immune and angiogenic responses. In particular, the binding of TSP-1 to CD47 influences several fundamental cellular functions, including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in the regulation of angiogenesis and lymphocyte clearance (Housset et al., 2015, 10.1089 / jop.2015.0023).

[0021] TSP-1, CD47, CD36, and LRP1 are all expressed on the vascular endothelium, RPE, and macrophages, the three cell types that play an important role in chorioretinal inflammation in diseases such as autoimmune uveitis (Fordham et al., 2012, 10.1038 / srep00512), but also in the pathogenesis of AMD (Brown et al., 2001 Trends Cell Biol 11(3): 130-135; Hollborn et al., 2004 Invest Ophthalmol Vis Sci 45(6): 2033-2038; Houssier et al., 2008, 10.1371 / journal.pmed.0050039; Calippe et al., 2017, 10.1016 / j.immuni.2017.01.006; Lavalette et al., 2019, 10.3389 / fimmu.2019.03032; Beguier et al., 2020, 10.1016 / j.immuni.2020.07.021).

[0022] In the outer retina, TSP-1 is one of the main factors contributing to immunosuppression in the subretinal space and thus to immune privilege.

[0023] Thbsl mice exhibit increased and prolonged subretinal inflammation after experimental induction of chorioretinitis with light- and laser-induced lesions (Ng et al., 2009, 10.1167 / iovs.08-2877; Chen et al., 2012, 10.1016 / j.ajpath.2011.09.020; Soriano-Romani et al., 2022, 10.3390 / ijms23105705; Touhami et al., 2022, 10.3390 / ijms23020681). This inflammation is similar to that occurring in a variety of peripheral diseases (extensive acute pneumonia, leukocytosis, pancreatitis, and inflammatory infiltrates in the lacrimal glands) (Lopez-Dee et al., 2011, 10.1155 / 2011 / 296069). Interestingly, Cd β^ mice, but not Cd36~ β, develop an age- and light-induced accumulation of subretinal macrophages similar to that of Thbsl β mice (Calippe et al., 2017, 10.1016 / j.immuni.2017.01.006). Furthermore, Thbsl β and Cc / 47 MC _ / transferred into the subretinal space of wild-type mice significantly resist the elimination observed with wild-type MCs (24h post-injection) and recombinant TSP-1 significantly accelerates the elimination of wild-type MCs, reverses the phenotype of Thbsl MCs but has no effect on Cd MCs, showing that the interaction of TSP-1 and CD47 is a mediator of the elimination of subretinal macrophages (Calippe et al., 2017, 10.1016 / j.immuni.2017.01.006) whose chronic accumulation is associated with some early and advanced forms of AMD (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002). However, several data sources suggest that TSP-1 / CD47 signaling is altered in AMD and associated with a non-resolving subretinal accumulation of MP in intermediate and advanced forms (wet AMD and geographic atrophy, AG) (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002)). It has also been shown that CD47 transcription (Augustin et al., 2023, 10.1186 / sl2974-023-02699-9) and TSP-1 immunostaining (Uno et al., 2006, 10.1136 / bjo.2005.074005) are reduced in AMD.

[0024] Therapeutically, recombinant TSP-1 has been shown to reduce subretinal inflammation and associated neovascularization (Calippe et al., 2017, 10.1016 / j.immuni.2017.01.006; Touhami et al., 2022, 10.3390 / ijms23020681) and photoreceptor degeneration (Ju et al., 2022, 10.1111 / bph.15303). Specific activation of CD47 using a TSP-1 CD47-binding peptide derivative has also been previously shown to have a similar effect (Calippe et al., 2017, 10.1016 / j.immuni.2017.01.006). On the other hand, CD36 deletion, rather than activation, significantly attenuates retinal inflammation and associated degeneration in mice (Lavalette et al., 2019, 10.3389 / fimmu.2019.03032). Similarly, CD47 activation decreases VCAM-1 expression and leukocyte adhesion, while CD36 activation increases them in uveitis models (Soriano-Romani et al., 2022, 10.3390 / ijms23105705).Furthermore, TGFR and PLCy activation could lead to deleterious fibrosis (Meng et al., 2016, 10.1038 / nrneph.2016.48) and increased inflammation (Liu et al., 2009, 10.1074 / jbc.M809198200; Koss et al., 2014, 10.1016 / j.tibs.2014.09.004), respectively.

[0025] The Inventors have previously proposed using particular derivatives of TSP-1 to treat low-grade chronic accumulation and inflammation (WO2017 / 194586).

[0026] The Inventors have now developed TSP-1-derived proteins, called tTSPs, which contain the CD47 activation C-terminal domain but lack the type 1 and type 2 repeat domains involved in the activation of CD36, TGFR and PLCy.

[0027] Their results show that these TSP-1 derived proteins are secreted, trimerized and suppress the expression of Osteopontin (OPN), which is known from previous research to be increased in AMD patients carrying the 10q26 risk haplotype, by human monocytes in vitro, in a manner similar to CD47 activating peptides described by Calippe's team (Calippe et al., 2017, 10.1016 / j.immuni.2017.01.006).

[0028] The Inventors have also shown that AAV2-mediated intravitreal transfection of tTSPs reduces MP infiltration in photosensitive Cx3crl mice, inhibits leukocyte infiltration and choroidal neovascularization in the laser-induced injury model, and highly significantly inhibits photoreceptor degeneration in albino mice subjected to weekly light exposure, repeated over several months.

[0029] These results, detailed in the experimental section that follows, demonstrate that gene therapy with a protein according to the invention can safely inhibit pathogenic inflammation in chorioretinal disease for prolonged periods.

[0030] Thus, the present invention relates to TSP-1 derived proteins, called tTSP, which mediate the elimination of mononuclear phagocytes via its receptor CD47 without activation of CD36, TGFR and PLCy, and their use in the treatment of pathologies involving chronic inflammation, such as age-related macular degeneration.

[0031] The term "protein" refers to a sequence of more than 100 amino acids and / or a multimeric entity. The proteins of the invention are not limited to a specific product length. The term "protein" does not exclude post-transcriptional modifications of the protein, for example, glycosylation, acetylation, phosphorylation, and others, as well as other modifications known in the art, whether of natural or artificial origin.

[0032] An "isolated protein" is a protein that has been identified and separated and / or recovered from a component of its natural environment.

[0033] The term "derived from", before a designated protein (e.g. TSP-1), refers to the origin of the protein.

[0034] The term "amino acid" refers to the 20 naturally occurring amino acids; these amino acids can be modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other non-proteinogenic amino acids, including 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. The term "amino acid" includes both D- and L-amino acids (stereoisomers).

[0035] The term "amino acid substitution" refers to the replacement in a polypeptide of one amino acid with another amino acid.

[0036] In one embodiment, an amino acid may be replaced by another amino acid having similar structural and / or chemical properties, for example, conservative amino acid substitutions. A "conservative amino acid substitution" may be made based on a similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged amino acids (acids) include aspartic acid and glutamic acid.Non-conservative substitutions involve exchanging an amino acid from one class for an amino acid from another class. For example, replacing an amino acid from one group (e.g., polar) with another amino acid from a different group (e.g., a basic group). Amino acid substitutions can be achieved using well-established genetic or chemical methods. Genetic methods include site-directed mutagenesis, PCR, gene synthesis, and similar techniques. It is also considered that methods for modifying the side chain of an amino acid by means other than genetic engineering, such as chemical modification, could be useful.

[0037] DETAILED DESCRIPTION

[0038] The present invention thus relates to a protein derived from TSP-1, called tTSP, comprising a formula (I):

[0039] X1-L1-X2 (I) where

[0040] - XI comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, of identity with SEQ. ID. No. 2 and necessarily includes a cysteine ​​at position 5 of SEQ. ID. No. 2;

[0041] - X2 comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, identity with SEQ ID No. 3 and necessarily comprises two WM fragments, one at positions 1-3 and the other at positions 87-89 of SEQ ID No. 3; and

[0042] - L1 is absent or a spacer peptide comprising between 1 and 100 amino acids; said TSP-1 derived protein being devoid of the type 1 repeat domain naturally present between positions 361 and 470 of native SEQ TSP-1. ID. No. 1.

[0043] The present invention also relates to a protein derived from TSP-1, called tTSP, of formula (I):

[0044] X1-L1-X2 (I) where

[0045] - XI comprises a sequence having at least 95%, and in order of preference 98%, 99%, and 100%, identity with SEQ ID No. 2 and necessarily includes a cysteine ​​at position 5 of SEQ ID No. 2; - X2 comprises a sequence having at least 95%, and in order of preference 98%, 99%, and 100%, identity with SEQ ID No. 3 and necessarily includes two WM fragments, one at positions 1-3 and the other at positions 87-89 of SEQ ID No. 3; and

[0046] - L1 is absent or a spacer peptide comprising between 1 and 100 amino acids; said TSP-1 derived protein being devoid of the type 1 repeat domain naturally present between positions 361 and 470 of native SEQ TSP-1. ID. No. 1.

[0047] The present invention also relates to a protein derived from TSP-1, called tTSP, comprising a formula (I):

[0048] X1-L1-X2 (I) where

[0049] - XI comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, of identity with SEQ. ID. No. 2 and necessarily comprises two cysteines, one at position 1 and the other at position 5 of SEQ. ID. No. 2;

[0050] - X2 comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, identity with SEQ ID No. 3 and necessarily comprises two WM fragments, one at positions 1-3 and the other at positions 87-89 of SEQ ID No. 3; and

[0051] - L1 is absent or a spacer peptide comprising between 1 and 100 amino acids; said TSP-1 derived protein being devoid of the type 1 repeat domain naturally present between positions 361 and 470 of native SEQ TSP-1. ID. No. 1.

[0052] The present invention finally relates to a protein derived from TSP-1, called tTSP, of formula (I):

[0053] X1-L1-X2 (I) where

[0054] - XI comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, of identity with SEQ. ID. No. 2 and necessarily comprises two cysteines, one at position 1 and the other at position 5 of SEQ. ID. No. 2;

[0055] - X2 comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, identity with SEQ ID No. 3 and necessarily comprises two WM fragments, one at positions 1-3 and the other at positions 87-89 of SEQ ID No. 3; and

[0056] - L1 is absent or a spacer peptide comprising between 1 and 100 amino acids; said TSP-1 derived protein being devoid of the type 1 repeat domain naturally present between positions 361 and 470 of native SEQ TSP-1. ID. No. 1.

[0057] According to a particular embodiment, said tTSP protein, derived from TSP-1, is also devoid of all or part of the type 2 repeat domain present between positions 471 and 672 of native SEQ TSP-1. ID. No. 1.

[0058] SEQ. ID. No. 2 corresponds to the fragment comprising the amino acids located from position 252 to position 295 of TSP-1 of SEQ. ID. No. 1.

[0059] According to one embodiment, XI comprises all or part of the fragment comprising the amino acids located from position 7 to position 290 of TSP-1 of SEQ. ID. No. 1, this fragment is designated SEQ. ID. No. 4.

[0060] According to one embodiment, XI comprises all or part of the fragment comprising the amino acids located from position 7 to position 290 of TSP-1 of SEQ. ID. No. 1, said fragment including all or part of SEQ. ID. No. 2. Preferably, XI comprises all or part of the fragment comprising the amino acids located from position 7 to position 290 of TSP-1 of SEQ. ID. No. 1, said fragment including all or part of SEQ. ID. No. 2 and the cysteine ​​at position 5 of SEQ. ID. No. 2.

[0061] According to one embodiment, XI comprises all or part of the fragment comprising the amino acids located from position 7 to position 290 of TSP-1 of SEQ. ID. No. 1, said fragment including SEQ. ID. No. 2 and the two cysteines of positions 1 and 5 of SEQ. ID. No. 2. Preferably, XI is a fragment of 50 to 360 amino acids, or of 75 to 300 amino acids, or of 100 to 150 amino acids.

[0062] According to this embodiment, XI can for example have a sequence having at least 95%, and in order of preference 98%, 99% and 100%, of identity with SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7 or even SEQ. ID. No. 8.

[0063] According to a preferred embodiment, XI has a sequence having at least 95%, and in order of preference 98%, 99% and 100%, of identity with SEQ. ID. No. 6.

[0064] SEQ. ID. No. 3 corresponds to the fragment comprising the amino acids from position 1019 to position 1107 of the native TSP-1 of SEQ. ID. No. 1. In one embodiment, X2 comprises all or part of the fragment comprising the amino acids from position 628 to position 1152 of the native TSP-1 of SEQ. ID. No. 1, said fragment including SEQ. ID. No. 3 and the two WM fragments present at positions 1-3 and 87-89 of SEQ. ID. No. 3; this fragment is designated SEQ. ID. No. 9.

[0065] Preferably, X2 is a fragment of 100 to 600 amino acids, for example 150 to 500 amino acids or even 200 to 300 amino acids.

[0066] The term "identity" refers to a measure of the identity of nucleotide or amino acid sequences. Generally, the sequences are aligned to achieve the highest order match. The notion of sequence "identity" has a meaning known to those skilled in the art and can be calculated using published techniques; see, for example: Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds, Humana Press, New Jersey, 1994. Various methods exist for measuring the identity or similarity between two polynucleotide or polypeptide sequences (Carillo and Lipton, SIAM J Applied Math, 1988, 48:1073).

[0067] If the spacer peptide L1 is absent, then XI and X2 are linked by a peptide bond.

[0068] According to one embodiment, the spacer peptide has a length of 3 to 50 amino acids, preferably 4 to 20 amino acids, more preferably 5 to 15 amino acids.

[0069] Examples of spacer peptides, without limitation, include Gly-rich spacers, such as poly-Gly spacers, Ser-rich spacers, spacers comprising Gly and Ser residues (also called "GS spacers"), Pro-rich spacers, and helical spacers.

[0070] In one embodiment, the amino acids of the spacer peptide are chosen from among 20 natural amino acids. In a preferred embodiment, amino acids 1 to 20 are chosen from Gly, Ala, Pro, Asn, Gin, Cys, Lys.

[0071] In one particular embodiment, the spacer peptide consists of a majority of sterically unhindered amino acids, such as Gly, Gly-Gly [(Gly)2], Gly-Gly-Gly [(Glyjî]... (Gly o, Ala, Gly-Ala, Ala-Gly, Ala-Ala, etc.). Other specific examples of linker elements are: (Gly)3Lys(Gly)4, (Gly AsnGlySerfGly (this structure provides a glycosylation site when produced by recombination in a mammalian cell system capable of glycosylating such sites), (Gly)3Cys(Gly)4, and GlyProAsnGly. In one particular embodiment, the spacer peptide is Gly-Gly-Gly-Gly-Gly-Gly-Gly. In another particular embodiment, the spacer peptide is a combination of Gly and d'Ala. In another particular embodiment, the spacer peptide is a combination of Gly and Lys.

[0072] According to one embodiment, the TSP-1 derived protein is chosen from the proteins of SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 12 or SEQ. ID. No. 13; preferably, the TSP-1 derived protein has SEQ. ID. No. 11.

[0073] According to one embodiment, the TSP-1 derived protein has SEQ. ID. No. 10.

[0074] In one embodiment, the TSP-1 derived tTSP protein of the invention is present as a multimer, preferably as a dimer or trimer, preferably as a trimer.

[0075] According to one embodiment, the protein according to the invention is obtained by protein synthesis methods or by recombinant DNA techniques well known to those skilled in the art.

[0076] According to a particular embodiment, the protein according to the invention is recombinant. The term "recombinant protein" refers to a protein produced by DNA recombination techniques. An example of such techniques is the case where the DNA encoding the expressed protein is inserted into a suitable expression vector, which is in turn used to transform a host cell to produce the DNA-encoded protein.

[0077] The TSP-1-derived protein of the invention may have modifications that make it more stable in an organism or more readily enters cells. Such modifications include, but are not limited to, N-terminal modification, C-terminal modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH, or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are described, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[0078] In one embodiment, the peptide bonds (-CO-NH-) within the peptide can be substituted, for example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)HC-OOC(R)-N-), ketomethylene bonds (-CO-CH2-), α-α-α bonds (-NH-N(R)-CO-) in which R represents any alkyl, for example a methyl, carbon-nitrogen bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), double bonds (-CH=CH-), amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), in which R is the "normal" side chain, naturally present on the carbon atom of the amino acid in question. These changes can occur at any of the bonds along the peptide chain.

[0079] In one embodiment, the natural aromatic amino acids Trp, Tyr and Phe can be replaced by non-natural synthetic acids such as phenylglycine, TIC, naphthylanine (Nol), methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

[0080] In one embodiment, the proteins of the invention may also be covalently or non-covalently associated with a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer; a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide. Other possible carriers include one or more water-soluble polymer attachments such as polyethylene glycol or polypropylene glycol. Other polymers still useful known in the technique include monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g. glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.In a preferred embodiment, the support is polyethylene glycol (PEG).

[0081] In one embodiment, the PEG group can have any suitable molecular weight and can be straight-chain or branched-chain.

[0082] In one embodiment, the average molecular weight of the PEG is preferably between approximately 2 kDa and approximately 100 kDa, more preferably between approximately 5 kDa and approximately 50 kDa, and most preferably between approximately 5 kDa and approximately 10 kDa. In one embodiment, the PEG groups are attached to the compounds of the invention via acylation, reductive alkylation, Michael addition, thiol alkylation, or other chemoselective conjugation / ligation methods via a reactive group on the PEG fragment (e.g., an aldehyde, amino, ester, thiol, ct-haloacetyl, maleimido, or hydrazino group) to a reactive group on the target compound (e.g., an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazino group).

