FR3135992A1 - Molecular signature of actinic lentigo associated with vesicle management - Google Patents

Abstract

Molecular signature of actinic lentigo associated with vesicle management The present invention relates to a method for characterizing an apparent or suspected skin pigment spot in a human being, comprising the comparison of expression levels, in skin samples from said spot and adjacent uninjured skin, of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, NAV3 and PCLO, and possibly the MREG gene. The invention also relates to methods for evaluating the effectiveness of a treatment of pigment spots, cosmetic methods of treating pigment spots, as well as various modulators of said genes and their use.

Description

Molecular signature of actinic lentigo associated with vesicle management

The present invention is in the cosmetic field, it relates to the skin. It is more generally part of the characterization of pigment spots on the skin and the treatment of these spots.

Skin color is mainly due to the presence of a pigment, melanin, in the epidermis. Melanin is synthesized by specific dendritic cells located in the basal layer of the epidermis, the melanocytes. Melanogenesis takes place in organelles, the melanosomes which, loaded with melanin, are transferred to neighboring epidermal cells, the keratinocytes, via dendrites. The color of the skin, or constitutive pigmentation, varies between individuals depending on the quantity of melanin produced as well as the chemical nature of melanins. Melanins are macromolecules formed from tyrosine (eumelanin) or tyrosine and cysteine (pheomelanin). The synthesis mechanisms involve enzymes, the main ones being tyrosinase and tyrosinase-associated protein (Tyrp-1). Naturally, skin pigmentation is stimulated by exposure to the sun, this is the phenomenon of tanning.

However, there are situations where the pigmentation process is altered, and which can lead to pigmentation defects, hypopigmentations (vitiligo, albinism) or conversely to excess pigmentation, hyperpigmentations. Among the benign hyperpigmentary disorders, characterized by an abnormal accumulation (apart from tanning) of melanin, we can cite actinic lentigo, melasma, the pigmentary after-effects of acne, post-inflammatory pigmentation, meadow dermatitis, or even dychromia. benign facial symptoms.

Actinic lentigo, also called senile or solar lentigo, senile spot, age spot, or commonly referred to as "senile filth", "cemetery daisies" or "cemetery flowers", is by far the most common pigmentary lesion. This type of lesion appears on areas of the skin that have been photoexposed, such as the face, the back of the hands, the upper limbs and in particular the dorsal side of the forearms, the back, and in particular the upper back. They generally affect individuals aged 40 and over.

Macroscopically, actinic lentigos are represented by benign pigmented macules of more or less dark brown color, the edges of which are clear but irregular. Very variable in size, they can extend from a few millimeters to more than two centimeters.

Despite its high frequency, few studies have been published on the physiology and genesis of actinic lentigo. On the basis of the rare existing histological studies, it is known that there is an increase in the overall epidermal melanin load and more particularly at the level of the basal layer. There is also an elongation of the epidermal ridges which can take on a club-like appearance in the papillary dermis (Montagna W, Hu F, Carlisle K. “A reinvestigation of solar lentigines”. Arch Dermatol. 1980; Ber Rahman, S. Bhawan, J . Lentigo. Int J Dermatol, 1996, Apr;35(4):229-39; Andersen WK, Labadie RR, Bhawan J. “Histopathology of solar lentigines of the face: a quantitative study” J Am Acad Dermatol. 1997 Mar; 36(3 Pt 1):444-7; Cario-Andre M, Lepreux, S, Pain C, Nizard C, Noblesse E, Taïeb A. “Prelesional vs. Lesional skin changes in senile lentigo” 2004). Finally, there may be anastomoses between adjacent ridges giving a bridge or network appearance.

Pigmentary incontinence with the presence of melanin and melanophages can also exist in the dermis.

Benign skin pigment disorders are generally considered unsightly. Due to the proven association between the appearance of actinic lentigo and chronic exposure to the sun, preventing their appearance generally involves the topical application of so-called photoprotective products such as sunscreens. In the case of “curative” treatments, numerous processes, mainly for cosmetic purposes, have therefore been developed in an attempt to eliminate them or reduce their presence. The elimination or reduction of the presence of these disorders is usually based on the application of depigmenting treatments, based on the reduction of melanin synthesis activity in melanocytes. Depigmenting molecules interfere with one or more stages of melanogenesis. One of the pathways of the main products used to date is based on the inhibition of tyrosinase, one of the key enzymes in the melanogenesis process. The goal of these treatments is to reduce or even stop pigment synthesis.

The main known depigmenting substances are hydroquinone and its derivatives, kojic acid, arbutin, iminophenols, ascorbic acid and its derivatives, the combination of carnitine and quinone, amino-phenol derivatives, and benzothiazole derivatives, natural extracts, corticosteroids.

We often also find associated with these active agents exfoliants to increase desquamation, and thus to more easily eliminate the melanin present in the stratum corneum.

Another non-cosmetic treatment method involves destroying the lesions by physical or chemical means using lasers or peels. However, these are quite cumbersome procedures that do not address the etiology of the disorder. In the majority of cases, actinic lentigo reappears some time after treatment.

Furthermore, there are major drawbacks to existing treatments. Depigmenting substances may present in particular a certain instability, low effectiveness at low concentrations, biological activity affecting other undesirable functions.

In addition, because they do not target the root cause that led to the dysregulation of pigmentation, there is great heterogeneity in response to different depigmenting treatments. Thus, for a given treatment, administered for example topically, a given benign skin pigment disorder, such as actinic lentigo or melasma, may improve or disappear, while another will not progress. This variability can be observed between lesions in different individuals but also in the same individual, between different lesions. There are therefore benign skin pigment disorders for which no topical treatment is effective to date.

There is therefore a need to find more effective and harmless treatments for this type of lesion.

To do this, it is necessary to identify the molecular deregulations and functional dysfunctions that may exist in this type of pigmentary disorder in order to be able to identify new targets and select active agents correcting these defects.

In the prior art, concerning the treatment of pigment spots and more particularly actinic lentigos, the stimulation at the genomic or protein level of molecules which are closely associated with melanocytes and melanogenesis (melanogenesis enzymes, melanosome protein, key paracrine factors of melanogenesis). It is found in the epidermis or dermis for the following proteins: Tyrosinase, TRP1, DCT, Pmel-17, POMC, ET1, ETBR, SCF, c-KIT, KGF, KGFR, Hepatocyte growth factor (HGF), MIA, TRPM1, Melan-A, Pink eye dilution, P53 and IL1α.

However, it has recently become known that melanogenesis does not represent the major biological mechanism involved in the appearance of pigment spots and in particular actinic lentigo (Warrick et al Br J Dermatol 2017, Aoki Br J Dermatol 2007).

The present inventors have demonstrated for the first time the involvement of genes linked to the management (in particular transport, fusion, accumulation, retention, degradation) of vesicles, preferably melanosomes, in pigmentary disorders. resulting in pigment spots on the skin.

In particular, it has been shown by the present inventors that certain genes linked to the management of vesicles, preferably melanosomes, have transcription levels that differ significantly between healthy skin and skin resulting from a pigment spot, putting thus highlighting the link between deregulation of the management of vesicles, preferably melanosomes, within the skin, preferably the epidermis, and modulation of pigmentation inducing in particular the appearance of pigment spots.

The melanosome is an intracellular organelle in the form of a vesicle, said vesicle being specialized in the production of melanin, a pigment protecting human skin from solar radiation.

It is produced in melanocytes. Surrounded by a lipid membrane, its shape varies from rounded to elongated. In human skin, melanosomes do not remain in the melanocytes, but are evacuated towards the keratinocytes along arms resembling the dendrites of neurons (the melanocytes are of neural origin), in which they move carried by the kinesins, along a system composed of microtubules, actin, intermediate filaments and associated proteins. This transport can be triggered or intensified by certain factors, notably by the intensity of ultraviolet radiation (tanning).