[0083] In one embodiment, the carbohydrate groups (oligosaccharides) are attached to sites known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues, while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the Asn-X-Ser / Thr sequence, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids, excluding proline. The structures of N- and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar commonly found in both is N-acetylneuraminic acid (called sialic acid). Sialic acid is usually the terminal residue of oligosaccharides bound to both N and O and, due to its negative charge, can confer acidic properties to the glycosylated compound.Such sites can be incorporated into the linker of the compounds of this invention and are preferably glycosylated by a cell during the recombinant production of the polypeptide compounds (for example, in mammalian cells such as CHO, BHK, COS). However, these sites can also be glycosylated by synthetic or semi-synthetic procedures known in the art.

[0084] The present invention also relates to a polynucleotide or nucleic acid encoding a protein derived from TSP-1 as described above. In one embodiment, the polynucleotide or nucleic acid is DNA.

[0085] The term "polynucleotide" refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotides" include, without limitation, single- and double-stranded DNA, or a mixture of single- and double-stranded DNA. The term can also refer to triple-stranded regions comprising RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA whose backbone is modified for increased stability or other reasons. Examples of "modified" bases include tritylated bases and unusual bases such as inosine.Various modifications can be made to DNA and RNA; thus, "polynucleotide" encompasses the chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.

[0086] In another embodiment, the polynucleotide of the invention is, for example, RNA in the form of messenger RNA (mRNA), possibly modified.

[0087] For example, the polynucleotide according to the invention can be chosen from the polynucleotides of SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 17 or SEQ. ID. No. 18 encoding respectively for the TSP-1 derived proteins of SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 12 or SEQ. ID. No. 13.

[0088] According to another embodiment, the TSP-1 derived protein according to the invention is expressed in situ by the cells of the organ to which they are to be administered for gene therapy purposes.

[0089] According to this embodiment, the polynucleotide encoding the TSP-1-derived protein according to the invention may be DNA or RNA, optionally modified, or may be cloned into a suitable expression vector such as a plasmid or a viral vector. When the polynucleotide encoding the TSP-1-derived protein according to the invention is in the form of DNA or RNA, optionally modified, it is preferably encapsulated in a synthetic vector such as a lipid nanoparticle (LNP); lipid vectors (liposomes) consisting of vesicles formed by lipids that can encapsulate optionally modified DNA or RNA encoding the TSP-1-derived protein according to the invention and that fuse with cell membranes to deliver the genetic material into the cells;polymer vectors such as cationic polymers (like polyethyleneimine, PEI) which are used to form complexes with DNA or RNA encoding the TSP-1 derived protein according to the invention and which can be internalized by cells; nanoparticles often made of gold, silica, or polymers, which can be used to transport and deliver genetic material into cells; peptide vectors such as cell-penetrating peptides (CPPs) which can be used to transport genetic material encoding the TSP-1 derived protein according to the invention into cells by crossing cell membranes; or dendrimer vectors which are branched polymers which can encapsulate genetic material encoding the TSP-1 derived protein according to the invention and deliver it into cells.

[0090] Thus, another object of the present invention is an expression vector comprising one or more polynucleotides encoding the TSP-1 derived protein according to the invention.

[0091] According to one embodiment, the expression vector is a plasmid or a viral vector comprising an expression cassette composed of a promoter, a polynucleotide encoding the TSP-1 derived protein according to the invention and a terminator.

[0092] According to another embodiment which applies more specifically to the administration of the TSP-1 derived protein according to the invention to the eye and in particular to the retina, the expression vector is a recombinant viral vector which can be chosen from lentiviruses, adenoviruses and adeno-associated viruses (AAVs) possibly transformed to reduce their immunogenicity.

[0093] Preferably, the viral vector is a recombinant adeno-associated virus (AAV).

[0094] AAVs have a protein coat that surrounds and protects a small, single-stranded DNA genome of approximately 4.8 kilobases (kb). AAVs belong to the parvovirus family and rely on co-infection with other viruses, primarily adenoviruses, for replication. Initially distinguished by serology, molecular cloning of AAV genes has led to the identification of hundreds of unique AAV strains in numerous species. Its single-stranded genome contains three genes: Rep (replication), Cap (capsid), and Aap (assembly). These three genes give rise to at least nine genetic products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are necessary for genome replication and packaging.

[0095] Thus, in the case of the use of AAV, the expression cassette encoding the TSP-1 derived protein according to the invention is placed between two ITRs.

[0096] Recombinant AAV (rAAV), lacking viral DNA, is essentially a protein-based nanoparticle designed to cross the cell membrane, where it can circulate and deliver its DNA cargo into the cell nucleus. In the absence of Rep proteins, ITR-flanked transgenes encoded in rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells.

[0097] More than 30 natural AAV serotypes are available. Numerous natural variants of the AAV capsid exist, allowing for the identification and use of AAVs with properties specifically suited to ocular cells. AAV viruses can be modified using conventional molecular biology techniques, enabling optimization of these particles for cell-specific delivery of nucleic acid sequences, minimizing immunogenicity, adjusting particle stability and lifespan, ensuring efficient degradation, precise delivery to the nucleus, and more.

[0098] Thus, the expression of the TSP-1 derived protein according to the invention can be obtained in retinal cells through the administration of recombinantly designed AAVs or artificial AAVs containing sequences encoding the TSP-1 derived protein according to the invention.

[0099] Various transfection techniques can be implemented for the administration of the polynucleotide encoding the TSP-1-derived protein or the expression vector according to the invention; a person skilled in the art will choose the most suitable technique depending on the form of the polynucleotide chosen:

[0100] - Direct injection of naked DNA or RNA: this method involves directly injecting DNA into targeted tissues (such as muscle or liver). Efficiency is often low, but it can be increased by other methods such as electroporation or binding to other molecules; possibly supplemented by iontophoresis or electroporation: after the DNA injection, electrical pulses are applied to increase the permeability of the cell membrane, thus facilitating the entry of genetic material into cells;

[0101] - transfection for liposomes (when injected in vivo, they fuse with cell membranes, allowing the entry of genetic material into cells), nanoparticles and viral vectors;

[0102] - Hydrodynamic injection: This technique involves the rapid injection of a large volume of solution containing expression vectors into the bloodstream. This method is often used to specifically target the liver; - Sonoporation: This method uses ultrasound which, in the presence of microbubbles, can temporarily disrupt cell membranes, facilitating the entry of expression vectors into cells;

[0103] - magnetofection: this method uses magnetic nanoparticles associated with the expression vector, which are guided towards target cells by an external magnetic field.

[0104] Another object of the invention is a composition comprising a TSP-1 derived protein or a polynucleotide encoding the TSP-1 derived protein or an expression vector according to the invention as described above.

[0105] In particular, the composition according to the invention may be a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

[0106] The term "pharmaceutically acceptable excipient" refers to an excipient that does not produce an adverse, allergic, or other adverse reaction when administered to an animal, preferably a human. It includes all solvents, dispersing media, coatings, antibacterial and antifungal agents, isotonic agents, absorption retardants, and so on. A pharmaceutically acceptable carrier or excipient refers to a nontoxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type. For human administration, preparations must meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biology standards.

[0107] Pharmaceutically acceptable excipients that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial mixtures of saturated vegetable fatty acid glycerides, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (for example, sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool grease.Preferably, the composition of the invention comprises a therapeutically effective amount of the TSP-1 derived protein or the polynucleotide encoding the TSP-1 derived protein or the expression vector according to the invention.

[0108] The term "therapeutically effective amount" refers to the level or quantity of an agent to be administered, without causing significant adverse or adverse effects on the target, to (1) delay or prevent the onset of inflammation; (2) slow or stop the progression, worsening, or deterioration of one or more symptoms of inflammation; (3) cause improvement in the symptoms of inflammation; (4) reduce the severity or incidence of inflammation; or (5) cure inflammation. A therapeutically effective amount may be administered before the onset of inflammation, for prophylactic or preventive action. Alternatively, or in addition, the therapeutically effective amount may be administered after the onset of inflammation, for therapeutic action or to maintain therapeutic action.

[0109] In one embodiment, the composition of the invention further comprises an additional preventive and / or therapeutic agent.

[0110] According to one embodiment, said additional preventive and / or therapeutic agent is another agent intended for the treatment of inflammation, in particular AMD.

[0111] It will be understood that the total daily use of the compound of the invention, the composition, the pharmaceutical composition, and the medicinal product of the present invention will be determined by the attending physician using sound medical judgment. The specific therapeutically effective dose level for a particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, body weight, general health, sex, and diet; the timing of administration, the route of administration, and the rate of excretion of the specific compound used; the duration of treatment; medications used in combination or concurrently with the specific compound used; and similar factors well known in the medical arts.For example, it is possible to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is obtained. The daily dosage of the products can vary within a wide range, from approximately 10 to approximately 10,000 mg per adult per day, preferably from 100 to approximately 5,000 mg, and more preferably from approximately 200 to approximately 2,000 mg per adult per day. Preferably, the compositions contain 10, 50, 100, 250, 500, 1,000, and 2,000 mg of the active ingredient for symptomatic dose adjustment to the patient being treated. A medicinal product typically contains from approximately 10 to approximately 10,000 mg of the active ingredient, preferably from 5 to approximately 5,000 mg, and more preferably from approximately 10 to approximately 2,000 mg of the active ingredient.An effective amount of the drug is usually provided at a dosage level ranging from 0.01 mg / kg to about 100 mg / kg of body weight per day, preferably about 0.05 mg / kg to 40 mg / kg of body weight per day, preferably still about 0.1 mg / kg to 20 mg / kg of body weight per day, more preferably about 0.2 mg / kg to 1 mg / kg of body weight per day.

[0112] The compositions of the present invention can be administered orally, parenterally, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, or by spraying or inhalation.

[0113] The term administration used here includes subcutaneous, intravenous, intramuscular, intraocular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0114] In one embodiment, the composition of the invention is in a form suitable for oral administration. Examples of forms suitable for oral administration include, but are not limited to, tablets, orodispersible tablets, effervescent tablets, powders, granules, pills (including coated tablets), capsules (including soft capsules), syrups, liquids, gels or other oral solutions, suspensions, gruels, liposomal forms and the like.

[0115] In one embodiment, the composition of the invention is in a form suitable for topical administration. Examples of forms suitable for topical administration include, but are not limited to, liquid, paste, or solid compositions, and more particularly in the form of aqueous solutions, drops, eye drops, ophthalmic solutions, dispersions, sprays, microcapsules, micro- or nanoparticles, polymer patches, or controlled-release patches. In a preferred embodiment, the composition of the invention is in the form of eye drops.

[0116] In one embodiment, the composition of the invention is in a form suitable for injection, such as, for example, for intraocular, intramuscular, subcutaneous, intradermal, transdermal, or intravenous injection or infusion. Examples of forms suitable for injection include, but are not limited to, solutions such as sterile aqueous solutions, dispersions, emulsions, suspensions, and solid forms suitable for use in preparing solutions or suspensions by adding a liquid prior to use, such as powder, liposomal forms, and the like. The sterile injectable forms of the compositions of this invention may be aqueous or oily suspensions. These suspensions may be formulated using techniques known in the art, employing suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic diluent or solvent acceptable for parenteral administration. Acceptable vehicles and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectable products, as are pharmaceutically acceptable natural oils, such as olive oil or castor oil, particularly in their polyoxyethylated forms.These oily solutions or suspensions may also contain a long-chain alcoholic diluent or dispersant, such as carboxymethylcellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifying or bioavailability-enhancing agents, commonly used in the manufacture of solid, liquid, or other pharmaceutically acceptable dosage forms, may also be used for formulation purposes. In one particular embodiment, the composition of the invention is in a form suitable for intraocular administration, preferably intraocular injection.For the purposes of this invention, "intraocular administration" means an injection directly into the eye, where "inside the eye" means any area within the eyeball, and generally includes, but is not limited to, all functional (e.g., vision-related) or structural tissues found within the eyeball, or the tissues or cell layers that partially or completely line the inside of the eyeball. Specific examples of such areas include the anterior chamber, posterior chamber, vitreous cavity, choroid, macula, and retina, as well as the blood vessels and nerves that supply blood or innervate a posterior ocular region or site.

[0117] In one embodiment, the interior of the eye means the posterior segment of the eye, including the posterior chamber, the vitreous cavity, the choroid, the macula and the retina, as well as the blood vessels and nerves that vascularize or innervate a posterior ocular region or site.

[0118] According to this embodiment, intraocular administration refers to administration into the posterior segment of the eye, preferably into the vitreous body, and intraocular administration is preferably an intravitreal injection.

[0119] In one embodiment, the composition of the invention comprises one or more pharmaceutically acceptable carriers for a formulation suitable for injection. In one embodiment, the composition of the invention is administered to the subject as required at least once a day, for example, once a day, twice a day, or three times a day.

[0120] The TSP-1-derived proteins according to the invention can be designated as CD47 activators or CD47 agonists.

[0121] For the purposes of this invention, the term "activator" means that the agent is capable of activating the biological activity of the target protein, directly or indirectly; in particular, a CD47 activating agent is one capable of activating the biological activity of CD47 directly or indirectly. As used herein, the term "CD47 agonists" refers to peptides capable of binding to the CD47 receptor and activating it to produce its biological activity, namely the elimination of mononuclear phagocytes. The present invention further relates to a TSP-1-derived protein, a polynucleotide, an expression vector, or a composition according to the invention for use in the prevention and / or treatment of inflammation.

[0122] For the purposes of this invention, "inflammation" is understood, as defined in Dorland's Medical Dictionary, as a localized response, triggered by injury or tissue destruction, that serves to destroy, dilute, or isolate both the harmful agent and the injured tissue, or caused by the accumulation of debris (such as, but not limited to, drusen). It is characterized by activation of resident macrophages, increased permeability of the microvascular system, and possibly leakage of blood components into the interstitial spaces and migration of leukocytes into the inflamed tissue. Macroscopically, it may be accompanied by familiar clinical signs of erythema, edema, hyperalgesia (tenderness), and pain, which may also be absent, as can be the case in chronic inflammation in age-related macular degeneration (AMD) or atherosclerosis.

[0123] In one embodiment, the TSP-1 derived protein, polynucleotide, expression vector, or a composition according to the invention are intended to be used in the treatment of inflammation, said inflammation being selected from the group comprising age-related macular degeneration (AMD), age-related maculopathy, uveitis, retinitis pigmentosa, Parkinson's disease, multiple sclerosis, Alzheimer's disease, obesity, atherosclerosis, allergies, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis, or psoriatic arthritis), asthma, graft-versus-host disease, peritonitis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome, lupus erythematosus, nephritis, and ulcerative colitis.

[0124] In one embodiment, the inflammation of the invention is acute inflammation. In another embodiment, the inflammation of the invention is chronic inflammation.

[0125] In one embodiment, the inflammation of the invention is a low-grade chronic inflammation that can be selected from the group including age-related diseases such as age-related macular degeneration (AMD) and age-related maculopathy; metabolic diseases, such as obesity and atherosclerosis; neurodegenerative diseases and cancers.

[0126] In one embodiment, the TSP-1-derived protein of the invention is intended for use in the treatment of inflammation associated with the accumulation of mononuclear phagocytes. Mononuclear phagocytes (MPs) comprise a family of cells including microglial cells (MCs), monocytes (Mos), and macrophages (Mcs).Inflammation associated with the accumulation of mononuclear phagocytes includes, but is not limited to, retinal inflammation, such as age-related macular degeneration (AMD), age-related maculopathy, uveitis, or retinitis pigmentosa; neurodegenerative diseases, such as Parkinson's disease, multiple sclerosis, or Alzheimer's disease; metabolic disorders, such as obesity or atherosclerosis; allergies; ankylosing spondylitis; arthritis, such as osteoarthritis, rheumatoid arthritis, or psoriatic arthritis; asthma; graft-versus-host disease; peritonitis; Crohn's disease; colitis; dermatitis; diverticulitis; fibromyalgia; hepatitis; irritable bowel syndrome; systemic lupus erythematosus; and nephritis. and ulcerative colitis.

[0127] In one embodiment, the inflammation according to the invention is chosen from the group comprising retinal inflammations, such as age-related macular degeneration (AMD), uveitis, retinitis pigmentosa or age-related maculopathy; neurodegenerative diseases, such as Parkinson's disease, multiple sclerosis or Alzheimer's disease; metabolic disorders, such as obesity or atherosclerosis.

[0128] In one embodiment, the inflammation according to the invention is non-autoimmune inflammation. Examples of non-autoimmune inflammatory diseases include, but are not limited to, renal, hepatic and pulmonary inflammation, atherosclerosis and metabolic syndrome, Behçet's disease and endometriosis.

[0129] In one embodiment, the inflammation according to the invention is autoimmune inflammation. Examples of autoimmune inflammatory diseases include, but are not limited to, uveitis, rheumatoid arthritis, systemic lupus erythematosus, celiac disease, scleroderma, psoriasis, inflammatory bowel disease, and Sjögren's syndrome. In one embodiment, the inflammation according to the invention is ocular inflammation. As used here, an inflammatory eye disease is inflammation affecting any part of the eye or surrounding tissues. Accordingly, an inflammation developing in the eyes, or in the optic nerve, blood vessels, muscles, or other tissues surrounding the eye, the resulting disease is an inflammatory eye disease.

[0130] In one embodiment, ocular inflammation is selected from the group comprising or consisting of age-related macular degeneration (AMD), uveitis, retinitis pigmentosa, age-related maculopathy, uveitis, scleritis, episcleritis, optic neuritis, keratitis, orbital pseudotumor, retinal vasculitis, and chronic conjunctivitis.

[0131] In one embodiment, the inflammation according to the invention is retinal inflammation.

[0132] For the purposes of this invention, "retinal inflammation" means inflammation of the retina or subretinal space, mediated by mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils. In one embodiment, the retinal inflammation of this invention includes age-related macular degeneration (AMD), age-related maculopathy, uveitis, and retinitis pigmentosa.