Thus, keratinocytes distribute melanosomes above their nucleus, protecting it from damage due to UV radiation. The number of melanosomes produced by the melanocyte, their size, their concentration of melanins and the nature of these (eumelanin or pheomelanin) , as well as the way the keratinocyte distributes them above its nucleus, are hereditary characteristics that influence the pigmentation of human skin.

Documents FR2974370, FR2974372, FR2974373, and FR2974371 although highlighting molecular deregulations or functional dysfunctions in actinic lentigo, in particular with the involvement of genes of the TGFβ-SMAD biological signaling pathway, genes participating in the remodeling of the extracellular matrix, genes coding for extracellular proteoglycans and glycoproteins, these do not disclose genes linked to the management of vesicles, in particular melanosomes.

Thus, the present invention has as its first object a method for characterizing an apparent or suspected skin pigment spot in a human being, comprising the comparison of the expression levels, in samples of skin from said spot and adjacent skin not injured, of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3 , RASIP1, ASGR1, CHMP4C, SVIP, LRMP, NAV3 and PCLO, and possibly the MREG gene.

The second object of the present invention is a method of evaluating the effectiveness of a treatment of skin pigment spots, comprising the comparison of the expression levels in a sample of skin resulting from such a stain, before and after treatment, at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1 , ASGR1, CHMP4C, SVIP, LRMP, NAV3 and PCLO, and possibly the MREG gene.

Another object of the present invention is an in vitro method for screening active agent for preventing and/or treating skin pigment spots, said method comprising the following steps:
i. bring into contact a cellular model representative of the skin with at least one active agent to be tested, or expose said cellular model to a physical treatment to be tested;
ii. carry out a measurement of the expression level of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1 , SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG, or the level of expression or activity of the expression product of said chosen gene, and
iii. compare said measurement to a reference measurement.

The invention also relates to a cosmetic method for treating or preventing non-pathological cutaneous pigment spots in human skin, comprising the application to the skin of a composition comprising at least one active agent modulating the level of expression or of the activity of a gene involved in the management of vesicles, preferably melanosomes, where said gene is chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29 , RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG.

The invention further relates to the cosmetic use of a modulator of the expression level or activity of the expression product of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG in the treatment of pigment spots non-pathological skin.

The present invention further relates to a method for preparing a cellular model capable of screening active agents to prevent and/or treat skin pigment spots, comprising the following steps:
i. collect keratinocytes and possibly melanocytes; Then
ii. transfect said keratinocytes taken in step i. using siRNA capable of repressing the level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG; and/or transfect said keratinocytes taken in step i. using an expression vector, preferably a plasmid, of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes, Then
iii. Cultivate the transfected keratinocytes in step ii. or co-culture the keratinocytes transfected in step ii. with the melanocytes collected in step i.

Another of its objects is a cellular model capable of screening active agents to prevent and/or treat skin pigment spots, capable of being obtained or directly obtained according to the preparation method according to the invention.

Another object of the present invention is a cellular model capable of screening active agents for preventing and/or treating skin pigment spots, comprising keratinocytes and optionally melanocytes, in which said keratinocytes have an expression level of at least a gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG , repressed and/or in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes ARC, NRN1, SYNPO2, RASIP1, and LRMP , overexpressed.

The invention further relates to an in vitro method for screening active agent for preventing and/or treating skin pigment spots, said method comprising the following steps:
i. bring into contact a cellular model comprising keratinocytes and possibly melanocytes, in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of JAKMIP2 genes , KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG, repressed and/or in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes, overexpressed with at least one active agent to be tested, or exposing said cellular model to a physical treatment to be tested ;
ii. carry out a quantitative or qualitative measurement of melanin and/or a measurement of the expression level of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP and PCLO, NAV3 and MREG, or the level of expression or activity of the product expression of said chosen gene, and comparing said measurement to a reference measurement.

Said genes mean human genes so called.

Definitions

By “level of expression of a gene”, we preferably mean the level of transcription and/or the level of translation of said gene, more preferably the level of transcription of said gene.

Concerning the evaluation of the level of transcription of the chosen gene, it can be carried out in different ways well known to those skilled in the art, directly or after reverse transcription. The level of transcription can in particular be assessed by using RNA chips or DNA chips sold commercially for this purpose. A possible evaluation method is described in the experimental part.

By “vesicle” we mean a more or less spherical structure formed of a biological membrane, in particular a lipid bilayer, closed on itself, present at the level of skin cells, preferably at the level of keratinocytes.

Preferably the vesicles are intracellular transport vesicles, even more preferably the vesicles are melanosomes.

By “management of vesicles” or “management of melanosomes” is meant within the meaning of the present invention, the transport, fusion, accumulation, retention, degradation of said vesicles or said melanosomes.

The names of the aforementioned genes as well as the functional biological families to which they belong are described in Table 1 below.

Subfamily Symbol Name Publications Intracellular traffic JAKMIP2 janus kinase and microtubule interacting protein 2 Cruz-Garcia Bichem J: The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells BOW activity-regulated cytoskeleton-associated protein Fila Neural plasticity 2021: mRNA Trafficking in the Nervous System: A Key Mechanism of the Involvement of Activity-Regulated Cytoskeleton-Associated Protein (Arc) in Synaptic Plasticity-
Wu Cell 2011: Arc/Arg3.1 Regulates an Endosomal Pathway Essential for Activity-Dependent b-Amyloid Generation
Pastuzin Cell 2017 The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer Kinesins KIF21A kinesin family member 21A Hirokawa Physiol Rev 2008 Intracellular Transport and Kinesin Superfamily Proteins, KIFs: Structure, Function, and Dynamics (Review)
Niwa Anatomical Science International 2015: Kinesin superfamily proteins and the regulation of microtubule dynamics in morphogenesis Axon growth NAV3 neuron navigator 3 Stringham Cell Adhesion & Migration 2009: Navigating the cell/UNC-53 and the navigators, a family of cytoskeletal regulators with multiple roles in cell migration, outgrowth and trafficking SLITRK6 SLIT and NTRK-like family. member 6 Aruga Mol Cell Neuroscience 2003: Identification and characterization of Slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth NRN1 neuritin 1 Zito Brain Struct functio n 2014: Neuritin 1 promotes neuronal migration - Di Giovanni Faseb J 2005: Neuronal plasticity after spinal cord injury: identification of a gene cluster driving neurite outgrowth Cytoskeletal organization SCIN splitter Trifano Can J Physiol Pharmacol. 1999: Scinderin and cortical F-actin are components of the secretory machinery- Trifaro Neurochem Res 2000: Scinderin, a Ca2+-dependent actin filament severing protein that controls cortical actin network dynamics during secretion; SYNPO2 synaptopodin 2 Kannan and Tang JCB 2015: Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation- Kingsbury Biochem 2013: Avian Synaptopodin 2 (Fesselin) Stabilizes Myosin Filaments and Actomyosin in the Presence of ATP Contractile activity TNNT1 troponin T type 1 (skeletal. slow) I Cohen Methods Achiev Exp Pathol 1979: The contractile system of blood platelets and its function- Tobacman Biophys J 2021:Troponin Revealed: Uncovering the Structure of the Thin Filament On-Off Switch in Striated Muscle- Wei GENE 2016: TNNT1, TNNT2, and TNNT3: Isoform genes, regulation, and structure–function relationships SNTB1 syntrophin, beta 1 (dystrophin-associated protein A1, 59kDa, basic component 1) Iwata Eur J Cell Biol 2004: Syntrophin is an actin-binding protein the cellular localization of which is regulated through cytoskeletal reorganization in skeletal muscle cells Rho/Rab family ARHGAP29 Rho GTPase activating protein 29 Post, et al. 2013 Rasip1 mediates Rap1 regulation of Rho in endothelial barrier function through ArhGAP29 - Biggs J Cell SCIENCE 2014: Interferon regulatory factor 6 regulates keratinocyte migration RAB3IP RAB3A interacting protein (rabin3) Brondyk Mol cell biol 1995: Interaction Cloning of Rabin3, a Novel Protein That Associates with the Ras-Like GTPase Rab3A- Hattula Method in Enzymol 2005: Purification and functional properties of a Rab8-specific GEF (Rabin3) in action remodeling and polarized transport RHOBTB3 Rho-related BTB domain containing 3 Espinosa Cell 2009:RhoBTB3: A Rho GTPase-family ATPase required for endosome to Golgi transport RASIP1 Ras interacting protein 1 Post, et al. 2013 Rasip1 mediates Rap1 regulation of Rho in endothelial barrier function through ArhGAP29 Endocytosis ASGR1 asialoglycoprotein receptor 1 Newman Cell 2021: Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyzes - Susan-Resiga JBC 2021: Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR cleaved by furin CHMP4C charged multivesicular body protein 4C Russel Mbio 2021:Novel Role for ESCRT-III Component CHMP4C in the Integrity of the Endocytic Network Utilized for Herpes Simplex Virus Envelopment - Tauro Methods 2012: Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863 -derived exosomes Autophagy Please small VCP/p97-interacting protein Wang PlosOne 2011: SVIP Induces Localization of p97/VCP to the Plasma and Lysosomal Membranes and Regulates Autophagy Vesicle management LRMP lymphoid-restricted membrane protein Behrens J Immonol 1994: Jaw1, A lymphoid-restricted membrane protein localized to the endoplasmic reticulum PCLO piccolo (presynaptic cytomatrix protein) leal Ortiz JCB 2008: Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis - Mukherjee PNAS 2010: Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis MREG melanoregulin Moore 1988: Mreg’s role in the suppression of Rab27a-Mlph-Myo5a–deficient phenotype over 30 years ago, O’Sullivan, 2004: melanosome center aggregation Ohbayashi, 2012: microtubules mediated retrograde transport as a cargo receptor

detailed description

Characterization method

The subject of the present invention is a method for characterizing an apparent or suspected skin pigment spot in a human being, comprising the comparison of the expression levels, in samples of skin from said spot and adjacent uninjured skin, at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, NAV3 and PCLO, and possibly the MREG gene.

Such a method makes it possible, among other things, to confirm the nature of the pigmentary spot in the case where the latter is already apparent, for example visually to the naked eye. The method also makes it possible to predict the appearance of a spot, when the latter is not yet observed but only suspected, or to conclude that skin is prone to the formation of skin spots, or subject to skin defects. pigmentation, for example when no stain is yet apparent.

The skin pigment spots concerned are preferably hyperpigmentary spots, or even so-called hyperpigmented spots, corresponding to an excess of pigment.

Benign hyperpigmentary disorders that can be envisaged in the context of the invention are in particular characterized by an abnormal accumulation (apart from tanning) of melanin, and can be chosen from actinic lentigo, melasma, the pigmentary aftereffects of acne, post-tanning pigmentation. inflammatory diseases, meadow dermatitis, or even benign facial dyschromia, pigmentation imperfections or irregularities making the complexion non-uniform, or the color of the skin non-uniform.

Preferably the pigment spots are actinic lentigo.

The pigment spots in question are preferably pigment spots on human skin.

The expression levels of at least one of the genes of the invention are measured at the level of the skin resulting from a proven or supposed blemish, and at the level of the adjacent uninjured skin. Preferably, levels are measured on skin samples taken from within the blemish and from an adjacent uninjured area. The samples are for example skin biopsies, shaves, ex vivo skin samples, suction bubbles. Regarding biopsies, a few millimeters in diameter are sufficient, for example a biopsy of 2 mm, or 3mm in diameter. We can also consider total excision of a lesion.

The uninjured area is preferably an adjacent area as close as possible to the spot but at a sufficient distance so that the sample taken does not contain any cells likely to belong to the pigment spot. Preferably, the adjacent uninjured area is an area having exposure to light and to the sun comparable to the area of the pigment spot.

Alternatively, the uninjured area may come from a symmetrical area on the other part of the subject, having a perfectly identical location; for example in the case of a stain on the left hand, the uninjured area may be the corresponding area on the right hand. In this case, the uninjured area is not strictly speaking an adjacent area. By uninjured, we mean an area which does not have a pigmentary spot or pigmentary irregularity, preferably an area which is homogeneous in terms of pigmentation.

Since the uninjured area serves as a reference, it must in all cases be as comparable as possible to the area of the stain, but free from pigmentary defects.

It is also important to note that the method according to the invention involves comparing the expression levels of at least one of the genes of the invention. Therefore, it may be sufficient to quantitatively or qualitatively evaluate the difference between the two levels of expression, without necessarily evaluating and quantifying each level of expression individually.

The method according to the invention is preferably carried out in vitro or ex vivo.

According to one embodiment of the method of the invention, the pigmentary spot is a non-pathological, benign spot, in particular as opposed to pathological lesions such as nevi; it may be an irregularity in the pigmentation of the skin.

Preferably, a method according to the invention comprises the comparison of the expression levels of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, SYNPO2, TNNT1, and LRMP.

Even more preferably, a method according to the invention comprises the comparison of the expression levels of at least two distinct genes among the genes of the invention, preferably of at least three distinct genes, or even five distinct genes. It is also possible to compare the expression levels of at least 6 genes, or even of at least 8 distinct genes.

When at least two genes are chosen, they are preferably chosen from the lists of the following 8 preferred genes: JAKMIP2, KIF21A, SLITRK6, SCIN, SYNPO2, TNNT1, and LRMP. All combinations two by two, among the 8 preferred genes, are preferential combinations for the implementation of the invention.

Implementation of the method according to the invention leads to the conclusion that the supposed or observed spot is a hyperpigmentary spot if the expression level is:
- higher in the sample of skin from the spot compared to the level in the sample of adjacent uninjured skin, if the gene is chosen from ARC, NRN1, SYNPO2, RASIP1, and LRMP, and/or
- lower in the sample of skin from the spot compared to the level in the sample of adjacent uninjured skin, if the gene is chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, NAV3 and PCLO, and possibly MREG.

Indeed, the present inventors have demonstrated the overexpression of the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes in the skin resulting from hyperpigmentary spots, in particular from actinic lentigo, compared to the level of expression in the adjacent uninjured skin. . They also demonstrated the underexpression of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, NAV3 and PCLO, and possibly MREG in the skin resulting from hyperpigmentary spots , in particular actinic lentigo, compared to the level of expression in the adjacent uninjured skin.

By “higher” or “lower” we mean a difference in expression levels that is statistically significant, above noise and reproducible. For example, the difference in expression level is at least 10%, that is to say if the expression level of a gene of the invention in uninjured skin is set to 1, the modulation rate is at least 1.1 for a gene overexpressed in the lesional skin and at most 0.9 for a gene underexpressed in the lesional skin.

In a first preferred embodiment, as part of this characterization method, the level of expression of one of the genes of the invention is compared within the skin of a Caucasian individual.

In a second preferred embodiment, as part of this characterization method, the level of expression of one of the genes of the invention is compared within the skin of an individual of Asian type.

Preferably, said individual is at least forty years old, preferably at least fifty years old, or even sixty years old. The individual considered, in the context of the present invention, is preferably female.

As previously detailed, the level of expression of one or more genes of the invention can be compared within the skin sample of said individual.