[0133] In one particular embodiment, age-related macular degeneration includes atrophic (or dry) AMD and neovascular (or wet) AMD.

[0134] In one embodiment, AMD is in an early stage. The early stage is characterized by the accumulation in and around the macula of extracellular deposits called drusen, associated with pigmented spots (alterations of the pigment epithelium).

[0135] In another embodiment, AMD is in an advanced stage. The advanced stage is characterized by unilateral or bilateral complications. Advanced AMD may be atrophic or wet AMD. In a particular embodiment, AMD is a late stage of the dry form of AMD (also called geographic AMD).

[0136] Non-infectious uveitis (UNI) is an eye disease in which the inflammatory response is often triggered by an autoimmune response against a self-antigen and an aberrant innate immune response triggered by microbes or danger signals (Caspi, 2010, 10.1172 / JCI42440; Mattapallil et al., 2015, 10.1167 / iovs.15-17280). This disease accounts for approximately 10% of all cases of total blindness. Corticosteroids are the mainstay of UNI treatment. However, some patients are refractory to corticosteroid-only therapy, and long-term treatment is associated with a high rate of side effects.In the UNI animal model, experimental autoimmune uveitis (UAE), tissue damage is associated with the presence of autoreactive T lymphocytes against retinal antigens, with a secondary infiltration of pro-inflammatory MPs largely involved in the tissue destruction observed in this pathology (Caspi, 2010,10.1172 / JCI42440; Mattapallil et al., 2015,10.1167 / iovs.l5-17280).

[0137] In one embodiment, the subject is affected by inflammation, preferably by inflammation associated with an accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils.

[0138] The term "subject" refers to a mammal, preferably a human. The term "mammal" refers to any mammal, including humans, domestic and farm animals, as well as zoo, sporting or companion animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.

[0139] In one particular embodiment, the subject suffers from retinal inflammation. In another embodiment, the subject is likely to develop inflammation, that is, to develop an accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils.

[0140] In one particular embodiment, the subject is at risk of developing retinal inflammation. In a preferred embodiment, the subject is at risk of developing age-related macular degeneration (AMD), age-related maculopathy, or retinitis pigmentosa. Examples of risk factors for developing AMD and age-related maculopathy include, but are not limited to, heredity, lifestyle factors such as smoking, sun exposure, or an unbalanced diet, age, high blood cholesterol, high blood pressure, etc. In one embodiment, the subject of the invention is an elderly person. As used herein, the term "elderly person" means that the subject is at least 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old.

[0141] In one particular embodiment, the subject is at risk of developing AMD due to the presence of a variant of complement factor H (CFH) and a minor haplotype (mH) of chromosome 10q26 explain the majority of the genetic risk of age-related macular degeneration (AMD) (Fritsche et al., 2016, 10.1038 / ng.3448). The AMD-associated mH haplotype differs from the common non-risk haplotype (cH) by several single nucleotide polymorphisms (SNPs) linked in a non-coding DNA sequence (Merle et al., 2023, 10.1016 / j.preteyeres.2022.101154), and increases the risk of developing early and late forms of AMD up to 10 times in homozygous mH (mH / mH) carriers (Fritsche et al., 2016, 10.1038 / ng.3448).These non-coding SNPs are located within the age-related maculopathy susceptibility 2 (ARMS2) gene and the HTRA1 (High-Temperature Requirement A Serine Peptidase 1) serine peptidase promoter, and are close to the gene encoding Pleckstrin Homology domain containing family A member 1 (PLEKHA1).

[0142] The present invention further relates to a method of treating inflammation in a subject in need, comprising administering to said subject a therapeutically effective amount of the TSP-1 derived protein, polynucleotide, expression vector or a composition of the invention.

[0143] In one embodiment, the method of the invention is intended for the treatment of inflammation associated with an accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils.

[0144] In a preferred embodiment, the method of the invention is intended for the treatment of age-related macular degeneration.

[0145] Another object of the present invention is a method for inducing CD47 activity in a subject requiring it, comprising administering to the subject an effective amount of the TSP-1 derived protein, polynucleotide, expression vector or composition of the invention.

[0146] Another object of the present invention is a method for eliminating the accumulation of mononuclear phagocytes in a subject in need, comprising administering to said subject a therapeutically effective amount of the TSP-1 derived protein or polynucleotide, expression vector or composition of the invention.

[0147] Another object of the present invention is a method for eliminating the accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils, thereby treating the inflammation associated with the accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils in a subject in need, comprising administering to said subject a therapeutically effective amount of the TSP-1 derived protein, polynucleotide, expression vector or composition of the invention.

[0148] The present invention also relates to a kit comprising at least one TSP-1 derived protein or a polynucleotide or an expression vector or a composition according to the invention.

[0149] In one embodiment, the kit of the invention further includes means for administering the TSP-1 derived protein or the polynucleotide or expression vector, or the composition of the invention to a subject in need thereof.

[0150] In one embodiment, the kit of the invention further includes instructions for carrying out this administration to said subject.

[0151] In one embodiment, the components of the kit of the invention can be administered separately, sequentially, simultaneously or in chronological order.

[0152] In one embodiment, the kit of the invention is used to treat (or to be used in the treatment of) inflammation.

[0153] FIGURES

[0154] [Fig 1] Structure and sequence of TSP-1 and tTSP

[0155] (A) Schematic representation of the TSP-1 protein domains and their respective ligands;

[0156] (B) Amino acid sequence of native TSP-1. The latency-associated peptide (LAP) binding sequences, CD36 and CD47, are underlined; in this figure, amino acid numbering begins 18 amino acids before the start of the protein as described in SEQ ID No. 1;

[0157] (C) Schematic representation of the protein domains of TSP-1, tTSP8, and tTSP12-derived proteins and their respective ligands. (D) Amino acid sequence of native TSP-1, first- and second-generation tTSP8 and tTSP12. The LAP, CD36, and CD47 binding sequences are highlighted.

[0158] [Fig 2] The tTSPs are secreted and trimerized

[0159] (A) Anti-TSP1 Western blot analysis of cell extracts and supernatants from human embryonic kidney cells (HEK 293) transfected two days earlier with 10 pg / ml of a blank plasmid, a plasmid encoding a 2nd generation tTSP8 with an N-terminal 6HIS-tag (6HIS-2 nd tTSP8) or a plasmid encoding a tTSP8 of 2 ème generation with a 6HIS-tag C-terminal (2 nd tTSP8-6HIS).

[0160] (B) Anti-flagM2 Western blot analysis of supernatants from HEK293 cells transfected two days earlier with 10 pg / ml of a plasmid encoding first-generation tTSP8 or native TSP-1, both containing an N-terminal FLAG. Half of the supernatants were incubated with the crosslinking agent bissulfosuccinimidyl suberate (BS3).

[0161] (C) Anti-TSP1 Western blot analysis of HEK 293 cell supernatants, cultured in a 24-well plate, transfected 7 days earlier with AAV2 virions encoding the 1st and 2nd generation of tTSP8 and tTSP12, the number of which is 150,000 or 75,000 as shown in the figure.

[0162] (D) Anti-TSP1 Western blot analysis of HEK 293 cell supernatants cultured in a 24-well plate, transfected 7 days earlier with 75,000 AAV2tTSP8 virus genomes (vg) / well or 75,000 AAV2.7m8-tTSP8 virions / well encoding the 2 ème tTSP8 generation. The WB was carried out under reduced and non-reduced conditions.

[0163] [Fig 3] tTSP inhibits osteopontin secretion by HTRA1-stimulated human monocytes

[0164] Quantification of osteopontin (OPN) concentrations by ELISA in supernatants of primary human monocytes incubated for 30 min with HTRA1 (5 pg / mL, R&D Systems), which induces OPN, followed by an 18-hour incubation with supernatants of HEK 293 cells transfected two days prior with 10 pg / ml of a blank plasmid or a plasmid encoding tTSP8. ème generation (n=4 independent wells, Mann-Whitney: HEK supernatant untransfected versus transfected by tTSP8 *p=0.0286).

[0165] [Fig 4] tTSP inhibits retinal MP activation in hyperinflammatory Cx3crl mice GFP / GFP / n vivo. (A) Schematic representation of the experiment: Cx3crl mice GFP / GFP and AP0E*2KI, aged two to three months, received an intravitreal injection of 5xl0 9 d'AAV2 blank control (cAAV) in the left eye and 5xl0 9 of AAVtTSP8 or AAVtTSP12 of the èregeneration in the right eye. After 14 days, the mice were adapted to darkness for 6 hours, pupils dilated and exposed to green LED light (from 2 a.m., 4500 Lux, JP Vezon equipment) for 4 days then maintained in normal 12h / 12h cyclic setup conditions for 6 days.

[0166] (B and C) Quantification of subretinal mononuclear phagocytes on choroid / RPE and retinal flat mounts stained by immunohistochemistry with anti-IBAl antibodies from Cx3crl mice GFP / GFP exposed to light (B) and APOE*2KI mice (C) (B: ​​n = 10 mice Wilcoxon paired test: cAAV versus AAV2tTSP8 *p=0.0176; C: n=7 mice Wilcoxon paired test: cAAV versus AAV2tTSP12 *p=0.0078).

[0167] (D) Schematic representation of the experiment: Cx3crl GFP / GFP Two- to three-month-old children received an intravitreal injection of 5x10 9AAV2 empty in the left eye and 5xl0 9 of AAVtTSP8 (first generation) in the right eye. After 14 days, the mice were adapted to darkness for 6 hours, with dilated pupils, and exposed to green LED light (from 2:00 AM, 4500 Lux, JP Vezon equipment) for 4 days, then maintained under normal 12h / 12h cyclic setup conditions for 3 days. The animals were exposed to three light exposure cycles and GFP + Retinal MPs were visualized in anesthetized and dilated animals using a Micron 4 background camera.

[0168] (E) Fundus images of the GFP + MPs of the right and left eyes of the 9 Cx3crl mice GFP / GFP . IVT: intravitreal injection, MP: phagocyte mononuclear cells, AAV: adenovirus-associated virus; Scale bar = 100 pm.

[0169] [Fig 5] tTSP inhibits MP accumulation and choroidal neovascularization in the laser injury model.

[0170] (A) Schematic representation of the experiment: Two- to three-month-old male C57BL6 / J mice received an intravitreal injection of 5xl0 9 AAV2 empty in the left eye and 5xl0 9 AAVtTSP8 or AAVtTSP12 (first generation) was injected into the right eye. After 14 days, six laser impacts for FACS analysis and four laser impacts for flat-panel evaluation were applied to each eye of anesthetized and dilated mice. Ten days after laser injury, the mice were sacrificed, and their posterior pole was analyzed by flow cytometry and flat-panel immunohistochemistry.

[0171] (B and C) Flow cytometry analysis of immune cells: CD45 lymphocytes + , CDllb + Ly6G neg MP, CD45 + CDllb neg Ly6G neg CDllb microglial cells + CD45 |OW Ly6G neg , CDllb infiltrating macrophages + CD45 high Ly6G negand CDllb monocytes + CD45 high Ly6G neg Ly6C high eyes injured by laser ten days before, as shown (n = 10 mice per group; nonparametric Mann-Whitney p-values ​​are shown in the graph).

[0172] (D) Quantification of subretinal mononuclear phagocytes IBAl + on flat mounts of RPE / choroid and retinal stained by immunohistochemistry with anti-IBA1 antibodies from mice injured with laser 10 days before (n = 32 and 28 impacts for eyes injected with cAAV and AAVtTSP8; Mann-Whitney cAAV versus AAVtTSP8 *p = 0.0325; n = 24 impacts for eyes injected with cAAV and AAVtTSP8; Mann-Whitney cAAV versus AAVtTSP8 *p<0.0001).

[0173] (E) Schematic representation of the experiment: two- to three-month-old male C57BL6 / J mice received an intravitreal injection of 5xl0 9 AAV2 empty in their left eyes and 5xl0 9d'AAVtTSP8 de 2 e generation in their right eyes. After 14 days, four laser impacts were applied to each eye of the anesthetized and dilated mice. In addition, the mice were exposed to 10% O2 hypoxia from day 3 to day 10. On day 10, subretinal infiltration of MP and CNV were quantified on flat mounts of RPE / choroid doubly labeled by immunohistochemistry with anti-IBAl and anti-CD102 antibodies.

[0174] (F) Quantification of subretinal MPs IBAl + and CD102 + CNV on double-labeled RPE choroidal flat mounts (n = 32 and 28 impacts for eyes injected by cAAV and AAVtTSP8; Mann-Whitney cAAV versus subretinal MP AAVtTSP8 *p<0.0001; CD102+ CNV size*p=0.0117).

[0175] [Fig 6] tTSP inhibits experimental autoimmune uveitis.

[0176] (A) Schematic representation of the experiment: 2-month-old female C57BL6 / J mice received a bilateral intravitreal injection of PBS containing pluronic acid (PBS / PA), or 5xl0 9 of AAV2tTSP8 in PBS / PA (n = 15 mice / group). The AAV2tTSP8 virus encoded for the 2 ème tTSP8 generation. After 14 days, EAU was induced by subcutaneous injection of 200 pg of interphotoreceptor retinoid-binding protein (IRBP)65i-67o emulsified in incomplete Freund's adjuvant and by intraperitoneal injection of 1 pg of purified Bordetella pertussis toxin. Clinical signs of EAU were assessed after ten and twelve days by fundus examination and cytometric analysis of retinal immune cells twelve days after EAU induction.

[0177] (B) Representative fundus images of PBS control eyes and AAV2tTSP8 injected eyes on day 12 and quantification of clinical retinal inflammation score on days 10 and 12. Each point represents one retina (n = 30 retinas / group, Mann-Whitney p-values ​​for group comparisons are shown in the figure).

[0178] (C) Flow cytometry analysis of CD45 immune cells + CD45 helper T cells + CDllb négatives CD3 + CD4 + CD45 microglial cells + CDllb + CD45 |OW Ly6G neg and monocyte-derived macrophages (MdM) CD45 high CDllb + Ly6G neg twelve days after induction of UNI (n = 30 retina / group, Mann-Whitney p-values ​​for group comparisons are shown in the figure).

[0179] [Fig 7] tTSP inhibits photoreceptor degeneration in albino mice exposed to repeated minor injuries.

[0180] (A) Schematic representation of the experiment: 2-month-old BALB / c mice received a bilateral intravitreal injection of either pluronic acid-containing PBS (PBS / PA) or 5xl0 9 of AAV2tTSP8 in PBS / PA (n = 5 mice / group). The viruses encoded for the 2 èmetTSP8 generation. After 14 days, mice were adapted to darkness for 6 hours, dilated, and exposed to green LED light for 8 hours, and then maintained under normal 12h / 12h cyclic setup conditions. Mice were exposed in groups of three per lamp, one mouse from each group, to minimize variability in exposure between groups. Seven days after light exposure, the thickness of the photoreceptor nuclear layer (outer nuclear layer) was measured by optical coherence tomography (OCT). After sacrifice on day 7, the retina was analyzed for the presence of immune cells by cytometry.

[0181] (B) Thickness measurements of the outer nuclear layer (ONL) composed of photoreceptor nuclei (n = 10 retinas / group; the p-value of a Mann-Whitney test for group comparison is shown in the figure). (C) Flow cytometry analysis of CD45 immune cells +: CD45 microglial cells + CDllb + CD45 |OW Ly6G neg and monocyte-derived macrophages (MdM) CD45 + CDllb + Ly6G neg seven days after exposure to light. The number of lymphocytes and neutrophils was negligible (n = 8 retinas / group, the p values ​​of the Mann-Whitney tests for group comparisons are shown in the figure).

[0182] (D) Schematic representation of the experiment: 2-month-old BALB / c mice received a bilateral intravitreal injection of PBS containing pluronic acid (PBS / PA), with 5xl0 9 of AAV2tTSP8 in PBS / PA, or with 5xl0 9 of AAV2.7m8-tTSP8 in PBS / PA (n = 8 mice / group). The viruses encoded for the 2 èmetTSP8 generation. After 14 days, mice were adapted to 6 hours of darkness, dilated, and exposed to green LED light once a week, and then maintained under normal 12h / 12h cyclic setup conditions. Mice were exposed in groups of three per lamp, one mouse from each group, to minimize variability in exposure between groups. To induce progressive photoreceptor degeneration, exposure time was slowly increased over time, starting at 2 hours in week 1 and increasing to 18 hours in week 15. Photoreceptor nuclear layer thickness (outer nuclear layer) was measured by OCT and averaged for the left and right eyes every 14 days.

[0183] (E) Thickness measurements of the outer nuclear layer (ONL) composed of photoreceptor nuclei over 15 weeks. Asterisks indicate times when the Mann-Whitney PBS p-value relative to AAV2tTSP8 is p < 0.05. Three graphs at weeks 1, 9, and 15 show the distribution of values ​​for each group.

[0184] (F) Schematic representation of the experiment: Two-month-old female BALB / c mice (five per group) were bilaterally injected with either PBS containing pluronic acid (PBS / PA, 0.001% Pluronic) or with 5 x 10 9vg of AAV2-tTSP8 in PBS / PA (second generation of tTSP8). Two weeks after intravitreal injection, BALB / c mice were exposed to intense white LED light (6000 Lux). Illumination was applied for 4 hours the first week and 6 hours the second week, followed by two weeks of 12 h / 12 ​​h light / dark cycles. (G) Quantification of A-wave amplitudes measured by electroretinography under scotopic conditions in mice treated with AAV2-tTSP8 or PBS (n = 5 mice / group; Mann-Whitney p-values ​​are shown in the figure).

[0185] [Fig 8] tTSP inhibits the accumulation of mononuclear phagocytes, RPE and photoreceptor degeneration, and restores photoreceptor function in the sodium iodate (NalO3) induced retinal degeneration / RPE model.