The described method is also a test method for predicting the formation of skin spots in a subject. According to this implementation, the level of expression of at least one, and preferably several, genes of the invention is compared in a skin sample to its level of expression in normal skin. A significant modulation of the level of expression of said gene(s) in relation to the level of expression of said gene(s) in normal skin makes it possible to conclude that the skin of the subject tested is prone to the formation of skin spots. .

By “level of expression in normal skin”, we mean either the level of expression of said gene(s) of the skin of the same subject, in a body area notoriously free of spots, for example areas not exposed to solar radiation, or areas not very prone to the formation of pigment spots, i.e. the average level of expression of said gene(s), in the skin of people without spots and preferably having the same skin type as the subject tested.

Method for evaluating the effectiveness of a treatment for skin pigment spots

The present invention also relates to a method for evaluating the effectiveness of a treatment of cutaneous pigment spots, comprising the comparison of the expression levels in a sample of skin resulting from such a stain, before and after treatment, at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, NAV3 and PCLO, and possibly MREG genes.

The treatment evaluated may be a treatment aimed at reducing pigment spots or any other modulation of pigmentation, in particular to even out the complexion, homogenize the color of the skin or combat dyschromia.

According to this evaluation method, the comparison of the expression levels of the chosen gene(s) is carried out within the skin resulting from a pigmented spot, or within a corresponding sample, before and after treatment. There is therefore no comparison to a level of expression within healthy, undamaged skin.

By the evaluation method as described, a given treatment is considered effective for the treatment of a hyperpigmentary spot if the level of expression is:
- lower after treatment compared to the expression level before treatment, if the gene is chosen from ARC, NRN1, SYNPO2, RASIP1, and LRMP, and/or
- higher after treatment compared to the expression level before treatment, if the gene is chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, NAV3 and PCLO, and optionally MREG.

A treatment will be considered to have no effect if the expression levels of the chosen gene, before and after treatment, are substantially identical, or if the differences observed are not significant.

When the expression levels of more than one gene are compared, the treatment is considered effective for the treatment of a pigment spot or irregularity if, for the majority of genes tested, and preferably for the all the genes tested, taken individually, the treatment is considered effective. For the other selected genes, the treatment should preferably have no effect, but not have the opposite effect.

According to another implementation, the method for evaluating the effectiveness of a treatment of skin pigment spots comprises the characterization of a pigment spot or irregularity according to the method of the invention, before and after treatment, and the comparison differences in expression levels observed between skin damaged by the spot or pigmentary irregularity and healthy skin.

According to this implementation of the evaluation of the effectiveness of a treatment, the treatment is considered effective if the difference between the expression levels of the gene(s) chosen in the damaged skin resulting from the spot compared to the healthy skin, preferably adjacent, is less after treatment compared to what it was before treatment.

When the expression levels of more than one gene are compared, the effectiveness of the treatment is preferably concluded when for the majority of the genes chosen, and preferably for all of the genes chosen, taken individually, we can conclude that treatment is effective.

Preferably, the comparison of the expression levels of the chosen gene(s) is carried out on skin samples taken from the pigment spot.

The treatment considered, evaluated by the method of the invention, is not limited to a particular type of treatment.

It may be a treatment with a chemical molecule, an active agent, a natural extract, in particular an essential oil, a nucleic acid, in particular an RNA such as siRNA or mRNA, a protein complex or any other molecule or combination. of molecules.

It may also involve treatment using physical means or waves, particularly electromagnetic waves.

This is preferably a topical treatment, but it is also envisaged to evaluate the effectiveness of treatments administered orally, by injection, or by any other means of administration.

The treatment tested may aim to reduce or make disappear a skin spot, to modulate the pigmentation of the skin, to even out the complexion, to homogenize the color of the skin or to reduce dyschromia.

Particularly preferred treatments in the context of this invention are cosmetic treatments, more particularly topical cosmetic treatments. In this case, the pigmentary spot considered is a non-pathological pigmentary spot or irregularity, for example it is an actinic, solar or senile lentigo.

By the methods of the invention, it is also possible to evaluate the effectiveness of the combination of several treatments. It is in fact possible to evaluate combinations which best enable the expression levels of one, several or all of the genes of the invention to be restored, as they are expressed in uninjured skin.

Using the evaluation methods described above, it is possible to evaluate the effectiveness of a new treatment considered, or also to quantify or qualify the effectiveness of already existing treatments against pigment spots, particularly hyperpigmental ones. In this way, combinations of treatments likely to be particularly effective, synergistic or complementary can also be considered.

As explained for the characterization methods according to the first aspect of the invention, the expression levels of more than one gene are preferably compared, preferably at least two, three, five, six, or eight genes. of the invention.

Particularly preferred genes or combinations of genes have already been explained with regard to the first aspect of the invention; the same genes or combinations are preferred in this aspect.

In particular, it is particularly preferred that the gene chosen is from the genes JAKMIP2, KIF21A, SLITRK6, SCIN, SYNPO2, TNNT1, and LRMP, and all two to two or three to three combinations of the genes in this list are particularly preferred. .

The skin sample preferably comes from a human being of Caucasian or Asian type, preferably at least forty years old, preferably at least fifty years old, or even at least sixty years old.

Preferably, in the context of the present invention and in particular for the evaluation method described, it concerns the treatment of hyperpigmentary spots, and most preferably actinic, solar or senile lentigos.

The method for evaluating the effectiveness of a treatment according to the present invention is preferably implemented in vitro or ex vivo.

It is desirable that the skin sample before treatment and after treatment be taken from the same pigment spot, or from a very close pigment spot if the size of the spot does not make it easy to obtain samples before and after treatment.

In vitro method for screening active agent to prevent and/or treat skin pigment spots

The present invention also relates to an in vitro method for screening active agent for preventing and/or treating skin pigment spots, said method comprising the following steps:
i. bring into contact a cellular model representative of the skin with at least one active agent to be tested, or expose said cellular model to a physical treatment to be tested;
ii. carry out a measurement of the expression level of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1 , SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG, or the level of expression or activity of the expression product of said chosen gene, and
iii. compare said measurement to a reference measurement.

By “cellular model representative of the skin” is meant within the meaning of the present invention a model comprising at least skin cells such as keratinocytes and possibly melanocytes.

The expression levels of at least one of the genes of the invention can be evaluated by reference or after normalization with the expression level of other genes, the expression level of which is assumed to be substantially identical within the spot and in the chosen area of uninjured skin. Such genes for normalization are well known to those skilled in the art and may depend on the area of the body where the spot is located. By way of example, the following genes can be cited as likely to be used for the normalization of the expression levels of the genes of the invention, coding for: ribosomal protein L13a (RPL13A), beta-2-microglobulin (B2M ), ribosomal protein S9 (RPS9), ribosomal protein S28 (RPS28), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Preferably, a reference measurement may be a quantitative or qualitative value relating to the level of expression of said gene(s), or of the level of expression or activity of the expression product of said gene(s). chosen(ies) determined in a sample in the absence of active agent or physical treatment tested.

Thus, a reference measurement can be obtained by repeating the steps of a process of the invention, and in particular steps i. and ii. of a method according to the invention as defined above, in the absence of active agents, or physical treatments to be tested.

A comparison of the test measurement to a reference measurement, and an observation of a deviation or absence of deviation between the two measurements, makes it possible to obtain information regarding the effect of the active agent or the physical treatment. tested.

The quantitative or qualitative determination of the level of expression of said gene(s), or of the level of expression or activity of the expression product of said gene(s) can be carried out by any method known to the man of the art, and in particular as described previously.

The cellular model can be of any type considered appropriate by those skilled in the art. These may include mono- or co-culture cell models, or three-dimensional models of reconstructed skin, or skin cultured ex-vivo. There are also cellular models representative of pigment spots, more particularly actinic lentigos, which can be used as part of this method.