[0186] (A) Schematic representation of gene therapy experiments with NalO3.

[0187] (B) Flow cytometry analysis of CD45 microglial cells + CDllb + CD45 med Ly6G“ and monocyte-derived macrophages (MdM) CD45 high CDllb + Ly6G“, four days after NalO3 injections (n= 9 eyes / group; p values ​​from paired Wilcoxon tests are shown in the figure).

[0188] (C) Quantification of A-wave amplitudes measured by electroretinography under scotopic conditions in mice treated with AAV2-tTSP8 and PBS in the contralateral eye. Mice that did not develop NalO3-induced RPE degeneration were excluded (n = 7 mice / group; paired Wilcoxon p-values ​​are shown in the figure).

[0189] (D) Representative images of phalloidin-stained RPE / choroid flat mounts, 17 days after NalO3 injections. Quantification of RPE atrophy and cone segment loss areas on RPE and retinal flat mounts (n = 12 mice / group; paired Wilcoxon p-values ​​are shown in the figure).

[0190] (E) Comparison of the protective effect of an empty virus versus AAV2-tTSP8 (n = 10 to 11; Mann-Whitney p-value shown in figure).

[0191] (F) Comparison of the protective effect of AAV2-tTSP8 in Cd47 mice + / + and Cd47“ / “ (n = 20 to 16; Mann-Whitney test p-value shown in figure).

[0192] (G) Quantification of RPE atrophy and cone segment loss areas on flat RPE and retinal mounts of eyes injected with 4N1K, compared to their contralateral eyes, (n = 4 mice / group; paired Wilcoxon p-values ​​showed no significant difference).

[0193] (H) Quantification of RPE atrophy and cone segment loss areas on flat RPE and retinal mounts of eyes injected with anti-CD47 B6H12 antibody, compared to their contralateral eyes, (n = 5 mice / group; paired Wilcoxon p-values ​​showed no significant difference).

[0194] EXAMPLES

[0195] I. Materials and methods

[0196] All of this work has been approved by the Ministry of National Education, Higher Education and Research.

[0197] tTSP design and production of tTSP AAV2 and AAV2.7m8 viruses

[0198] The different tTSP sequences were designed with Snapgene and cloned using Gibson technology in mammalian expression (Genecust) or the AAV plasmid (Vectorbuilder).

[0199] Two general designs of a number of shortened TSP-1 constructions were retained: tTSP8, which is defined by the excision of repeating domains of type 1 and 2 only, and tTSP12, furthermore devoid of the terminal N domain (Fig. 1A-C).

[0200] In the 1st generation of constructs (called tTSP8-l of SEQ. ID. No. 12 and tTSP12-l of SEQ. ID. No. 10), the 312 AAs encoding the type 1 and type 2 repeated domains from D379 to G690 (inclusive) of the native sequence were removed.

[0201] In the 2 ème generation (called tTSP8-2 of SEQ. ID. No. 13 and tTSP12-2 of SEQ. ID. No. 11), the 333 AA from R314 to P646 (inclusive, the positions indicated are in reference to figure IB) have been removed.

[0202] The tTSP8 constructs start from N19. The first-generation tTSP12 construct starts from G271. The second-generation tTSP12 construct also starts from N19, but the region from G25 to S233 (209AA) has been removed (Figure 1D) (all indicated positions refer to Figure IB). Two generations of AAV viruses encoding the different tTSP constructs were used.

[0203] For viruses of the èreFor the second generation, a 933-base-pair immediate-early enhancer / chicken |3-actin (CAG) promoter, a HAVT20 leader peptide sequence, and a FLAG sequence followed by the tTSP sequence were cloned between the inverted terminal repeat (ITR) domain of an AAV vector plasmid and then packaged into AAV2 viruses. For the second generation, the AAV vector plasmids were the same, but the signal peptide was changed to the natural Thbsl sequence, the FLAG sequence was deleted, the adjusted tTSP sequence was not optimized in terms of codons, and a groundhog hepatitis virus post-transcriptional regulatory element (WPRE) was added to enhance tTSP expression. These second-generation plasmids were then encapsidated into AAV2 or AAV2.7m8 viruses.

[0204] Recombinant AAV2 (l ère and 2 èmegeneration) were produced by Sirion. Recombinant AAV2.7m8 were produced in-house as previously described using the co-transfection method and purified by ultracentrifugation on an iodixanol gradient (Garita-Hernandez et al., 2021, 10.3389 / fncel.2021.648210).

[0205] Production of tTSP8 and tTSP12 in HEK293 cells

[0206] HEK293 cells (human embryonic kidney cell line, transformed Ad5 DNA; American Type Culture Collection) were cultured in 24-well plates and transfected with various TSP or tTSP plasmids using Lipofectamine2000 reagent and cultured for 48 hours according to the manufacturer's instructions. In other experiments, the wells were transduced with 150,000 or 75,000 viral genomes as shown in Figure IC and cultured for seven days. Supernatants were collected, centrifuged to agglomerate cell debris, and proteins were extracted in lysis buffer (10 mM HEPES pH 7, 100 mM NaCl, 2 mM EDTA, 0.5% NP-40).

[0207] tTSP Western blot analysis

[0208] The presence of TSP or tTSP in the supernatant and total lysates at the expected molecular weight was confirmed by Western blot with anti-TSP1 (1:500; 399,300; Life Technologies) and anti-DYKDDDDK Tag (D6WD5) (FlagM2) (1:500; 14793; Cell Signaling). For the experiment shown in Figure 2, the proteins were crosslinked with BS3 (bis(sulfosuccinimidyl) suberate) (21280; ThermoFisher Scientific) according to the manufacturer's protocol.

[0209] The samples were then loaded onto 4–15% mini-Protean TGX Tris-Glycine SDS-PAGE buffer (Bio-Rad) or 3–8% NuPAGE Tris-acetate gels (ThermoFisher Scientific) under reducing or non-reducing conditions and transferred to 0.2 µm Trans-Blot Turbo nitrocellulose membranes (Bio-Rad). The membranes were blocked for one hour at room temperature in lxTBS (10 mM Tris, pH 8.0, 150 mM NaCl) supplemented with 5% (w / v) skimmed milk powder. The primary antibodies were incubated overnight at 4°C. Secondary antibodies coupled to HRP were used: an anti-rabbit goat antibody for DYKDDDDK Tag (111-035-003, Jackson ImmunoResearch, West Grove, PA) and an anti-mouse goat antibody for TSP-1 (115-035-003, Jackson ImmunoResearch, West Grove, PA). Between and after antibody incubations, the membranes were thoroughly washed in TBS-T (TBS containing 2.5% Tween-20).Western transfers were visualized using the enhanced chemiluminescence method (ECL prime Western Blotting detection reagent, Amersham).

[0210] Isolation of human blood monocytes, culture and ELISA (Enzyme-linked immunosorbent assay) analysis

[0211] Human peripheral blood mononuclear cells were obtained from the blood of a healthy volunteer after centrifugation on a Ficoll gradient (GE Healthcare). CD14 + Mos were isolated by negative selection using the EasySep human monocyte enrichment cocktail (StemCell Technologies). The purified monocytes were seeded into 96-well plates at a rate of 150,000 cells / well and incubated with recombinant human HTRA1 (5 pg / mL, R&D Systems) for 30 minutes before the culture medium was replaced with supernatants from untransfected cells and HEK 293 cells transfected with tTSP8.ème generation. OPN concentrations were determined in a 24-hour medium using the Quantikine ELISA Human Osteopontine (OPN) kit (DOSTOO, R&D Systems).

[0212] Mouse

[0213] Cx3crl mice GFp / GFP and APOE*2KI were obtained from Jackson Laboratories; all mice were either negative or backcrossed to eliminate Pde6b mutations rdl , Gnat2 c Pf 13 and Crbl rd8 .

[0214] C57BL6 / J and BALB / c mice were obtained from Charles River or Janvier Labs depending on the experiments.

[0215] Mice were kept in specific pathogen-free conditions in a 12h / 12h light / dark cycle (100 to 500 Ix), unless otherwise indicated, without additional cover in the cage and with water and a normal diet available at will.

[0216] Intravitreal Injections: Animals were anesthetized by intraperitoneal injection of ketamine (50 mg / kg) and xylazine (10 mg / kg), and the eyes were also anesthetized locally by topical application of oxybuprocaine hydrochloride. The eyes were injected intravitreally 14 days prior to photocoagulation or light exposure using glass capillaries (Eppendorf) and a microinjector (UMP3, Precision Instruments World), with either 3 µL of PBS containing 0.001% plurionic acid alone (P188) or, in addition, AAV2.7m8 empty virions or virions encoding the indicated tTSPs. 9 virus genomes (vg) were injected per eye.

[0217] Laser lesion model, intravitreal injections and hypoxia

[0218] Laser coagulation was performed using a Vitra laser mounted on a surgical microscope (532 nm, 450 mW, 50 ms, and 250 pm) as previously described (Levy et al., 2015, 10.1523 / JNEUROSCI.2468-15.2015). Four (flat mount) to six (flow cytometry) impacts (532 nm, 450 mW, 50 ms, and 250 pm) per eye were applied to the mid-periphery, equidistant from the optic nerve and other impacts. Mice were sacrificed at the specified times, and the immunostained RPE / Choroid flat mounts were analyzed. Ambient hypoxia (10% O2 / 90% N2) was administered using an oxycycler for the specified durations. Control mice breathing normoxic air were housed in the same room under identical conditions.

[0219] Light Challenge Model

[0220] Female mice aged two to three months were adapted to darkness for 6 hours, with their pupils dilated using 1% atropine (Novartis) and exposed to different light intensities depending on the experiment:

[0221] Cx3crl mice GFP / GFP(Fig. 4) were exposed to green LED light (from 2 a.m., 4500 Lux, JP Vezon Equipment) for 96 hours and then returned to normal animal facility lighting conditions until their sacrifice. BALB / c albino mice, for an initial manipulation aimed at measuring the thickness of the photoreceptor layer and performing a flow cytometry analysis (Fig. 7B and 7C), were exposed to green LED light (from 2 a.m., 4500 Lux, JP Vezon Equipment) for 8 hours and then returned to normal animal facility lighting conditions until their sacrifice. For the chronic experiment, (Fig.7D) The mice were exposed to 4500 Lux green LED light once a week for 2h (week 1), 4h (week 2), 6h (week 3), 6h (week 4), 6h (week 5), 6h (week 6), 8h (week 7), 8h (week 8), 12h (week 9), 12h (week 10), 12h (week 11), 12h (week 12), 18h (week 13), 18h (week 14), 18h (week 15) and then maintained under normal 12h / 12h cyclic setup conditions for the remainder of the week as previously described (Sennlaub et al., 2013, 10.1002 / emmm.201302692). For functional measurements by electroretinogram (Fig. 7G), BALB / c mice were exposed to 6000 Lux white light once a week for 4 h (first week), 6 h (second week), and 12 h (third and fourth weeks) before sacrifice. Fundus and OCT imaging and MP counting were performed as described below.

[0222] Induction of experimental autoimmune uveitis (EAU)

[0223] For active induction of EAU, mice were immunized subcutaneously with 200 pg of IRBP65i-67o (LAQGAYRTAVDLESLAQT) (GeneCust) emulsified in Freund's incomplete adjuvant (Sigma-Aldrich) (1:1 w / v) additionally containing 3.3 mg / ml of Mycobacterium tuberculosis H37Ra (BD Difco). Mice received an intraperitoneal injection of 1 pg of purified Bordetella pertussis toxin (Sigma-Aldrich) (Mattapa I li I et al., 2015, 10.1167 / iovs.15-17280). Clinical signs of EAU were assessed ten and twelve days after EAU induction by fundus examination using a MicronlV and MicronV system (Phoenix-Micron, Oregon, USA) (see below) and scored on a scale of 0 to 5 according to the extent of signs of inflammation, as described (Xu et al., 2008, 10.1111 / j.1474-9726.2007.00351.x). Retinal immune cell flow cytometry was performed twelve days after EAU induction.

[0224] Immunorevelation of IBAl and CD102 on flat mount of RPE / choroid and retina

[0225] After fixation in a 4% paraformaldehyde solution, the retinas and choroids were dissected, washed, and incubated with anti-IBA-1 (1:400, 019-19741, Fujifilm Wako) and anti-CD102 (for laser experiments only; 1:200, 553325, BD Pharmingen) in PBS containing 0.1% Triton X-100 for 12 hours at room temperature under gentle rocking. After a few washes in PBS, the samples were incubated for 2 hours at room temperature with appropriate Alexa Fluor® conjugated secondary antibodies (1:500) in PBS and counterstained with Hoechst (1:1000, 33258, Thermo Fisher Scientific). The preparations were rinsed and mounted on glass slides with Fluoromount aqueous mounting medium (Sigma-Aldrich). The preparations were observed under a fluorescence microscope (DM5500, Leica), and the surface area covered by CD102+CNV was measured on photographs, and the average size of CNV was calculated.IBA-1+ MPs on the RPE were counted within a 500 µm diameter around CD102+ neovascularizations. IBA-1 cells. + were counted on flat mounts of whole RPE / choroidals and on the outer segment side of retinal photoreceptors for the light exposure model.

[0226] Optical coherence tomography (OCT) and fundus imaging in mice (Augustin et al., 2023, 10.1186 / sl2974-023-02699-9)

[0227] For OCT and fundus imaging, pupils were dilated with tropicamide (Mydriaticum, Théa, France) and phenylephrine (Neosynephrine, Europhta, France), and animals were anesthetized with isoflurane (5% to induce and 2% to maintain anesthesia). SD-OCT scans were performed using a Bioptigen SDIOS-HHP SD-OCT imaging system equipped with a lens for imaging the mouse posterior segment (Bioptigen 840 nm HHP; Bioptigen, North Carolina, USA) or a MicronV system (Phoenix-Micron, Oregon, USA). The eyes were kept hydrated with 9% NaCl during the procedure. Image acquisitions were performed using Bioptigen acquisition software and the InVivoVue clinic scans were processed with the open source FIJI (ImageJ) software to measure retinal layers.Fundus images and GFP background images were acquired using a MicronlV and MicronV system (Phoenix-Micron, Oregon, USA) equipped with excitation and emission filters for GFP. Retinal immune cells were isolated and analyzed by flow cytometry.

[0228] The retinas were dissected and homogenized in 500 pL of PBS with TL liberase at 0.8 Wünsch units / mL (Sigma-Aldrich) for 30 min at 37°C and 5% CO2. The retinal homogenate was washed with PBS and the pellet containing the immune cells was resuspended in 100 pL of PBS containing 1 pL of Viobility 405 / 520 fixable dye (Miltenyi). Cells were washed and labeled with 50 pL of primary antibody mixtures: (1) anti-CD45-VioBlue (REA737), anti-MHCII-FITC (REA813), anti-CD1b-PE (REA592), anti-Ly-6C-PE-Vio770 (REA796), anti-CD3-APC (REA641), and anti-Ly-6g-APC-Vio770 (REA526) (Miltenyi), or (2) anti-CD45 (REA737), anti-CD1b (REA592), anti-CD3 (REA641), and anti-CD4 (REA604) (Miltenyi), all diluted 1:50 in PBS. After labeling, cells were fixed in 1% paraformaldehyde. For compensation parameters, single-stain cell controls with the corresponding antibodies were used.Fluorescence intensities were measured using a MACSQuant analyzer (Miltenyi) and the data were analyzed using FlowJo software.

[0229] Statistical analyses

[0230] GraphPad Prism 9 software was used for data analysis and graphical representation. All values ​​are reported as mean ± SEM. Statistical analyses were performed using the Mann-Whitney U test to compare mean values. The n and p values ​​are indicated in the figure captions.

[0231] IL Results

[0232] The tTSPs are secreted and trimerized

[0233] Analysis of cell extracts and supernatant from HEK 293 cells transfected 2 days earlier with either a blank plasmid or a plasmid encoding tTSP8-2 (2 èmegeneration, with a HIS 6 N- or C-terminal tag) by Western blot with an anti-TSP-1 antibody demonstrates that a band of a size of about 100 kDa appears only in the supernatants of HEK cells transfected with tTSP8 (figure 2A).

[0234] Furthermore, analysis of supernatants from HEK293 cells transfected with the tTSP8 plasmid (in this case, a first-generation tTSP8 containing FlagM) reveals a band of approximately 300 kDa when the proteins were crosslinked with bissulfosuccinimidyl suberate (BS3) prior to reducing conditions (Fig. 2B). This band corresponds to the trimerized protein.

[0235] A plasmid encoding native FlagM-TSP-1 served as a positive control (Fig. 2B). HEK293 cells were also transfected 6 days prior with AAV2s encoding either first-generation or second-generation tTSP8. In both cases, these cells secreted tTSP8 into their supernatants by Western blotting (Fig. 2C), but the yield of second-generation tTSPs was higher than that of first-generation constructs. Finally, Western blotting of the supernatants of HEK293 cells transfected with AAV2 or AAV2.7m8 encoding tTSP8 (transfected 6 days prior) revealed bands approximately three times larger under unreduced conditions than under reduced conditions. These results demonstrate that tTSPs are translated, secreted, and trimerized.

[0236] tTSP inhibits osteopontin secretion by HTRA1-stimulated human monocytes. Previous research has shown that HTRA1 and OPN levels are elevated in AMD patients carrying the 10q26 risk haplotype. Furthermore, HTRA1 impairs the ability of TSP1 to activate CD47, which represses OPN secretion by monocytes. Conversely, exogenous addition of HTRA1 to primary human monocytes increases OPN secretion, and activation of CD47 using recombinant TSP1 or activating peptides decreases its secretion in the supernatant (Beguier et al., 2020, 10.1016 / j.immuni.2020.07.021).