Such cellular models do not need to mimic pigment spots (or lentigo) in their entirety but must mimic biological events, morphological or pigmentary characteristics observed in pigment spots, particularly lentigo. Such in vitro models are well known to those skilled in the art.

Preferably, said cellular model is a model comprising keratinocytes and melanocytes.

According to a preferred implementation of the in vitro method described above, it comprises the comparison of the expression levels of at least two genes, preferably of at least three, five, six, or eight genes of the invention, as explained for the other evaluation methods according to the invention.

The invention further relates to an in vitro method for screening active agent for preventing and/or treating skin pigment spots, said method comprising the following steps:
i. bring into contact a cellular model comprising keratinocytes and possibly melanocytes, in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of JAKMIP2 genes , KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG, repressed and/or in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes, overexpressed with at least one active agent to be tested, or exposing said cellular model to a physical treatment to be tested ;
ii. carry out a quantitative or qualitative measurement of melanin and/or a measurement of the expression level of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the JAKMIP2, ARC, KIF21A genes , SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG, or the level of expression or activity of the expression product of said chosen gene, and comparing said measurement to a reference measurement.

For the purposes of the present invention, the term “repressed gene” means a gene whose expression level is lower than the average expression level of said gene in the skin of people devoid of pigment spots.

For the purposes of the present invention, “overexpressed gene” means a gene whose expression level is higher than the average expression level of said gene in the skin of people without pigment spots.

By “qualitative or quantitative measurement of melanin” is meant within the meaning of the present invention both the determination of the presence of melanosomes, and/or the organization of melanosomes and/or the morphology of melanosomes in keratinocytes in particular at the using an optical or electronic microscope, but also the determination of melanin by techniques well known to those skilled in the art, or even the marking of protein(s) characteristic of the presence of melanosomes such as for example the PMEL 17 protein and its immunofluorescence assay(s).

Said cells in which the level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG, is repressed, is still called “knock-out” cells.

The repression or overexpression of the level of expression of one or more of said aforementioned genes can in particular be carried out by any method known to those skilled in the art.

By way of example, such cells can be obtained by transfection and homologous recombination of a nucleic acid fragment inserted into or taking the place of the gene expressing the amino acid sequence whose expression is to be suppressed. or overexpress.

Also, it may be possible to suppress the expression of a given gene by transfection, for example using a lentivirus, into the cell of a nucleic acid sequence coding for an interfering RNA specific for the mRNA from the gene whose expression is to be suppressed.

Preferably, the level of expression of at least one of said aforementioned genes is repressed by transfection into cells, preferably keratinocytes, of siRNA.

Furthermore, it may be possible to overexpress the expression of a given gene by transfection, for example using an expression vector, preferably a plasmid comprising the nucleic acid sequence of the given gene as well as 'a promoter sequence for the expression of said gene,

All these molecular biology techniques making it possible to repress or overexpress the expression of a gene are known to those skilled in the art and do not require specific development.

Cellular model

The present invention also relates to a cellular model capable of screening active agents for preventing and/or treating cutaneous pigment spots, comprising keratinocytes and optionally melanocytes, in which said keratinocytes present a level of expression of at least one gene involved. in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG, repressed and /or in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes, overexpressed.

For the purposes of the present invention, the term “repressed gene” means a gene whose expression level is lower than the average expression level of said gene in the skin of people devoid of pigment spots.

For the purposes of the present invention, “overexpressed gene” means a gene whose expression level is higher than the average expression level of said gene in the skin of people without pigment spots.

Preferably, said gene(s) is(are) repressed by transfection of siRNA into said keratinocytes.

Cell model preparation method

The present invention also relates to a method for preparing a cellular model capable of screening active agents to prevent and/or treat skin pigment spots, comprising the following steps:
i. collect keratinocytes and possibly melanocytes; Then
ii. transfect said keratinocytes taken in step i. using siRNA capable of repressing the level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1 , ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG; and/or transfect said keratinocytes taken in step i. using an expression vector, preferably a plasmid, in particular a plasmid, of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes ARC, NRN1, SYNPO2, RASIP1, and LRMP, then
iii. Cultivate the transfected keratinocytes in step ii. or co-culture the keratinocytes transfected in step ii. with the melanocytes collected in step i.

Preferably, human keratinocytes and melanocytes are obtained in particular from explants originating from surgical waste.

Culture media used to cultivate keratinocytes and melanocytes in step iii. are well known to those skilled in the art.

Preferably, the keratinocytes and melanocytes are co-cultured for a period of 48 hours to 72 hours, preferably 72 hours.

Preferably, the keratinocytes are cultured for a period of 48 hours to 72 hours, preferably 72 hours.

The present invention further relates to a cellular model capable of being obtained or directly obtained by the aforementioned preparation process.

Cosmetic method of treating or preventing non-pathological cutaneous pigment spots in human skin

According to another aspect, the present invention also relates to a cosmetic method for treating or preventing non-pathological cutaneous pigment spots in human skin, comprising the application to the skin of a composition comprising at least one active agent modulating the level of expression or activity of at least one gene involved in the management of vesicles, preferably melanosomes, where said gene is chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG.

Non-pathological skin pigment spots are benign spots, the elimination of which is only desirable for aesthetic reasons and not for therapeutic reasons. Pigmentation irregularities include imperfections at the pigment level making the complexion non-uniform or the skin color non-homogenous.

The application of said composition is preferably carried out on healthy skin.

The present inventors have shown the important role of the genes of the invention within the dermis at the level of pigment spots, and in particular the link between deregulation of the level of expression of these genes and the appearance of pigment spots. Therefore, suspending or reducing the modulation of these genes can make it possible to reduce or abolish the deregulations observed and thus make it possible to restore a situation at the level of the genes involved in the management of vesicles, preferably of melanosomes, in particular of the transport of melanosomes to the within keratinocytes compatible with the absence of pigment spots, therefore with a uniform complexion and homogeneous skin color.

For the same reasons as explained with regard to the other aspects of the invention, more than one gene is preferably chosen within the genes of the invention, for example at least two genes, at least three, five, six , eight.

According to one implementation, the cosmetic method aims to modulate the level of expression of all of the genes of the invention.

The cosmetic method according to the invention aims to restore expression levels close to those observed in healthy skin, adjacent for example to a pigment spot.

Particularly preferred genes are JAKMIP2, KIF21A, SLITRK6, SCIN, SYNPO2, TNNT1, LRMP, NAV3 and MREG.

According to a preferred implementation, said pigmentary spot is a hyperpigmentary spot, for example actinic, senile or solar lentigo. In such a case, the modulation sought in the cosmetic methods according to the invention is a total, partial or temporary inhibition of the expression of at least one gene chosen from the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes. and/or an increase, possibly temporary, in the expression of at least one gene chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG.

Preferably, the inhibition is not a total inhibition, but a partial one, tending to reduce the level of expression of the chosen gene without entirely inhibiting its expression.

By “increase or decrease in the level of expression”, it is included the increase or decrease in the level of transcription of said genes, and the increase or decrease in the level of translation of said genes, as well as the increase or decrease of the activity of the proteins encoded by these genes.

A cosmetic method according to the present invention therefore comprises the application of a cosmetic composition comprising in particular at least one active agent chosen from a chemical molecule, a natural extract, a nucleic acid, a peptide, or a treatment modulating the level of expression or activity of the expression product of at least one of the genes of the invention.

The cosmetic composition is preferably applied topically.

Preferably, the modulation is carried out using a modulator of one of the genes of the invention. For example, a cosmetic method according to the invention will advantageously include the application of a compound chosen from caffeine, calcitriol, folic acid, hyaluronic acid, palm oil, sesame oil, curcumin, and mixtures thereof.

Preferably, a cosmetic method according to the invention is implemented after the characterization of the pigmentary spot that it is desired to treat by a method according to the first aspect. Indeed, this first step makes it possible to characterize the spot and therefore to detect genes whose expression level is strongly modulated between the area of the spot and an uninjured area, preferably adjacent. It is then possible to adapt a treatment which makes it possible to act on the gene(s) of the invention which are differentially modulated within this spot, by specifically applying modulators for said genes.

Thus, even more preferably, the cosmetic method of treating or preventing non-pathological cutaneous pigment spots on human skin comprises the following steps:
i. characterize an apparent or suspected skin pigment spot in a human being according to the characterization method according to the invention;
ii. deduce from step i. if said apparent or suspected spot is a skin pigment spot;
iii. if said spot is identified as being a skin pigment spot in step ii., treating said spot with a cosmetic composition comprising an active agent allowing the modulation of the level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP and PCLO, NAV3 and MREG.

Whatever the treatment considered, the cosmetic method according to the invention can also include the application of one or more additional active agents aimed at reinforcing the desired effects, for example, any substance described as depigmenting, keratolytic and/or desquamating agents. , antioxidants, chemical or physical UV sunscreens, anti-inflammatory and/or soothing agents, deoxyribonucleic acids and their derivatives.

The cosmetic method according to the present invention is also applicable in the case of preventing the appearance of pigment spots or other irregularities in the complexion or color of the skin, and in particular hyperpigmented spots such as actinic lentigo.

Cosmetic use of modulator in the prevention and/or treatment of non-pathological skin pigment spots

The present invention also relates to the cosmetic use of a modulator of the expression level or activity of the expression product of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG in prevention and/or treatment non-pathological skin pigment spots.

For the treatment of hyperpigmentary spots, the modulator used is an inhibitor of at least one gene chosen from ARC, NRN1, SYNPO2, RASIP1, and LRMP, and/or an activator of at least one gene chosen from JAKMIP2 , KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG. Preferably, the inhibitor is a partial inhibitor leading to a reduction in the level of expression or a reduction in the activity of the expression product of said gene, without entirely abolishing the expression or activity of this gene. .

Such modulators may in particular be caffeine, calcitriol, folic acid, hyaluronic acid, palm oil, sesame oil, curcumin, and mixtures thereof, for use in the cosmetic treatment of spots. hyperpigmentary.

Well-known modulators are also antisense molecules, siRNAs, and microRNAs.

Preferred modulators in the context of the present invention include caffeine, calcitriol, folic acid, hyaluronic acid, palm oil, sesame oil, curcumin, and mixtures thereof.

It is also possible to use a combination of at least two of these modulators.

Modulators can be used in combination with other products, active agents or excipients. Preferably, they are packaged in a form suitable for topical application, for example in the form of an ointment, a cream, particularly in the form of an emulsion, lotion or ointment.

The various implementations detailed in the section relating to cosmetic methods according to the invention are applicable to the uses for the cosmetic applications described above.

The pigment spots considered are preferably hyperpigmentary spots and most preferably actinic, solar or senile lentigos.

Example 1: Transcriptional study

Material and methods

Clinical selection

2 independent studies were carried out: a study in France on 15 female volunteers, phototypes II to IV, aged 51 to 67 years and a study in Japan on 12 female volunteers, phototypes III to IV, aged 57 to 70 years old.

Actinic lentigos are chosen on the back of the hand, at least 3mm in size.

They are characterized by epiluminescence. This exam allows:

1°) to verify the clinical diagnosis of the lesion (actinic lentigo exclusively) according to the criteria of patterns of the dermo-epidermal junction (pigmented network structure, “fingerprint” type) to differentiate from ephelides (absence of pigmented structure in network, homogeneous pigmentation and jagged edges) and flat seborrheic keratoses (presence of microcysts (“milia-like cysts) or pseudo-cysts, blunt to jagged edges, pseudofollicular openings and irregular “fingerprint” type areas) [Menzies et al 1996; Stolz et al 2002; Marghoob et al 2005]

2°) to delimit homogeneous areas in terms of structure/pattern inside these lesions where skin biopsies will be taken,

3°) to establish a phenotypic score based on a quantitative image analysis with the specific software which was developed on Matlab® (SQA software, CMLA, ENS Cachan, UMR CNRS 8536).

Two 3mm diameter biopsies are taken from one of the hands of each patient. For each volunteer, one of the biopsies corresponds to the actinic lentigo (AL) lesion and the other to an adjacent area of non-lesional (PN) skin (also verified by epiluminescence).

The 3mm biopsies are placed, upon collection, in an RNA stabilization solution (RNAlater, Qiagen reference 76154) for 16 to 24 hours at 4°C. The next day the samples are placed at -20°C until the homogenization and extraction stages.

RNA extraction

After thawing, the samples are cut with a scalpel to facilitate homogenization, then transferred to the lysis buffer (Qiagen lysis buffer, RLT + 10% beta-Mercapthoethanol). For the France study, homogenization is carried out in a potter (Fisher Labosi ref A6391000) with RNase free polypropylene pistons (Fisher Labosi ref A1419753). For the Japan study, homogenization is carried out with the TissueRuptor from Qiagen (ref 990890).

RNA was extracted with the RNeasy micro kits (Qiagen ref: 74004) following the supplier's instructions. For the France study, RNA quantification is carried out using a ribogreen assay (Molecular Probes ref R11490). For the Japan study, RNA quantification is carried out by spectrophotometry (Nanodrop 8000).

The quality of the RNA is validated with an Agilent 2100 bioanalyzer which gives the ratio of the intensities of 28S to 18S ribosomal RNAs as well as an RNA Integrity Number (RIN) which takes into account the degradation of the RNAs. Good quality RNA has a ratio >1.5 and an RIN >7.

Probe preparation and hybridization

A reverse transcriptase (RT) reaction is carried out to obtain the corresponding cDNAs. For the France study, a cDNA amplification step is carried out.

The cDNAs are then marked with fluorochromes (Affymetrix labeling kit) and hybridized on Affymetrix® DNA chips to reveal the level of expression of all the genes in the human genome. (Affymetrix U133A 2.0 type DNA biochips containing 54,000 probes, thus allowing the study of the expression of 47,000 transcripts, including 38,500 characterized genes).

Affymetrix RMA software was used to generate the raw files and to perform quality checks on both studies before data analysis. After revealing the specific hybridizations and processing the raw data (extraction, subtraction of background noise, normalization), gene expression is compared between healthy skin and damaged skin.

For the France study, 2 Affymetrix HG_U133 Plus 2 chips were hybridized per sample. Patients are only retained for further analysis if the 2 Affymetrix chips have correct hybridization quality. 13 patients out of the initial 15 could be analyzed. Statistical analysis was carried out on the average of duplicates. Likewise, for the Japan study, the results of the hybridization of the probes on the Affymétrix® HG_U133 Plus 2 chips (1 chip per sample) are obtained and controlled using the Expression Console® software. Several checks are carried out to validate the quality of the hybridization on each chip. At the end of these quality controls, the hybridization results of the 12 chips are kept for differential analysis.