[0237] To assess whether tTSP8 activates human CD47, primary human monocytes were incubated with HTRA1 for 30 minutes, then HTRA1 was removed and the cells were incubated for 24 hours with untransfected HEK supernatants or with HEK supernatants transfected 48 hours prior with plasmids encoding tTSP8. ème generation. ELISA quantifications of 24-hour supernatants from these cultured human monocytes revealed a significant decrease in OPN secretion when the monocytes were incubated with the supernatant from HEKs transfected with tTSP8 (Fig. 3). These data indicated that tTSP8 secreted by HEKs represses OPN secretion from primary human monocytes, which is CD47-dependent (Beguier et al., 2020, 10.1016 / j.immuni.2020.07.021).

[0238] tTSP inhibits retinal MP activation in hyperinflammatory Cx3crl GFP / GFP mice jn vjv0

[0239] Physiologically, the subretinal space is devoid of immune cells, including resident microglial cells (MCs) (Combadiere et al., 2007, 10.1172 / JCI31692; Levy et al., 2015, 10.15252 / emmm.201404524), due to potent immunosuppressive pro-apoptotic factors produced by the RPE that eliminate infiltrating leukocytes (Griffith et al., 1995, 10.1126 / science.270.5239.1189; Levy et al., 2015, 10.15252 / emmm.201404524). High levels of apolipoprotein E, observed in subretinal MPs from AMD patients, Cx3crl-deficient mice, and humanized transgenic mice expressing the AMD-risk APOE2 isoform (APOE*2KI mice), have been shown to induce age-related chronic accumulation of pathogenic subretinal MPs (Sennlaub et al., 2013, 10.1002 / emmm.201302692; Levy et al., 2015, 10.15252 / emmm.201404524; Levy et al., 2015, 10.1523 / JNEUROSCI.2468-15.2015).The accumulation of subretinal microparticles (MPs) in these mice can also be accelerated by light-induced stress. The intensity of the light exposure model used here was calibrated to induce substantial subretinal MP infiltration in Cx3crl mice. GFp / GFP and APOE*2KI are prone to inflammation, but not in controls (Sennlaub et al., 2013, 10.1002 / emmm.201302692; Levy et al., 2015, 10.1523 / JNEUROSCI.2468-15.2015).

[0240] Cx3crl mice GFP / GFP and APOE*2KI aged two to three months received an intravitreal injection of 5xl0 9 AAV2 empty in the left eye and 5xl0 9 of AAV2tTSP8 or AAVtTSP12 of the èregeneration in their right eyes. After 14 days, the mice were adapted to darkness for 6 hours, with dilated pupils, and exposed to green LED light (from 2 a.m., 4500 lux, JP Vezon equipment) for 4 days and then maintained under normal 12h / 12h cyclic light exposure conditions for 6 days (Sennlaub et al., 2013, 10.1002 / emmm.201302692) (Fig. 4A). Quantification of IBA-1+ subretinal MPs on immunostained RPE / choroidal and retinal flat mounts revealed a significant decrease in MPs in the right eyes transfected with AAVtTSP8 compared to the left control eyes treated with AAV from Cx3crl mice GFp / GFP exposed to light (Fig. 4B). Similarly, the right eyes of APOE*2KI mice exposed to light and previously treated with AAVtTSP12 revealed a significant decrease in MP IBA1 accumulation. +compared to control left eyes treated with empty AAV2 (Fig. 4C).

[0241] In a second series of experiments, Cx3crl mice GFp / GFP Two- to three-month-old mice received an intravitreal injection of 5 x 10⁹ of empty AAV2 into their left eyes and 5 x 10⁹ of AAV2tTSP8 or AAVtTSP12 (first generation) into their right eyes. After 14 days, the mice were exposed to three light cycles, consisting of four days of exposure to green LED light, as described above, and three days of normal 12 h / 12 ​​h cycle light (Fig. 4D). This protocol induced GFP agglomeration. + MP around retinal vessels in the left control eyes injected with AAV in all animals. Injection of AAVtTSP8 into the right eye completely prevented this phenotype of perivascular GFP accumulation. +MP in the right eye (Fig. 4E). Taken together, these data demonstrated that tTSP8 and tTSP12 inhibit subretinal inflammation in Cx3crl deficient mice and A POE*2KI mice (which express the AMD-risk APOE2 isoform), which have been shown to develop pathogenic subretinal inflammation due to high levels of APOE also observed in human AMD (Levy et al., 2015, 10.15252 / emmm.201404524; Levy et al., 2015, 10.1523 / JNEUROSCI.2468-15.2015). Furthermore, transduction with tTSP8 using AAVtTSP8 completely repressed the perivascular accumulation of MP induced by three consecutive light exposures in AAV-injected empty control eyes.

[0242] tTSP inhibits MP accumulation and choroidal neovascularization in the laser injury model

[0243] Functional studies in animal models have shown that subretinal MP accumulation plays a key role in the neovascularization that characterizes wet AMD (Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002). Laser-induced choroidal neovascularization (CNV), a commonly used model of wet AMD, induces subretinal MP infiltration, with peak recruitment three to four days after lesion induction, followed by a resolution phase of inflammation characterized by a decrease in the number of MPs and the formation of choroidal neovascularizations (CNV) (Lavalette et al., 2011, 10.1016 / j.ajpath.2011.01.013). Depletion of circulating Mo (Sakurai et al., 2003, 10.1167 / iovs.03-0097; Caicedo et al., 2005, 10.1016 / j.exer.2005.01.013; Beguier et al., 2020, 10.1016 / j.immuni.2020.07.021) and inhibition of monocyte (Mo) recruitment (Sakurai et al., 2003, 10.1167 / iovs.03-0097; Tsutsumi et al., 2003 10.1189 / jlb.0902436; Luhmann et al., 2009, 10.1167 / iovs.09-3462; Liu et al., 2013, 10.1371 / journal.pone.0072935; Robbie et al., 2016.

[0244] 10.1016 / j.neurobiolaging.2015.12.019) inhibit CNV formation very significantly, demonstrating the pro-angiogenic role of MPs infiltration. It is important to note that infiltrating MPs are observed in the damaged tissue in close contact with the developing CNV, but also in the subretinal space, adjacent to physiologically immunosuppressive RPE cells (Lavalette et al., 2011, 10.1016 / j.ajpath.2011.01.013; Levy et al., 2015, 10.15252 / emmm.201404524; Calippe et al., 2017, 10.1016 / j.immuni.2017.01.006; Mathis et al., 2017, lO.llll / acel.12540). In an initial experiment, two- to three-month-old C57BL6 / J mice received an intravitreal injection of 5 x 10 9 AAV2 empty in the left eye and 5x10 9 of AAVtTSP8 or AAVtTSP12 (of the èregeneration) in the right eye. After 14 days, six laser impacts were applied to each eye of anesthetized and dilated mice (Fig. 5A). Using flow cytometry and a gating strategy adapted from O'Koren et al. (O'Koren et al., 2016, 10.1038 / srep20636), the leukocyte population was analyzed in the posterior segments of the right and left eyes, ten days after laser injury. CD45 leukocytes + , the CDllb MPs + Ly6G neg CD45 lymphocytes + CDllb neg Ly6G neg , the MC CDllb + CD45 low Ly6G neg , the infiltrating CDllb macrophages + CD45 high Ly6G neg and CDllb monocytes + CD45 high Ly6G neg Ly6C highall were significantly reduced in right eyes treated with AAVtTSP8 compared to left control eyes treated with empty AAV2 (Fig. 5B). A similar immune cell reduction effect was observed in eyes treated with AAVtTSP12 compared to their controls, except that the less pronounced reduction in MCs did not reach a statistically significant level (Fig. 5C).

[0245] Quantification of subretinal MPs IBA-l + on the choroidal and retinal flat mounts immunostained RPE revealed a significant decrease in MPs in right eyes treated with AAVtTSP8 and AAVtTSP12 compared to left control eyes treated with empty AAV2 (Fig. 5D).

[0246] In a second series of experiments, second-generation AAV2tTSP8 was tested for its anti-inflammatory capacity. To further mimic AMD, mice were exposed to 10% Ch hypoxia from day 3 to day 10 (Fig. 5E). Indeed, hypoxia has long been considered an important trigger of AMD. Late-onset AMD, particularly wet AMD, is preceded by choriocapillary atrophy, reduced ocular blood flow, and drusen deposits that impede oxygen delivery from the choroid to the RPE and photoreceptors (Stefansson et al., 2011, 10.1016 / j.preteyeres.2010.09.003). From a systemic point of view, AMD is associated with hypertension, atherosclerosis, cardiovascular diseases (Chakravarthy et al., 2010, 10.1186 / 1471-2415-10-31) and emphysema (Klein et al., 2003, 10.1016 / S0161- 6420(03)00599-2) which are all associated with hypoxia (Feigl, 2009, 10.1016 / j.preteyeres.2008.11.004).Systemic hypoxia has been shown to reduce TSP-1 expression in MPs and lead to exaggerated choroidal neovascularization (Touhami et al., 2022, 10.3390 / ijms23020681). Quantification of the number of subretinal MPs IBA-1. + Impact measurements and measurements of the area covered by CD102 + CNV on EPR / choroidal and immunostained flat mounts revealed a significant decrease in the number of subretinal MPs and the size of CNVs in right eyes treated with AAVtTSP8 compared to left control eyes treated with empty AAV2 (Fig. 5F).

[0247] Taken together, these data demonstrate that first- and second-generation tTSP8 and tTSP12 reduce inflammation in the eyes of laser-injured wild-type mice, including the specific accumulation of subretinal microparticles. Furthermore, the associated choroidal neovascularization is inhibited.

[0248] tTSP inhibits experimental autoimmune uveitis.

[0249] To evaluate whether tTSP8 gene therapy would inhibit experimental autoimmune uveitis (EAU), injections of either 3 lp of PBS containing pluronic acid (PBS / PA 0.001% Pluronic (P188)) or 5 lp of 9 AAV2tTSP8 virus was injected bilaterally via intravitreal injection into 2-month-old female C57BL6 / mice. Each group consisted of 15 mice. The AAV2tTSP8 virus encoded the 2 èmetTSP8 generation. After 14 days, EAU was induced by subcutaneous injection of 200 pg of interphotoreceptor retinoid-binding protein (IRBP) 65i-67o emulsified in an incomplete Freund adjuvant and by intraperitoneal injection of 1 pg of purified Bordetella pertussis toxin (Mattapallil et al., 2015, 10.1167 / iovs.15-17280) (Fig. 6A). Fundus examination ten days after EAU induction revealed clinical signs of uveitis in 13 / 30 of the eyes injected with PBS, with a mean clinical score of 0.57, but only 2 / 30 of the eyes and a significantly lower mean clinical score of 0.06 in AAV2tTSP8 (Fig. 6B). At twelve days, clinical signs of uveitis were detected in 22 / 30 of the eyes that received an injection of PBS with a mean clinical score of 1.53, while 18 / 30 of the eyes injected with AAV2tTSP8 developed only mild signs of retinal inflammation with a significantly lower clinical score of 0.733 (Fig. 6B).

[0250] Consequently, cytometric analysis revealed that the CD45 immune cells + , the T helper CD45 + CDllb neg CD3 + CD4 + , CD45 microglial cells + CDllb + CD45 |OW Ly6G neg and monocyte-derived macrophages (MdM; the number of monocytes and neutrophils was negligible at this stage of the disease) CD45 + CDllb + Ly6G neg , were all significantly reduced in eyes treated with AAVtTSP8 compared to control eyes treated with PBS (control) (Fig. 6C).

[0251] tTSP protects the photoreceptors of albino mice against degeneration induced by repeated light exposure.

[0252] Functional studies on animal models have shown that subretinal accumulation of mononuclear phagocytes (PM) plays a critical role in photoreceptor degeneration, characteristic of geographic atrophy (GA) (Charles-Messance et al., 2020, 10.1186 / sl2974-019-1655-5; Hu et al., 2015, 10.1523 / JNEUROSCI.3955-14.2015; Eandi et al., 2016, 10.7554 / eLife.16490; Guillonneau et al., 2017, 10.1016 / j.preteyeres.2017.06.002). In particular, PMs also participate in photoreceptor degeneration in the light-induced degeneration model in the BALB / c albino mouse (Scholz et al., 2015, 10.1186 / sl2974-015-0431-4; Elbaz-Hayoun et al., Mol Vis. 2019, 25:479-488; Bell et al., 2015, 10.1016 / j.exer.2015.04.009).

[0253] To assess whether tTSP8 gene therapy could inhibit photoreceptor degeneration, we first injected two groups of five 2-month-old female BALB / c mice bilaterally with either PBS containing pluronic acid (PBS / PA; 0.001% Pluronic P188) or with 5 x 10 9 vg of AAV2-tTSP8 in PBS / PA (second generation of tTSP8). After 14 days, mice were adapted to darkness for 6 hours with dilated pupils, then exposed to green LED light (from 2 a.m., 4500 lux, JP Vezon equipment) for 8 hours, before being returned to normal rearing conditions with a 12 h / 12 ​​h light / dark cycle (Fig. 7A). OCT imaging and quantification of outer nuclear layer (ONL) thickness after 7 days revealed significant ONL preservation in AAV2-tTSP8-treated eyes (Fig. 7B). Flow cytometry analysis showed a similar number of CD45 microglial cells+ CDllb + CD45 faible Ly6G nég , but a significant reduction in CD45 monocyte-derived macrophages (MdM) + CDllb + Ly6G nég Monocyte and neutrophil counts were negligible at this stage of the disease model (Fig. 7C). Next, we intravitreal injections were administered bilaterally to 2-month-old female BALB / c mice, either with PBS containing pluronic acid (PBS / PA; 0.001% Pluronic P188) or with 5 x 10 9 vg of AAV2-tTSP8 in PBS / PA, i.e. with 5 x 10 9AAV2-7m8-tTSP8 virus was introduced into PBS / PA. Each group consisted of 8 mice. Both viruses encoded the second generation of tTSP8. After 14 days, the mice were adapted to darkness for 6 hours with dilated pupils, then exposed to green LED light (starting at 2:00 AM, 4500 Lux, JP Vezon equipment) once a week, but otherwise maintained under normal rearing conditions with a 12-hour light / dark cycle. The mice were exposed in groups of three per lamp (one mouse from each group) to minimize variability in exposure between groups. To induce progressive degeneration of the photoreceptors, the duration of exposure was progressively increased over time, from 2 h in week 1 to 18 h in week 15. The thickness of the ONL was measured at the upper pole by OCT and averaged between the left and right eyes every 14 days (Fig. 7D).While pre-light OCT measurements did not reveal significant differences in ONL thickness among the three groups, the ONL of PBS-treated control animals was significantly thinner after the third light exposure compared to tTSP8-treated animals, and these differences increased with each OCT assessment (Fig. 7E). Although AAV2-tTSP8 and AAV2-7m8-tTSP8 provided similar protection against light-induced photoreceptor degeneration for 7 weeks, the effect of AAV2-7m8-tTSP8 subsequently diminished progressively.

[0254] To evaluate whether tTSP8 gene therapy improves photoreceptor function in the light injury model, we bilaterally injected 2-month-old female BALB / c mice (five per group) with either PBS containing pluronic acid (PBS / PA, 0.001% Pluronic) or with 5 x 10 9vg of AAV2-tTSP8 in PBS / PA (second generation tTSP8). For functional studies, we adapted the light exposure paradigm to induce more severe but still inflammation-dependent degeneration. Two weeks after intravitreal injection, BALB / c mice were exposed to intense white LED light (6000 Lux). Illumination was applied for 4 hours the first week and 6 hours the second week, followed by two weeks of 12-hour / 12-hour light / dark cycles (Fig. 7F). Electroretinograms performed after four weeks showed a marked improvement in A-wave amplitude at scotopic flash intensities of 3 and 10 cd-s / m². 2 , indicating functional protection in eyes treated with AAV2-tTSP8 compared to their control eyes injected with PBS (Fig. 7G).

[0255] ADDITIONAL EXAMPLES

[0256] I. Materials and methods

[0257] Animals

[0258] Two-month-old male C57BI6 / J mice and female BALB / c mice were obtained from Janvier Labs, while the Cd47“ / “ mice were obtained from Charles River. All mice were tested for the rd8 mutation responsible for retinal degeneration. The animals were kept in conditions free of specific pathogens, under a 12 h / 12 ​​h light cycle unless otherwise specified, with access to water and a standard diet ad libitum. All mice were 8 weeks old at the start of the experimental period.

[0259] Intravitreal injections

[0260] Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg / kg) and xylazine (8 mg / kg) diluted in physiological saline (0.9% NaCl). Corneal anesthetized locally by instillation of oxybuprocaine eye drops, and corneal moisture was maintained using a transparent ophthalmic gel (Lubrithal). Intravitreal injections were performed using glass microcapillaries (Eppendorf) and a microinjector.

[0261] In experiments with AAV2-tTSP8, mice received an intravitreal injection of AAV (5 x 10 9 vg in 3 µl of PBS containing 0.001% pluronic acid) in one eye and of PBS + 0.001% pluronic acid (3 µl) in the contralateral eye. For the empty viral vector controls, the mice received an intravitreal injection of an empty viral vector (5 x 10 9vg in 3 µl of PBS containing 0.001% pluronic acid) in one eye and PBS + 0.001% pluronic acid (3 µl) in the contralateral eye. All viruses were injected four weeks before induction of the retinal degeneration / RPE model with sodium iodate (NalO3).

[0262] In experiments with alternative CD47 ligands, mice received 3 pl of 4N1K (Genepep, France) from SEQ. ID. No. 19 (200 pM in sterile PBS) or anti-CD47 antibody B6H12 (ThermoFisher Scientific, 16-0479-85; 3 pl at 50 pg / ml in sterile PBS) by intravitreal injection in one eye, and 3 pl of sterile PBS in the contralateral eye.