Generation of lists of genes differentially expressed between damaged skin and healthy skin
- Filter on the expression level: only ProbeSets with an expression level ≥ 2 ⁶ (64) in at least one condition (PN or LA) are retained. After this filter, 24393 ProbeSets are retained for the France study and 26943 ProbeSets are retained for the Japan study.
- Supervised analysis:
t-test: A linear T-test was applied to the data with the help of ArrayStudio software to compare the gene expression profile of the actinic lentigo biopsy group with that of the non-lesional skin biopsy group. A list of 1973 ProbeSets modulated significantly (p < 0.05) in actinic lentigo compared to non-lesional skin was thus established for the France study and a list of 3084 ProbeSets modulated significantly in actinic lentigo compared to non-lesional skin for the Japan study.
- Unsupervised analysis:
i. Removal of the patient effect: in order to remove the patient effect observed in the results of the differential analyses, the following algorithm was carried out: for each patient and each probeset, 1) subtraction of the average expression in log2 of the two conditions (lesional, non-lesional), 2) addition of the average log2 expression of all patients and conditions.
ii. Differential analysis: an NMF (Non Negative Matrix Factorization) analysis made it possible to identify 3296 ProbeSets in the France study and 3288 ProbeSets in the Japan study which make it possible to differentiate the group of actinic lentigo biopsies from the group of non-actinic skin biopsies. lesional (p<0.05).
- Filter on modulation and union of lists: the lists of differentially expressed probesets were first filtered on the level of modulation: geometric mean of corrected folds 1.25 ≤|FC|< 1.5 (p value < 0.05 ) and |FC| ≥ 1.5 (p value < 0.05) of gene expression. The two lists obtained through supervised analysis (t-test) and unsupervised analysis (NMF) were then joined to generate the final list of genes differentially expressed in actinic lentigo compared to non-lesional skin. Thus 266 probeSets (196 genes) are differentially expressed in actinic lentigo compared to non-lesional skin in the France study. 332 probeSets (245 genes) are differentially expressed in actinic lentigo compared to non-lesional skin in the Japan study.

Comparison of the two studies

The lists of genes differentially expressed in actinic lentigo compared to non-lesional skin in the two studies were compared. 178 probesets, corresponding to 136 genes, are common to both studies, that is to say that 136 genes are modulated in the same direction in actinic lentigo compared to healthy skin both in the France study and in the Japan study.

Concerning the other genes, after detailed analysis of the modulations of these genes, it turns out that most of them are also modulated in the same direction of variation in the two populations.

Following this analysis, the signature common to the two studies therefore includes 245 genes significantly modulated in both studies.

Results: List of genes of interest - lentigo molecular signature associated with vesicle/melanosome management

All of these genes were subdivided into several functional families using a targeted bibliographic analysis. A completely original gene family in a pigmentary disorder/actinic lentigo is found associated with a particular biological function "Transport and Management of vesicles" and implies a defect in the management of vesicles, including the specific vesicles responsible for pigmentation, melanosomes. In particular, the MREG (melanoregulin) gene is significantly underexpressed in both studies, and is associated with the retrograde transport of melanosomes from the periphery of the cell to the nucleus (Ohbayashi N et al. 2012) and plays a role in pigmentation ( Moore K.J. et al. 1988).

The genes, grouped in the functional family named “Transport and Management of melanosomes” are listed below in Table 1. This table groups together the MREG gene as well as the 19 other genes associated with “Transport and Management of melanosomes” from the analysis described previously of the 245 genes of the strong and weak signature common to the two studies:
- Genes over-expressed in actinic lentigo compared to non-lesional skin: ARC, NRN1, SYNPO2, RASIP1, LRMP.
- Genes underexpressed in actinic lentigo compared to non-lesional skin: JAKMIP2, KIF21A, NAV3, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, MREG.

The genes of this family are mainly underexpressed in actinic lentigo.

Subfamily Symbol Name FC Japan P
value FC France P
value Intra-cellular traffic JAKMIP2 janus kinase and microtubule interacting protein 2 -1.93 7.52E-05 -1.69 2.40E-03 BOW activity-regulated cytoskeleton-associated protein 1.50 4.92E-06 1.39 9.00E-03 Kinesins KIF21A kinesin family member 21A -1.60 6.67E-07 -1.64 2.81E-02 Axon growth NAV3 neuron navigator 3 -1.78 9.49E-07 -1.78 6.80E-05 SLITRK6 SLIT and NTRK-like family. member 6 -1.93 4.00E-04 -1.73 3.00E-04 NRN1 neuritin 1 1.46 2.00E-04 1.59 1.00E-03 Cytoskeletal organization SCIN splitter -1.80 2.00E-04 -1.54 1.00E-04 SYNPO2 synaptopodin 2 1.97 1.68E-05 1.63 4.00E-04 Contractile activity TNNT1 troponin T type 1 (skeletal. slow) -1.72 4.57E-05 -1.71 1.10E-03 SNTB1 syntrophin, beta 1 (dystrophin-associated protein A1, 59kDa, basic component 1) -1.51 4.92E-06 -1.41 2.00E-04 Rho/Rab family ARHGAP29 Rho GTPase activating protein 29 -1.50 7.34E-06 -1.42 5.40E-03 RAB3IP RAB3A interacting protein (rabin3) -1.81 2.44E-06 -1.48 2.00E-04 RHOBTB3 Rho-related BTB domain containing 3 -1.55 4.66E-05 -1.30 3.70E-03 RASIP1 Ras interacting protein 1 1.67 2.82E-06 1.45 2.00E-04 Endocytosis ASGR1 asialoglycoprotein receptor 1 -1.80 3.82E-05 -1.47 1.74E-06 CHMP4C charged multivesicular body protein 4C -1.45 2.79E-06 -1.51 1.23E-05 Autophagy Please small VCP/p97-interacting protein -1.55 5.12E-05 -1.321 9.00E-03 Vesicle management LRMP lymphoid-restricted membrane protein 1.82 1.88E-02 1.71 3.40E-03 PCLO piccolo (presynaptic cytomatrix protein) -1.37 4.00E-04 -1.56 1.00E-04 MREG melanoregulin -1.18 1.60E-03 -1.20 4.00E-04

Table 1: List of genes grouped in subfamilies all associated with the functional family regulating vesicles/melanosomes. The first column indicates the subfamily of genes linked to the organization of vesicles/melanosomes, the second column the gene symbol, the third column details the name of the gene. Columns 4 and 6 indicate the mean modulation rate (Fold-change (FC)), geometric mean of individual modulation rates across all study patients in Actinic Lentigo) compared to adjacent non-lesional skin (NLP) in the France study and in the Japan study, respectively. Columns 5 and 7 indicate the p-values associated with these modulations in the France study and the Japan study, respectively.

Example 2: Screening method: silencing and quantification of melanosomes – Demonstration of the effect of MREG on the organization of melanosomes.

Material and methods

Experimental coculture model: 2D vitro experimental system of keratinocytes loaded with melanin

Normal human keratinocytes and melanocytes are obtained from samples of surgical waste, respectively for the keratinocytes from mammaplasty from an 18-year-old female subject with weakly pigmented skin, and for the melanocytes from the foreskin of a 2-year-old male donor from moderately pigmented skin. The keratinocytes are cultured in MEM-7F proliferative medium: MEM (Gibco), containing 10% fetal bovine serum (Sigma), 1% Sodium Pyruvate (Gibco), 1% non-essential amino acids (Gibco), 1% of L-Glutamine (Gibco), and supplemented with antibiotics/antimycotics and 7 growth factors (EGF 10 ng/ml, cholera toxin 10-10 M, hydrocortisone 0.4 μg/ml, adenine 180 μM, transferrin 5 μg/ml , Triiodothyonine 2 nM, insulin 5 μg/ml). They are thawed at a density of 3000 cells/cm2, and amplified for 7 days at 37°C, 5% CO2 in the presence of feeder cells (3T3 murine fibroblasts treated with mitomycin C). The melanocytes are cultured in M2 medium: DMEM/F12 (1:1), supplemented with “Melanocyte Growth Medium Supplement Mix” (PromoCell), 1% L-Glutamine (Gibco), and antibiotics/antimycotics. They are thawed to a density of 10,000 cells/cm2, and amplified for 7 days at 37°C, 5% CO2.