[0263] Model of retinal degeneration / RPE induced by sodium iodate (NalO3)

[0264] Sodium iodate solutions in sterile PBS were prepared on the day of injection. Mice received an intraperitoneal injection of sodium iodate solution (20 mg / kg), either 4 weeks after intravitreal injection for experiments with AAV, or immediately after intravitreal injection for experiments with CD47 ligands.

[0265] Electroretinography (ERG)

[0266] After overnight dark adaptation, the pupils were dilated using tropicamide (0.5%, Mydriaticum, Théa Laboratories) and phenylephrine (5%, Neosynephrine, Europhta Laboratories). The corneas were anesthetized by instillation of oxybuprocaine eye drops (Théa Laboratories), and the animals were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg / kg) and xylazine (8 mg / kg) diluted in physiological saline (0.9% NaCl). Electroretinograms (ERGs) were recorded using a Lab Cradle system equipped with a white LED stimulation (6500 K) of the Color Dome Ganzfeld type (Diagnosys). Gold wire loop corneal electrodes were placed on both eyes, a reference electrode was placed subcutaneously between the eyes, and a ground electrode was positioned subcutaneously at the base of the tail.A transparent ophthalmic gel (Lubrithal) was applied to both eyes to maintain corneal moisture and good contact with the corneal electrode during recording. The mice were kept on a heating plate to maintain normal body temperature. Scotopic retinal function was explored using flashes of varying intensities, spaced 60 seconds apart. The resulting responses were amplified and filtered using a 0–300 Hz bandpass filter with a single-channel DC- / AC- amplifier. The collected traces were then processed with Espion V6 software (Diagnosys). A-wave amplitudes were measured from the baseline to the peak of the first negative deflection.

[0267] Immunohistochemistry on flat mounts

[0268] The eyes were collected 17 days after the sodium iodate injection. They were fixed in 4% PFA (methanol-free, diluted in PBS) for 45 minutes at room temperature, then rinsed and stored in PBS. After removal of the anterior segment, lens, and scleral connective tissue, four curvature relaxation incisions were made, and the retinas and RPE / choroid / sclera complexes were carefully separated. The retinas and RPE / choroid / sclera complexes were then incubated overnight at room temperature in PBS + 1% TritonX-100 containing rabbit anti-IBAl antibody (Wako, dilution 1:400), AlexaFluor-542 conjugated phalloidin (Invitrogen, dilution 1:100), and AlexaFluor-647 conjugated peanut agglutinin (PNA) (Thermo Fisher, dilution 1:100).After three washes with PBS, the retinas and RPE / choroid / sclera complexes were incubated for 2 hours at room temperature in PBS containing the anti-rabbit secondary antibody conjugated to AlexaFluor-488 (Thermo Fisher, 1:500 dilution) and Hoechst (Sigma Aldrich, 1:1000 dilution). After three further washes with PBS, the tissues were mounted flat and covered with a coverslip containing aqueous mounting medium (Sigma Aldrich), then imaged using a fluorescence microscope (Leica DM5500). Images of the entire photoreceptor surface and the RPE were acquired for subsequent analysis.

[0269] Image analysis

[0270] All image analyses were performed using FIJI software (ImageJ). Pathological areas were measured using the FIJI Measure tool. The cone loss area was defined as the area lacking cone labeling by PNA, the RPE loss area was defined as the area exhibiting an absence of the phalloidin-labeled RPE mosaic network, and the dysmorphic RPE area was defined as the area where the phalloidin-labeled mosaic network consisted of irregular, hypertrophic, or dysmorphic cells, rather than the uniform hexagonal mosaic of a healthy RPE.

[0271] Statistical analyses

[0272] Data analysis and graphical representation were performed using GraphPad Prism 9 (GraphPad Software). All values ​​are presented as mean ± SEM. Statistical analyses were conducted using the Mann-Whitney U test for comparisons of means and the Wilcoxon signed-rank test for paired comparisons. The values ​​of n and p are indicated in the figure captions.

[0273] IL Results

[0274] tTSP inhibits the accumulation of mononuclear phagocytes, RPE and photoreceptor degeneration, and restores photoreceptor function in the sodium iodate (NalO3) induced retinal degeneration / RPE model.

[0275] In the sodium iodate (NalO3)-induced retinal degeneration model, primary oxidative damage to the retinal pigment epithelium (RPE) is followed by the recruitment and activation of mononuclear phagocytes (PMs), leading to photoreceptor degeneration. Recent data suggest that PM accumulation contributes to the worsening of retinal pathology, with infiltrating PMs implicated in the exacerbation of damage affecting both the RPE and photoreceptors (Mulfaul et al., 2020, 10.1016 / j.celrep.2020.01.064; Moriguchi et al., 2018, 10.1167 / iovs.17-23532). These observations indicate that, in this model, PMs play a major role in amplifying tissue damage rather than acting as repairing mediators.

[0276] To evaluate whether tTSP8 gene therapy could attenuate RPE and photoreceptor degeneration, 2-month-old male C57BI6 / J mice were treated according to a paired-eye regimen, receiving in one eye an injection of PBS containing pluronic acid (PBS / PA, 0.001% Pluronic P188), and in the contralateral eye 5 x 10 9vg of AAV2-tTSP8 in PBS / PA (second-generation tTSP8). This design was chosen to provide internal control, given the well-known variability of the NalO3 model between individuals. After a 4-week period, allowing optimal transgene expression, mice received an intraperitoneal injection of NalO3 (20 mg / kg in PBS), which induces RPE degeneration within a few days (Anderson et al., 2024, 10.1016 / j.exer.2023.109772). Inflammatory cell infiltration was assessed at 4 days, retinal function by ERG at 7 days, and retina / RPE-choroid morphology at 17 days after NalO3 administration (Fig. 8A). Flow cytometry analysis revealed a significant reduction in CD45 microglia. + CDllb + CD45medLy6G“ and monocyte-derived macrophages (MdMs) CD45highCDllb +Ly6G“, while the numbers of monocytes and neutrophils remained negligible at this stage of the pathological model (Fig. 8B).

[0277] At 7 days, electroretinography showed a significant improvement in A-wave amplitude for three of the four scotopic flash intensities, indicating functional protection in AAV2-tTSP8-treated eyes compared to contralateral eyes (Fig. 8C).

[0278] Phalloidin-labeled RPE-choroid flat mounts showed central RPE atrophy, bordered by a transitional zone of dysmorphic but still present RPE, itself surrounded by a RPE with normal morphology. Quantification of RPE atrophy on these flat mounts, combined with measurement of corresponding areas of cone segment loss on peanut lectin-labeled retinal flat mounts, revealed highly significant preservation of the RPE and cones in AAV2-tTSP8-treated eyes compared to contralateral eyes injected with the control, in all mice (Fig. 8D).

[0279] Overall, these data show that treatment with AAV2-tTSP8 protects against NalO3-induced RPE and photoreceptor degeneration and preserves visual function.

[0280] Empty AAV2 virus does not alter NalO3-induced RPE degeneration, and the protective effect of tTSP is partly dependent on CD47.

[0281] The observed effect of AAV2-tTSP8 could be attributed to the AAV2 vector itself rather than the transgene. Furthermore, given that tTSP8 contains a heparin-binding domain (HBD) at the NH2-terminal position, interacting with cell surface heparan sulfate proteoglycans and the low-density lipoprotein-related receptor (LRP1 / CD91), in addition to the CD47 activating domain, tTSP8 could also exert effects independent of CD47.

[0282] To better separate the mechanisms underlying the protective effects of tTSP8, we first injected Cd47 mice + / + (C57BL6 / J) with 5 x 10 9 vg of AAV2-tTSP8 or empty vector, then compared the effects of AAV2-tTSP8 (5 x 10 9 (vg) in Cd47 mice + / +and Cd47“ / “. For each mouse, the contralateral eye received PBS containing pluronic acid as a control. RPE atrophy was quantified, and protection was expressed as a percentage reduction in atrophic area compared to control eyes, according to the formula: Protection (%) = (CTLmm 2 - tTSPmm 2 ) / CTLmm 2 x 100. Our results demonstrate that treatment with empty vectors had no protective effect, which differs significantly from the robust protection observed in animals treated with AAV2-tTSP8 (Fig. 8E).

[0283] AAV2-tTSP8 gene therapy in Cd47 mice + / +Compared to Cd47“ / “ mice, a significant reduction in the percentage of protection was revealed in the absence of Cd47. These results indicate that tTSP8-mediated protection depends largely on CD47 activation, but also suggest the involvement of CD47-independent mechanisms, potentially via interactions of the N-terminal domain of tTSP8 with LRP1 (Fig. 8F).

[0284] Overall, our results demonstrate that the protective effect of AAV2-tTSP8 is not primarily mediated by the viral capsid, and that CD47 activation plays a key role in the underlying mechanism.

[0285] The CD47 agonist, 4N1K, as well as the anti-CD47 antibody B6H12, do not protect mice against NalO3-induced retinal degeneration / RPE.

[0286] The TSP-1-derived peptide 4N1K, corresponding to the sequence KRFYVVMWKK (SEQ ID No. 19), is capable of activating CD47, although it requires molar concentrations approximately 50 times higher than those required for TSP-1 itself (Martinez-Torres et al., 2015, 10.1371 / journal.pmed.1001796; McDonald et al., 2003, 10.1021 / bi0341408). To assess whether this CD47 agonist could influence the outcome of NalO3-induced RPE and photoreceptor degeneration, we administered 3 µL of 200 pM 4N1K on the day of model induction, following intraperitoneal injection of NalO3. Quantification of RPE atrophy size and cone segment loss areas on flat RPE and retina mounts stained with phalloidin and peanut agglutinin revealed no significant effect of 4N1K treatment compared to contralateral eyes injected with PBS (Fig. 8G).

[0287] Similarly, intravitreal injection of 3 µl of anti-CD47 B6H12 antibody (50 pg / ml) — known to block the CD47-mediated "don't eat me" signal (Antonsen et al., 2024, 10.1002 / eji.202350824), but also capable of inducing T-cell apoptosis when immobilized (e.g., cross-linked or plate-fixed) in vitro (Leclair et al., 2018, 10.1038 / s41419-018-0601-2) — at the time of intraperitoneal NalO3 administration, did not alter the size of RPE atrophy or the areas of cone segment loss, as assessed on phalloidin- and agglutinin-stained RPE and retina flat mounts peanut, compared to contralateral eyes (Fig. 8H).

[0288] Taken together, our results indicate that, unlike AAV2-tTSP8 therapy, neither 4N1K nor anti-CD47 antibody treatment altered the progression of degeneration in this model.

[0289] LIST OF SEQUENCES

[0290] SEQ. ID. N°l - full TSPl

[0291] NRIPESGGDNSVFDIFELTGAARKGSGRRLVKGPDPSSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKG

[0292] FLLLASLRQMKKTRGTLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEEALLATGQWK

[0293] SITLFVQEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIAKGGVNDNFQGVLQNVRFVFGTTP

[0294] EDILRNKGCSSSTSVLLTLDNNVVNGSSPAIRTNYIGHKTKDLQAICGISCDELSSMVLELRGLRTIVTTL

[0295] QDSIRKVTEENKELANELRRPPLCYHNGVQYRNNEEWTVDSCTECHCQNSVTICKKVSCPIMPCSNA

[0296] TVPDGECCPRCWPSDSADDGWSPWSEWTSCSTSCGNGIQQRGRSCDSLNNRCEGSSVQTRTCHIQ

[0297] ECDKRFKQDGGWSHWSPWSSCSVTCGDGVITRIRLCNSPSPQMNGKPCEGEARETKACKKDACPIN

[0298] GGWGPWSPWDICSVTCGGGVQKRSRLCNNPTPQFGGKDCVGDVTENQICNKQDCPIDGCLSNPC

[0299] FAGVKCTSYPDGSWKCGACPPGYSGNGIQCTDVDECKEVPDACFNHNGEHRCENTDPGYNCLPCPP

[0300] RFTGSQPFGQGVEHATANKQVCKPRNPCTDGTHDCNKNAKCNYLGHYSDPMYRCECKPGYAGNGI

[0301] ICGEDTDLDGWPNENLVCVANATYHCKKDNCPNLPNSGQEDYDKDGIGDACDDDDDNDKIPDDRD

[0302] NCPFHYNPAQYDYDRDDVGDRCDNCPYNHNPDQADTDNNGEGDACAADIDGDGILNERDNCQYV

[0303] YNVDQRDTDMDGVGDQCDNCPLEHNPDQLDSDDSDRIGDTCDNNQDIDEDGHQNNLDNCPYVPN

[0304] ANQADHDKDGKGDACDHDDDNDGIPDDKDNCRLVPNPDQKDSDGDGRGDACKDDFDHDSVPDI

[0305] DDICPENVDISETDFFRRFQMIPLDPKGTSQNDPNWVVRHQGKELVQTVNCDPGLAVGYDEFNAVDF

[0306] SGTFFINTERDDDYAGFVFGYQSSSRFYVVMWKQVTQSYWDTNPTRAQGYSGLSVKVVNSTTGPGE

[0307] HLRNALWHTGNTPGQVRTLWHDPRHIGWKDFTAYRWRLSHRPKTGFIRVVMYEGKKIMADSGPIY

[0308] DKTYAGGRRLGLFVFSQEMVFFSDLKYECRDP

[0309] SEQ. ID. N°2 (fragment to replace the amino acids in the position 252 in the position

[0310] 295 de TSP-1 de SEQ. ID. N°l).

[0311] CGISCDELSSMVLELRGLRTIVTTLQDSIRKVTEENKELANELR

[0312] SEQ. ID. No. 3 (fragment comprising amino acids from position 1019 to position 1107 of native TSP-1 of SEQ. ID. No. l).

[0313] VVMWKQVTQSYWDTNPTRAQGYSGLSVKVVNSTTGPGEHLRNALWHTGNTPGQVRTLWHDPRH

[0314] IGWKDFTAYRWRLSHRPKTGFIRVVM

[0315] SEQ. ID. No. 4: fragment comprising the amino acids located from position 7 to position 290 of TSP-1 of SEQ. ID. No.l.

[0316] GGDNSVFDIFELTGAARKGSGRRLVKGPDPSSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKGFLLLAS

[0317] LRQMKKTRGTLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEALATGQWKSITLFV QEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIAKGGVNDNFQGVLQNVRFVFGTTPEDILR

[0318] NKGCSSSTSVLLTLDNNVVNGSSPAIRTNYIGHKTKDLQAICGISCDELSSMVLELRGLRTIVTTLQDSIR KVTEENKEL

[0319] SEQ. ID. No. 5 : XI of tTSP12-l,

[0320] GISCDELSSMVLELRGLRTIVTTLQDSIRKVTEENKELANELRRPPLCYHNGVQYRNNEEWTVDSCTEC

[0321] HCQNSVTICKKVSCPIMPCSNATVPDGECCPRCWPSDSA

[0322] SEQ. ID. N°6 : XI de tTSP12-2

[0323] SSTSVLLTLDNNVVNGSSPAIRTNYIGHKTKDLQAICGISCDELSSMVLELRGLRTIVTTLQDSIRKVTEE

[0324] KEELANELR

[0325] SEQ. ID. N°7: XI of tTSP8-l

[0326] NRIPESGGDNSVFDIFELTGAARKGSGRRLVKGPDPSSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKG

[0327] FLLLASLRQMKKTRGTLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEEALLATGQWK

[0328] SITLFVQEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIAKGGVNDNFQGVLQNVRFVFGTTP

[0329] EDILRNKGCSSSTSVLLTLDNNVVNGSSPAIRTNYIGHKTKDLQAICGISCDELSSMVLELRGLRTIVTTL

[0330] QDSIRKVTEENKELANELRRRPPLCYHNGVQYRNNEEWTVDSCTECHCQNSVTICKKVSCPIMPCSNA TVPDGECCPRCWPSDSA

[0331] SEQ. ID. N°8. : XI de tTSP8-2

[0332] NRIPESGGDNSVFDIFELTGAARKGSGRRLVKGPDPSSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKG

[0333] FLLLASLRQMKKTRGTLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEEALLATGQWK

[0334] SITLFVQEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIAKGGVNDNFQGVLQNVRFVFGTTP

[0335] EDILRNKGCSSSTSVLLTLDNNVVNGSSPAIRTNYIGHKTKDLQAICGISCDELSSMVLELRGLRTIVTTL

[0336] QDSIRKVTEENKELANELR

[0337] SEQ. ID. N°9: fragment containing amino acids at position 628 to position 1152 of the native SEQ TSP-1. ID. N°1

[0338] PRNPCTDGTHDCNKNAKCNYLGHYSDPMYRCECKPGYAGNGIICGEDTDLDGWPNENLVCVANAT

[0339] YHCKKDNCPNLPNSGQEDYDKDGIGDACDDDDDNDKIPDDRDNCPFHYNPAQYDYDRDDVGDRC

[0340] DNCPYNHNPDQADTDNNGEGDACAADIDGDGILNERDNCQYVYNVDQRDTDMDGVGDQCDNC

[0341] PLEHNPDQLDSDDSDRIGDTCDNNQDIDEDGHQNNLDNCPYVPNANQADHDKDGKGDACDHDDD

[0342] NDGIPDDKDNCRLVPNPDQKDSDGDGRGDACKDDFDHDSVPDIDDICPENVDISETDFRFRFQMIPL

[0343] DPKGTSQNDPNWVVRHQGKELVQTVNCDPGLAVGYDEFNAVDFSGTFFINTERDDDYAGFVFGYQ

[0344] SSSRFYVVMWKQVTQSYWDTNPTRAQGYSGLSVKVVNSTTGPGEHLRNALWHTGNTPGQVRTLW HDPRHIGWKDFTAYRWRLSHRPKTGFI RVVMYEGKKIMADSGPIYDKTYAGGRRLGLFVFSQEMVFFS DLKYECRDP

[0345] SEQ. ID. N°10 : tTSP12-l

[0346] GISCDELSSMVLELRGLRTIVTTLQDSIRKVTEENKELANELRRPPLCYHNGVQYRNN EEWTVDSCTEC HCQNSVTICKKVSCPI MPCSNATVPDGECCPRCWPSDSAEDTDLDGWPNENLVCVANATYHCKKD NCPN LPNSGQEDYDKDGIGDACDDDDDNDKIPDDRDNCPFHYNPAQYDYDRDDVGDRCDNCPYN HNPDQADTDNNGEGDACAADI DGDGILNERDNCQYVYNVDQRDTDMDGVGDQCDNCPLEHNPD

[0347] QLDSDSDRIGDTCDN NQDIDEDGHQNN LDNCPYVPNANQADHDKDGKGDACDHDDDN DGIPDD KDNCRLVPNPDQKDSDGDGRGDACKDDFDHDSVPDI DDICPENVDISETDFRRFQMI PLDPKGTSQ NDPNWVVRHQGKELVQTVNSDPGLAVGYDEFNAVDFSGTFFINTERDDDYAGFVFGYQSSSRFYVV M WKQVTQSY W DTN PTR AQG YSG LS V KVV KSTTG PG EH LR N ALW HTG NTPGQV RTLW HDPRHIG

[0348] WKDFTAYRWRLSHRPKTGFIRVVMYEGKKIMADSGPIYDKTYAGGRGLLFVFSQEMVFFSDLKYECR DP

[0349] SEQ. ID. N°ll : tTSP12-2

[0350] NRI PESSSTSVLLTLDNNVVNGSSPAI RTNYIGH KTKDLQAICGISCDELSSMVLELRGLRTIVTTLQDSI R KVTEEN KELANELRRNPCTDGTHDCN KNAKCNYLGHYSDPMYRCECKPGYAGNGIICGEDTDLDGW PN EN LVCVANATYHCKKDNCPNLPNSGQEDYDKDGIGDACDDDDDNDKIPDDRDNCPFHYNPAQY DYDRDDVGDRCDNCPYNHN PDQADTDNNGEGDACAADI DGDGI LN ERDNCQYVYNVDQRDTDM DGVGDQCDNCPLEHNPDQLDSDSDRIGDTCDNNQDI DEDGHQNN LDNCPYVPNANQADHDKDG KGDACDH DDDNDGI PDDKDNCRLVPNPDQKDSDGDGRGDACKDDFDHDSVPDI DDICPENVDISE TDFRRFQMI PLDPKGTSQNDPNWVVRHQGKELVQTVNCDPGLAVGYDEFNAVDFSGTFFINTERD DDYAGFVFGYQSSSRFYVVMWKQVTQSYWDTNPTRAQGYSGLSVKVVNSTTGPGEHLRNALWHT

[0351] GNTPGQVRTLWH DPRHIGWKDFTAYRWRLSHRPKTGFIRVVMYEGKKI MADSGPIYDKTYAGGRL GLFVFSQEMVFFSDLKYECRDP

[0352] SEQ. ID. N°12 : tTSP8-l

[0353] NRI PESGGDNSVFDI FELTGAARKGSGRRLVKGPDPSSPAFRI EDANLI PPVPDDKFQDLVDAVRAEKG FLLLASLRQMKKTRGTLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEEALLATGQWK SITLFVQEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIAKGGVN DNFQGVLQNVRFVFGTTP EDI LRNKGCSSSTSVLLTLDNNVVNGSSPAI RTNYIGH KTKDLQAICGISCDELSSMVLELRGLRTIVTTL

[0354] QDSI RKVTEEN KELANELRRPPLCYHNGVQYRNN EEWTVDSCTECHCQNSVTICKKVSCPIMPCSNA TVPDGECCPRCWPSDSAEDTDLDGWPNEN LVCVANATYHCKKDNCPNLPNSGQEDYDKDGIGDAC DDDDDNDKIPDDRDNCPFHYNPAQYDYDRDDVGDRCDNCPYNHNPDQADTDNNGEGDACAADI

[0355] DGDGILNERDNCQYVYNVDQRDTDMDGVGDQCDNCPLEHNPDQLDSDSDRIGDTCDNNQDIDED

[0356] GHQNNLDNCPYVPNANQADHDKDGKGDACDHDDDNDGIPDDKDNCRLVPNPDQKDSDGDGRG

[0357] DACKDDFDHDSVPDIDDICPENVDISETDFRRFQMIPLDPKGTSQNDPNWVVRHQGKELVQTVNSD

[0358] PGLAVGYDEFNAVDFSGTFFINTERDDDYAGFVFGYQSSSRFYVVMWKQVTQSYWDTNPTRAQGY

[0359] SGLSVKVVKSTTGPGEHLRNALWHTGNTPGQVRTLWHDPRHIGWKDFTAYRWRLSHRPKTGFIRV

[0360] VMYEGKKIMADSGPIYDKTYAGGRLGLFVFSQEMVFFSDLKYECRDP

[0361] SEQ. ID. N°13 : tTSP8-2

[0362] NRIPESGGDNSVFDIFELTGAARKGSGRRLVKGPDPSSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKG

[0363] FLLLASLRQMKKTRGTLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEEALLATGQWK

[0364] SITLFVQEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIAKGGVNDNFQGVLQNVRFVFGTTP

[0365] EDILRNKGCSSSTSVLLTLDNNVVNGSSPAIRTNYIGHKTKDLQAICGISCDELSSMVLELRGLRTIVTTL

[0366] QDSIRKVTEENKELANELRRNPCTDGTHDCNKNAKCNYLGHYSDPMYRCECKPGYAGNGIICGEDTD

[0367] LDGWPNENLVCVANATYHCKKDNCPNLPNSGQEDYDKDGIGDACDDDDDNDKIPDDRDNCPFHY

[0368] NPAQYDYDRDDVGDRCDNCPYNHNPDQADTDNNGEGDACAADIDGDGILNERDNCQYVYNVDQ

[0369] RDTDMDGVGDQCDNCPLEHNPDQLDSDSDRIGDTCDNNQDIDEDGHQNNLDNCPYVPNANQAD

[0370] HDKDGKGDACDHDDDNDGIPDDKDNCRLVPNPDQKDSDGDGRGDACKDDFDHDSVPDIDDICPE

[0371] NVDISETDFRRFQMIPLDPKGTSQNDPNWVVRHQGKELVQTVNCDPGLAVGYDEFNAVDFSGTFFI

[0372] NTERDDDYAGFVFGYQSSSRFYVVMWKQVTQSYWDTNPTRAQGYSGLSVKVVNSTTGPGEHLRNA

[0373] LWHTGNTPGQVRTLWHDPRHIGWKDFTAYRWRLSHRPKTGFIRVVMYEGKKIMADSGPIYDKTYA

[0374] GGRLGLFVFSQEMVFFSDLKYECRDP

[0375] SEQ ID N°14 = new code TPS-1

[0376] 1 atggggctgg cctggggact aggcgtcctg ttcctgatgc atgtgtgtgg caccaaccgc

[0377] 61 attccagagt ctggcggaga caacagcgtg tttgacatct ttgaactcac cggggccgcc

[0378] 121 cgcaaggggt ctgggcgccg actggtgaag ggccccgacc cttccagccc agctttccgc

[0379] 181 atcgaggatg ccaacctgat cccccctgtg cctgatgaca agttccaaga cctggtggat

[0380] 241 gctgtgcggg cagaaaaggg tttcctcctt ctggcatccc tgaggcagat gaagaagacc

[0381] 301 cggggcacgc tgctggccct ggagcggaaa gaccactctg gccaggtctt cagcgtggtg

[0382] 361 tccaatggca aggcgggcac cctggacctc agcctgaccg tccaaggaaa gcagcacgtg

[0383] 421 gtgtctgtgg aagaagctct cctggcaacc ggccagtgga agagcatcac cctgtttgtg

[0384] 481 caggaagaca gggcccagct gtacatcgac tgtgaaaaga tggagaatgc tgagttggac 541 gtccccatcc aaagcgtctt caccagagac ctggccagca tcgccagact ccgcatcgca

[0385] 601 aaggggggcg tcaatgacaa tttccagggg gtgctgcaga atgtgaggtt tgtctttgga

[0386] 661 accacaccag aagacatcct caggaacaaa ggctgctcca gctctaccag tgtcctcctc

[0387] 721 acccttgaca acaacgtggt gaatggttcc agccctgcca tccgcactaa ctacattggc

[0388] 781 cacaagacaa aggacttgca agccatctgc ggcatctcct gtgatgagct gtccagcatg

[0389] 841 gtcctggaac tcaggggcct gcgcaccatt gtgaccacgc tgcaggacag catccgcaaa

[0390] 901 gtgactgaag agaacaaaga gttggccaat gagctgaggc ggcctcccct atgctatcac

[0391] 961 aacggagttc agtacagaaa taacgaggaa tggactgttg atagctgcac tgagtgtcac

[0392] 1021 tgtcagaact cagttaccat ctgcaaaaag gtgtcctgcc ccatcatgcc ctgctccaat

[0393] 1081 gccacagttc ctgatggaga atgctgtcct cgctgttggc ccagcgactc tgcggacgat

[0394] 1141 ggctggtctc catggtccga gtggacctcc tgttctcga gctgtggcaa tggaattcag

[0395] 1201 cagcgcggcc gctcctgcga tagcctcaac aaccgatgtg agggctcctc ggtccagaca

[0396] 1261 cggacctgcc acattcagga gtgtgacaag agatttaaac aggatggtgg ctggagccac

[0397] 1321 tggtccccgt ggtcatcttg ttctgtgaca tgtggtgatg gtgtgatcac aaggatccgg

[0398] 1381 ctctgcaact ctcccagccc ccgatgaac gggaaaccct gtgaaggcga agcgcgggag

[0399] 1441 accaaagcct gcaagaaaga cgcctgcccc atcaatggag gctggggtcc ttggtcacca

[0400] 1501 tgggacatct gttctgtcac ctgtggagga ggggtacaga aacgtagtcg tctctgcaac

[0401] 1561 aaccccacac cccagtttgg aggcaaggac tgcgttggtg atgtaacaga aaaccagatc

[0402] 1621 tgcaacaagc aggactgtcc aattgatgga tgcctgtcca atccctgctt tgccggcgtg

[0403] 1681 aagtgtacta gctaccctga tggcagctgg aaatgtggtg cttgtccccc tggttacagt

[0404] 1741 ggaaatggca tccagtgcac agatgttgat gagtgcaaag aagtgcctga tgcctgcttc

[0405] 1801 aaccacaatg gagagcaccg gtgtgagaac acggaccccg gctacaactg cctgccctgc

[0406] 1861 cccccacgct tcaccggctc acagcccttc ggccagggtg tcgaacatgc cacggccaac

[0407] 1921 aaacaggtgt gcaagccccg taacccctgc acggatggga cccacgactg caacaagaac

[0408] 1981 gccaagtgca actacctggg ccactatagc gaccccatgt accgctgcga gtgcaagcct

[0409] 2041 ggctacgctg gcaatggcat catctgcggg gaggacacag acctggatgg ctggcccaat

[0410] 2101 gagaacctgg tgtgcgtggc caatgcgact taccactgca aaaaggataa ttgccccaac

[0411] 2161 cttcccaact cagggcagga agactatgac aaggatggaa ttggtgatgc ctgtgatgat

[0412] 2221 gacgatgaca atgataaaat tccagatgac agggacaact gtccattcca ttacaaccca

[0413] 2281 gctcagtatg actatgacag agatgatgtg ggagaccgct gtgacaactg tccctacaac

[0414] 2341 cacaacccag atcaggcaga cacagacaac aatggggaag gagacgcctg tgctgcagac 2401 attgatggag acggtatcct caatgaacgg gacaactgcc agtacgtcta caatgtggac

[0415] 2461 cagagagaca ctgatatgga tggggttgga gatcagtgtg acaattgccc cttggaacac

[0416] 2521 aatccggatc agctggactc tgactcagac cgcattggag atacctgtga caacaatcag

[0417] 2581 gatattgatg aagatggcca ccagaacaat ctggacaact gtccctatgt gcccaatgcc

[0418] 2641 aaccaggctg accatgacaa agatggcaag ggagatgcct gtgaccacga tgatgacaac

[0419] 2701 gatggcattc ctgatgacaa ggacaactgc agactcgtgc ccaatcccga ccagaaggac

[0420] 2761 tctgacggcg atggtcgagg tgatgcctgc aaagatgatt ttgaccatga cagtgtgcca

[0421] 2821 gacatcgatg acatctgtcc tgagaatgtt gacatcagtg agaccgattt ccgccgattc

[0422] 2881 cagatgattc ctctggaccc caaagggaca tcccaaaatg accctaactg ggttgtacgc

[0423] 2941 catcagggta aagaactcgt ccagactgtc aactgtgatc ctggactcgc tgtaggttat

[0424] 3001 gatgagttta atgctgtgga cttcagtggc accttcttca tcaacaccga aagggacgat

[0425] 3061 gactatgctg gatttgtctt tggctaccag tccagcagcc gcttttatgt tgtgatgtgg

[0426] 3121 aagcaagtca cccagtccta ctgggacacc aaccccacga gggctcaggg atactcgggc

[0427] 3181 ctttctgtga aagttgtaaa ctccaccaca gggcctggcg agcacctgcg gaacgccctg

[0428] 3241 tggcacacag gaaacacccc tggccaggtg cgcaccctgt ggcatgaccc tcgtcacata

[0429] 3301 ggctggaaag atttcaccgc ctacagatgg cgtctcagcc acaggccaaa gacgggtttc

[0430] 3361 attagagtgg tgatgtatga agggaagaaa atcatggctg actcaggacc catctatgat

[0431] 3421 aaaacctatg ctggtggtag actaggttg tttgtctct ctcaagaat gtgttctc

[0432] 3481 tctgacctga atacgaatg tagagatccc taa

[0433] SEQ. ID. No. 15 : polynucleotide tTSP12-l

[0434] 1 ggcatctcct gtgatgagct gtccagcatg gtcctggaac tcaggggct gcgcaccatt

[0435] 61 gtgaccacgc tgcaggacag catccgcaaa gtgactgaag agaacaag gttggccaat

[0436] 121 gagctgaggc ggcctccct atgctatcac aacggagttc agtacagaaa taacgaggaa

[0437] 181 tggactgttg atagctgcac tgagtgtcac tgtcagact cagttaccat ctgcaaaag

[0438] 241 gtgtcctgcc ccatcatgcc ctgctccaat gccacagttc ctgatggaga atgctgtcct

[0439] 301 cgctgttggc ccagcgactc tgcggaggac acgacctgg atggctggcc caatgagac

[0440] 361 ctggtgtgcg tggccaatgc gacttaccac tgcaaaagg atattgcc caaccttccc

[0441] 421 aactcagggc aggagacta tgacagat ggaattggtg atgctgtga tgatgacgat

[0442] 481 gacaatgata aaattccaga tgacagggac aactgtccat tccattacaa cccagctcag

[0443] 541 tatgactatg acagagatga tgtgggagac cgctgtgaca actgtcccta caaccacaac

[0444] 601 ccagatcagg cagacacaga caacaatggg gaaggagacg cctgtgctgc agacattgat 661 ggagacggta tcctcaatga acgggacaac tgccagtacg tctacaatgt ggaccagaga

[0445] 721 gacactgata tggatggggt tggagatcag tgtgacaact gtcctctgga acacaatccg

[0446] 781 gaccaactgg atagtgacag cgacagaatt ggcgatacat gcgacaacaa tcaggatatc

[0447] 841 gacgaagatg ggcatcagaa caacctggat aactgtccct atgttcctaa cgccaatcag

[0448] 901 gccgatcacg ataaagacgg caaaggtgat gcctgtgatc atgacgacga caacgatggg

[0449] 961 attccagatg acaaggataa ttgccgtctt gtcccaaatc ccgaccagaa ggattccgac

[0450] 1021 ggtgatggaa gaggtgatgc gtgcaaagac gatttcgacc atgattccgt gccagacatc

[0451] 1081 gacgacatat gccctgaaaa tgtggacatt agcgagacag actttcgccg gtttcagatg

[0452] 1141 attcccctgg atcccaaagg cacgagtcag aatgatccta actgggtggt aaggcatcaa

[0453] 1201 ggcaaggagc tggtgcaaac cgtgaactca gaccctggac ttgcagtagg atatgacgag

[0454] 1261 ttcaacgctg tcgacttttc tggcacattc ttcatcaata ccgagcgaga tgatgactac

[0455] 1321 gcggggtttg tattcgggta tcagtcctct tcacgctttt acgttgtcat gtggaaacag

[0456] 1381 gtgactcagt cctactggga tactaaccca actcgggcac agggatacag cgggttgtcc

[0457] 1441 gttaaggtcg tcaagagcac aacaggacct ggcgaacatc tgcgaaatgc actgtggcac

[0458] 1501 actggcaata ctccaggaca agttcgcacc ctttggcacg atcccagaca catcggctgg

[0459] 1561 aaagacttta ccgcctatcg gtggaggctc tcacacaggc ccaaaaccgg tttcatacgg

[0460] 1621 gtggtgatgt acgagggaaa gaagatcatg gccgacagtg ggccgatcta cgataagacc

[0461] 1681 tatgctggcg gtagactcgg cctgtttgtg ttctctcagg agatggtgtt cttcagcgac

[0462] 1741 ttgaagtacg aatgcaggga cccctaa

[0463] SEQ. ID. N°16 : polynucléotide tTSP12-2

[0464] 1 aacagaatcc ctgagagcag cagcacaagc gtgctgctga ccctggacaa caacgtggtc

[0465] 61 aatggcagca gccctgccat ccggaccaat tacatcggcc acaagaccaa ggacctgcag

[0466] 121 gccatctgtg gcatcagctg tgatgagctg agcagcatgg tgctggaact gagaggcctg

[0467] 181 cggaccatcg tgaccacact gcaggacagc atccggaaag tgaccgagga aaacaaagag

[0468] 241 ctggccaacg agctgcggag aaacccttgt accgatggca cccacgactg caacaagaac

[0469] 301 gccaagtgca actacctggg ccactacagc gaccccatgt acagatgcga gtgcaagcct

[0470] 361 ggctatgccg gcaacggcat catctgcggc gaggacacag atctggacgg ctggcccaat

[0471] 421 gagaacctcg tgtgtgtggc caacgccacc taccactgca agaaggacaa ctgccccaac

[0472] 481 ctgcctaaca gcggccaaga ggactacgac aaggatggaa tcggcgacgc ctgcgacgac

[0473] 541 gacgatgaca acgacaagat ccccgacgac cgggacaatt gccccttcca ctacaacccc

[0474] 601 gctcagtacg actacgatag ggacgacgtg ggcgatagat gcgataactg cccctacaat 661 cacaaccccg accaggccga caccgacaac aatggcgaag gcgacgcatg tgccgccgat

[0475] 721 attgatggcg acggcatcct gaacgagcgg gacaactgtc agtacgtgta caacgtggac

[0476] 781 cagcgggaca ctgacatgga cggcgttggc gaccagtgcg ataattgtcc cctggaacac

[0477] 841 aatcccgatc agctggacag cgactccgac aggatcggcg atacctgtga caacaatcag

[0478] 901 gacatcgacg aggacggcca ccagaacaac ctggataatt gcccttacgt gcccaacgcc

[0479] 961 aatcaggccg accacgataa ggacggaaag ggcgacgctt gtgaccacga tgacgacaat

[0480] 1021 gatggcatcc ccgatgacaa ggataactgc agactggtgc ccaatcctga ccagaaggat

[0481] 1081 agcgacggcg acggaagagg ggatgcctgc aaggacgact tcgatcacga tagcgtgccc

[0482] 1141 gacatcgatg acatctgccc cgagaacgtg gacatcagcg agacagactt ccggcggttc

[0483] 1201 cagatgatcc ctctggatcc taagggcacc agccagaacg accctaactg ggtcgtcaga

[0484] 1261 caccagggca aagaactggt gcagaccgtg aactgcgatc ctggactggc cgtgggctac

[0485] 1321 gacgagttca atgccgtgga tttcagcggc accttcttca tcaacaccga gcgggatgac

[0486] 1381 gactacgccg gcttcgtgtt tggctaccag agcagctccc ggttctacgt ggtcatgtgg

[0487] 1441 aagcaagtga cccagagcta ctgggacaca aaccccacaa gagcccaggg ctactctggc

[0488] 1501 ctgtctgtga aggtggtcaa cagcacaaca ggccctggcg agcacctgag aaatgccctg

[0489] 1561 tggcacacag gcaacacccc aggacaagtt cggacactgt ggcacgaccc tagacacatc

[0490] 1621 ggctggaagg acttcaccgc ctacagatgg cggctgagcc acaggcctaa gaccggcttt

[0491] 1681 atccgggtcg tgatgtacga gggcaagaaa atcatggccg acagcggccc catctacgat

[0492] 1741 aagacatatg ccggcggaag gctgggcctg ttcgtgttct ctcaagagat ggtgttcttc

[0493] 1801 agcgacctga agcgacctg cagggacccc taa

[0494] SEQ. ID. No. 17: tTSP8-l polynucleotide

[0495] 1 aaccgcattc cagagtctgg cggagacaac agcgtgtttg acatctttga actcaccggg

[0496] 61 gccgcccgca aggggtctgg gcgccgactg gtgaagggcc ccgacccttc cagcccagct

[0497] 121 ttccgcatcg aggatgccaa cctgatcccc cctgtgcctg atgacaagtt ccaagacctg

[0498] 181 gtggatgctg tgcgggcaga aaagggtttc ctccttctgg catccctgag gcagatgag

[0499] 241 aagacccggg gcacgctgct ggccctggag cggaagacc actctggcca ggtcttcagc

[0500] 301 gtggtgtcca atggcaaggc gggcaccctg gacctcagcc tgaccgtcca aggaaagcag

[0501] 361 cacgtggtgt ctgtggaga agctctcctg gcaaccggcc agtggagag catcaccctg

[0502] 421 tttgtgcagg aagacagggc ccagctgtac atcgactgtg aaaagatgga gaatgctgag

[0503] 481 ttggacgtcc ccatccaaag cgtcttcacc agagacctgg ccagcatcgc cagactccgc

[0504] 541 atcgcaaagg ggggcgtcaa tgacaatttc cagggggtgc tgcagaatgt gaggtttgtc 601 tttggaacca caccagaaga catcctcagg aacaaaggct gctccagctc taccagtgtc

[0505] 661 ctcctcaccc ttgacaacaa cgtggtgaat ggttccagcc ctgccatccg cactaactac

[0506] 721 attggccaca agacaaagga cttgcaagcc atctgcggca tctcctgtga tgagctgtcc

[0507] 781 agcatggtcc tggaactcag gggcctgcgc accattgtga ccacgctgca ggacagcatc

[0508] 841 cgcaaagtga ctgaagagaa caaagagttg gccaatgagc tgaggcggcc tcccctatgc

[0509] 901 tatcacaacg gagttcagta cagaaataac gaggaatgga ctgttgatag ctgcactgag

[0510] 961 tgtcactgtc agaactcagt taccatctgc aaaaaggtgt cctgccccat catgccctgc

[0511] 1021 tccaatgcca cagttcctga tggagaatgc tgtcctcgct gttggcccag cgactctgcg

[0512] 1081 gaggacacag acctggatgg ctggcccaat gagaacctgg tgtgcgtggc caatgcgact

[0513] 1141 taccactgca aaaaggataa ttgccccaac cttcccaact cagggcagga agactatgac

[0514] 1201 aaggatggaa ttggtgatgc ctgtgatgat gacgatgaca atgataaaat tccagatgac

[0515] 1261 agggacaact gtccattcca ttacaaccca gctcagtatg actatgacag agatgatgtg

[0516] 1321 ggagaccgct gtgacaactg tccctacaac cacaacccag atcaggcaga cacagacaac

[0517] 1381 aatggggaag gagacgcctg tgctgcagac attgatggag acggtatcct caatgaacgg

[0518] 1441 gacaactgcc agtacgtcta caatgtggac cagagagaca ctgatatgga tggggttgga

[0519] 1501 gatcagtgtg acaactgtcc tctggaacac aatccggacc aactggatag tgacagcgac

[0520] 1561 agaattggcg atacatgcga caacaatcag gatatcgacg aagatgggca tcagaacaac

[0521] 1621 ctggataact gtccctatgt tcctaacgcc aatcaggccg atcacgataa agacggcaaa

[0522] 1681 ggtgatgcct gtgatcatga cgacgacaac gatgggattc cagatgacaa ggataattgc

[0523] 1741 cgtcttgtcc caaatcccga ccagaaggat tccgacggtg atggaagagg tgatgcgtgc

[0524] 1801 aaagacgatt tcgaccatga ttccgtgcca gacatcgacg acatatgccc tgaaaatgtg

[0525] 1861 gacattagcg agacagactt tcgccggttt cagatgattc ccctggatcc caaaggcacg

[0526] 1921 agtcagaatg atcctaactg ggtggtaagg catcaaggca aggagctggt gcaaaccgtg

[0527] 1981 aactcagacc ctggacttgc agtaggatat gacgagttca acgctgtcga cttttctggc

[0528] 2041 acattcttca tcaataccga gcgagatgat gactacgcgg ggtttgtatt cgggtatcag

[0529] 2101 tcctcttcac gcttttacgt tgtcatgtgg aaacaggtga ctcagtccta ctgggatact

[0530] 2161 aacccaactc gggcacagggattackagcggg ttgtccgtta aggtcgtcaa gagcacaaca

[0531] 2221 ggacctggcg aacatctgcg aaatgcactg tggcacactg gcaatactcc aggacaagtt

[0532] 2281 cgcacccttt ggcacgatcc cagacacatc ggctggaaag actttaccgc ctatcggtgg

[0533] 2341 aggctctcac acaggcccaa aaccggtttc atacgggtgg tgatgtacga gggaaagaag

[0534] 2401 atcatggccg acagtgggcc gatctacgat aagacctatg ctggcggtag actcggcctg 2461 tttgtgttct ctcaggagat ggtgttcttc agcgacttga agtacgaatg cagggacccc

[0535] 2521 city

[0536] SEQ. ID. N°18 : polynucleotide tTSP8-2

[0537] 1 aacagaatcc ctgagtctgg cggcgacaac agcgtgttcg acatctttga actgaccggc

[0538] 61 gctgcccgga aaggatctgg aagaaggctg gtcaagggcc ccgatcctag cagccctgcc

[0539] 121 ttcagaatcg aggacgccaa tctgatccct cctgtgcctg acgacaagtt ccaggacctg

[0540] 181 gtggatgccg tgcgggccga gaaaggattt ctgctgctgg cctctgcg gcagatgaag

[0541] 241 aaaaccagag gcaccctgct ggccctggaa agaaaggatc acagcggcca ggtgttcagc

[0542] 301 gtggtgtcca atggaaaggc cggcacactg gatctgagcc tgacagtgca gggaaagcag

[0543] 361 cacgtggtgt ctgtggaaga agctctgctg gctaccggcc agtggaagtc tatcaccctg

[0544] 421 ttcgtgcaag aggacagagc ccagctgtac atcgactgcg agaagatgga aaacgccgag

[0545] 481 ctggacgtgc ccatccagag cgtgttcaca agagatctgg cctctatcgc ccggctgaga

[0546] 541 atcgctaaag gcggcgtgaa cgataacttc cagggcgtgc tgcagaacgt ccgcttcgtg

[0547] 601 tttggcacca cacctgagga catcctgcgg aacaagggct gtagcagcag cacaagcgtg

[0548] 661 ctgctgacac tggacaacaa cgtggtcaac ggcagcagcc cagccatccg gacaaattac

[0549] 721 atcggccaca agaccaagga cctgcaggcc atctgtggca tcagctgtga tgagctgagc

[0550] 781 agcatggtgc tggaactgag aggcctgcgg accatcgtga ccacactgca ggacagcatc

[0551] 841 cggaaagtga ccgaggaaaa caaagagctg gccaacgagc tgcggagaaa cccttgtacc

[0552] 901 gatggcaccc acgactgcaa caagaacgcc aagtgcaact acctgggcca ctacagcgac

[0553] 961 cccatgtaca gatgcgagtg caagcctggc tatgccggca acggcatcat ctgcggcgag

[0554] 1021 gacacagatc tggacggctg gcccaatgag aacctcgtgt gtgtggccaa cgccacctac

[0555] 1081

[0556] 1141

[0557] 1201 gacaattgcc ccttccacta caaccccgct footcgact acgataggga cgacgtgggc

[0558] 1261 gacagatgtg ataactgccc ctacaatcac aaccccgacc aggccgacac cgacaacaat

[0559] 1321 ggcgaaggcg acgcatgtgc cgccgatatt gatggcgacg gcatcctgaa cgagcgggac

[0560] 1381 aactgtcagt acgtgtacaa cgtggaccag cgggacactg acatggatgg cgtgggagat

[0561] 1441

[0562] 1501 atcggcgata cctgcgataa caaccaggac atcgacgagg acggccacca gaacaacctg

[0563] 1561 gataactgtc cttacgtgcc caacgccaat caggccgacc acgataagga cggaaaggc

[0564] 1621 gacgcttgtg accacgatga cgacaatgat ggcatccccg atgacaagga taactgcaga 1681 ctggtgccca atcctgacca gaaggatagc gacggcgacg gaagagggga tgcctgcaag

[0565] 1741 gacgacttcg atcacgatag cgtgcccgac atcgatgaca tctgccccga gaacgtggac

[0566] 1801 atcagcgaga cagacttccg gcggttccag atgatccctc tggatcctaa gggcaccagc

[0567] 1861 cagaacgacc ctaactgggt cgtcagacac cagggcaaag aactggtgca gaccgtgaac

[0568] 1921 tgcgatcctg gactggccgt gggctacgac gagttcaatg ccgtggattt cagcggcacc

[0569] 1981 ttcttcatca acaccgagcg ggatgacgac tacgccggct ttgtgttcgg ctaccagagc

[0570] 2041 agctcccggt tctacgtggt catgtggaag caagtgaccc agagctactg ggacacaaac

[0571] 2101 cccacaagag cccagggcta ctctggcctg tctgtgaagg tggtcaacag cacaacaggc

[0572] 2161 cctggcgagc acctgagaaa tgccctgtgg cacacaggca acacccctgg acaagttcgg

[0573] 2221 acactgtggc acgaccctag acacatcggc tggaaggact tcaccgccta cagatggcgg

[0574] 2281 ctgagccaca ggcctaagac cggctttatc cgggtcgtga tgtacgaggg caagaaaatc

[0575] 2341 atggccgaca gcggccccat ctacgataag acatatgccg gcggaaggct gggcctgttt

[0576] 2401 gtgttctctc aagagatggt gttcttcagc gacctgaagt acgagtgtcg ggaccctaa

[0577] SEQ. ID. N 19: TSP-1-derived 4N1K peptide

[0578] KRFYVVMWKK

Claims

DEMANDS 1. Protein derived from TSP-1 comprising a formula (I): X1-L1-X2 (I) where - XI comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, of identity with SEQ. ID. No. 2 and necessarily includes a cysteine ​​at position 5 of SEQ. ID. No. 2; - X2 comprises a sequence having at least 95%, and in order of preference 98%, 99% and 100%, identity with SEQ ID No. 3 and necessarily comprises two WM fragments, one at positions 1-3 and the other at positions 87-89 of SEQ ID No. 3; and - L1 is absent or a spacer peptide comprising between 1 and 100 amino acids; said TSP-1 derived protein being devoid of the type 1 repeat domain naturally present between positions 361 and 470 of native SEQ TSP-1. ID. No.

1.

2. TSP-1 derived protein of formula (I) according to claim 1, characterized in that it is also devoid of all or part of the type 2 repeat domain present between positions 471 and 672 of native TSP-1 of SEQ. ID. No.

1.

3. TSP-1 derived protein of formula (I) according to claim 1 or claim 2, characterized in that XI comprises all or part of the fragment of SEQ. ID. No.

4.

4. TSP-1 derived protein of formula (I) according to any one of the preceding claims, characterized in that X2 comprises all or part of the fragment of SEQ. ID. No. 9 including SEQ. ID. No. 3 and the two WM fragments present at positions 1-3 and 87-89 of SEQ. ID. No.

3.

5. TSP-1 derived protein of formula (I) according to any one of the preceding claims, said protein having the sequence SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 12 or SEQ. ID. No. 13, preferably SEQ. ID. No.

11. 68 6. Polynucleotide encoding a protein derived from TSP-1 according to any one of the preceding claims.

7. Expression vector comprising one or more polynucleotides according to claim 6.

8. Composition comprising a protein derived from TSP-1 or a polynucleotide or an expression vector according to any one of the preceding claims.

9. Proteins derived from TSP-1 according to any one of claims 1 to 5, for use in the prevention and / or treatment of inflammation, preferably inflammation associated with an accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils; more preferably, retinal inflammation, such as age-related macular degeneration (AMD), age-related maculopathy, uveitis or retinitis pigmentosa; neurodegenerative diseases, such as Parkinson's disease, multiple sclerosis or Alzheimer's disease; metabolic disorders, such as obesity or atherosclerosis; allergies; ankylosing spondylitis; arthritis, such as osteoarthritis, rheumatoid arthritis or psoriatic arthritis; asthma, graft-versus-host disease; peritonitis, Crohn's disease; colitis; dermatitis; diverticulitis; fibromyalgia; hepatitis;Irritable bowel syndrome; systemic lupus erythematosus; nephritis; or ulcerative colitis.

10. Polynucleotide according to claim 6, for use in the prevention and / or treatment of inflammation, preferably inflammation associated with an accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils; more preferably, retinal inflammation, such as age-related macular degeneration (AMD), age-related maculopathy, uveitis or retinitis pigmentosa; neurodegenerative diseases, such as Parkinson's disease, multiple sclerosis or Alzheimer's disease; metabolic disorders, 69 such as obesity or atherosclerosis; allergies; ankylosing spondylitis; arthritis, such as osteoarthritis, rheumatoid arthritis or psoriatic arthritis; asthma, graft-versus-host disease; peritonitis, Crohn's disease; colitis; dermatitis; diverticulitis; fibromyalgia; hepatitis; irritable bowel syndrome; systemic lupus erythematosus; nephritis; or ulcerative colitis.

11. Composition according to claim 8 for their use in the prevention and / or treatment of inflammation, preferably inflammation associated with an accumulation of mononuclear phagocytes and / or other leukocytes such as lymphocytes and neutrophils; more preferably, retinal inflammation, such as age-related macular degeneration (AMD), age-related maculopathy, uveitis or retinitis pigmentosa; neurodegenerative diseases, such as Parkinson's disease, multiple sclerosis or Alzheimer's disease; metabolic disorders, such as obesity or atherosclerosis; allergies; ankylosing spondylitis; arthritis, such as osteoarthritis, rheumatoid arthritis or psoriatic arthritis; asthma, graft-versus-host disease; peritonitis, Crohn's disease; colitis; dermatitis; diverticulitis; fibromyalgia; hepatitis; irritable bowel syndrome;Systemic lupus erythematosus; nephritis; or ulcerative colitis.

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