The in vitro keratinocyte/melanocyte co-culture model allows contact between keratinocytes and melanocytes, so that the melanocytes transfer the melanosomes to the keratinocytes as in normal skin. The co-culture model consists of seeding the two cell types on either side of a porous membrane, in pods/inserts immersed in the culture medium. . Keratinocytes are seeded in DMEM-7F or KGM-Gold culture medium during transfection for 48 hours. The melanocytes are then seeded 48 hours after the keratinocytes, in DMEM-7F-M2 medium (culture (MEM-7F supplemented with “Melanocyte Growth Medium Supplement Mix” at a rate of 0.833 μl of Mix per ml of medium). After 3 days of co-culture, the melanocytes are detached from the membrane. The keratinocytes are then analyzed directly.

This model allows the monitoring of melanosomes and melanin over time within keratinocytes.

Silencing of the MREG target gene
- Bringing the sample into contact with the siRNA:
The transfection of siRNAs to silence the expression of the MREG protein (NM_018000.2) is carried out from a pool of 3 siRNA sequences (Invitrogen Stealth RNAi siMREG 04 HSS124804, siMREG 97 HSS183197, siMREG 98 HSS183198). The corresponding control sequence is denoted LowGC (Stealth RNAi Negative control Low GC Invitrogen 12935-200).
Keratinocytes in suspension are transfected with 20 nM siRNA using Lipofectamine RNAiMax (Invitrogen), then seeded in KGM-Gold medium (Lonza) on the porous pod. The keratinocytes are again transfected with 20 nM of siRNA using Lipofectamine RNAiMax (Invitrogen) in KGM-Gold medium for 24 hours after adhesion to the pod. The next day the medium is changed to DMEM-7F, and the melanocytes are seeded the following day in DMEM-7F-M2.
- Western Blot and qPCR analysis of MREG silencing
To validate the silencing of MREG expression, the mRNA and protein expression levels were measured by qPCR and Western-blot, respectively. After 5 days of transfection, an average decrease of 99% in the level of expression of the mRNA, and of 97% in the level of expression of the protein is observed.

Analysis and quantification of the organization of melanosomes in keratinocytes
- Immunofluorescence marking:
Melanosomes in keratinocytes are analyzed by immunofluorescence by marking the PMEL17 protein. The keratinocytes are fixed in methanol -20°C on ice, then incubated for 1 hour in the blocking solution (PBS containing 5% Goat Serum, Dako), and labeled for 1 hour at room temperature with the primary antibody directed against PMEL17 (Mouse NKI/beteb antibody, Monosan, dilution 1/20). After rinsing with PBS-Tween 0.05%, the cells are incubated with the secondary antibodies (Goat anti-Mouse Alexa Fluor 488, dilution 1/150, Life Technologies). The nuclei are then marked for 15 minutes with Hoesch (Life Technologies 1/5000). After rinsing in PBS, the membranes are cut, then mounted between slides and coverslips (Fluoromount G mounting medium, Southern Biotech). The cells are observed at 63x immersion with a Leica DMIRB4000B inverted epifluorescence microscope in the 2 channels at 488nm for the melanin vesicles labeled PMEL17, and 405nm for the nuclei in Hoesch.
- Image analysis using ImageJ:
The images are automatically analyzed with an ImageJ macro to segment the cells, and the melanin vesicles in order to measure the locations of the melanin vesicles labeled in green (488nm) with PMEL17 in the cytoplasm.
The major steps in the analysis of each image are: the segmentation of the nuclei then the cells, the segmentation of the vesicles, and finally the measurement and quantification of the following characteristics: size/area of the vesicles, number of vesicles per cell, density of vesicles. /µm², relative distance of each vesicle from the nucleus of the cell, and percentage of vesicles present in a perinuclear zone.
Segmentation of the nuclei: the following steps were applied to segment the nuclei in the 405nm channel: filtering with a Gaussian filter of sigma=10, subtraction of the fluorescence background with a rolling ball=200px, thresholding of the mask of the nuclei via intensity marking at 405nm manually, particle analysis (minimum size 1500px), separation of objects with a watershed process, manual correction of the nucleus mask if necessary, recording of ROInuclei.
Cellular segmentation: from the segmentation of the nuclei, the cell is individualized. A watershed approach is used. For each nucleus a cellular region is initialized on the exterior contour of the nucleus. This region is enlarged by iteratively adding the pixels touching the region starting from the darkest pixels to the most intense pixels. A particle analysis without size filtration is carried out to save the ROIcell.
Segmentation of melanin vesicles: melanin vesicles labeled PMEL17 in the 488nm channel are segmented by applying a median filter of radius 10, then subtraction of this filter on the image, segmentation by thresholding with the “triangle dark” algorithm , a resegmentation by watershed. An analysis of the particles is carried out with a threshold of 3px for the smallest, and the coordinates of the vesicles are saved in the ROIvesicles.
- Quantification of the distance to the nucleus of melanin vesicles in keratinocytes
Several parameters can be measured from these different objects: nuclei/cells/vesicles, in particular the size/area of the vesicles, the number of vesicles per cell, the density of vesicles/µm², the relative distance of each vesicle from the nucleus of the cell, and the percentage of vesicles present in a perinuclear zone. In order to calculate distances between vesicles between the cytoplasmic membrane and the nuclear membrane, a map of the distances to the cytoplasmic membrane, as well as a map of the distances to the nuclear membrane are created with the “Distance map” algorithm. The ratio of these two maps is calculated as follows (distance to the nucleus)/(distance to the nucleus + distance to the cytoplasmic membrane). It allows you to have the entire nuclear zone at value 0, and the cytoplasmic membrane at value 1. The perinuclear zone is defined as the region between 0 and 0.35, i.e. the nucleus and an area of 35% around the nucleus.

Results: Demonstration of the effect of MREG on the organization of melanosomes

The organization of melanin vesicles is analyzed by immunofluorescence with PMEL17 marking for keratinocytes after 3 days of contact and loading with melanin on the co-culture model in a pod with normal melanocytes. The organization of the vesicles is compared for untreated normal keratinocytes denoted NT, keratinocytes silenced with 20nM of the control sequence called LowGC, and silenced with 20nM for the melanoregulin MREG protein denoted siMREG.

In order to quantify the effect of modulation via MREG silencing on the distribution of melanin around the nucleus in keratinocytes, the percentage of PMEL17-labeled vesicles present in the perinuclear zone is measured in siMREG keratinocytes and in the LowGC control case. In MREG silencing only 42.8% of the vesicles reach the region close to the nucleus compared to 55.4% in the control case. This shows that the reduction in MREG expression leads to a significant dispersion of melanin vesicles in keratinocytes, which are found less close to the nucleus.

Example 3: Interest modulators on the signature

Several chemical modulators are known to modulate some of the actinic lentigo molecular signature genes associated with vesicle/melanosome handling. In order to restore activity, the interest for the treatment of actinic lentigo is to have inhibitors of the expression level of at least one gene chosen from ARC, NRN1, SYNPO2, RASIP1, and LRMP, or activators of the expression level of at least one gene chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG. Several molecules of interest are grouped in the table below.

Molecules
Gene/protein modulators Genoa modulator modulation direction meaning in relation to the signature Caffeine RAB3IP increase opposite Calcitriol NAV3 increase opposite TNNT1 increase opposite SNTB1 increase opposite CHMP4C increase opposite MREG increase opposite Folic acid NRN1 decreases opposite CHMP4C increase opposite Hyaluronic Acid ARHGAP29 increase opposite Palm oil KIF21A increase opposite TNNT1 increase opposite Sesame oil NAV3 increase opposite NRN1 decreases opposite Curcumin LRMP decreases opposite

Claims (17)

Method for characterizing an apparent or suspected skin pigment spot in a human being, comprising measuring and then comparing the expression levels measured, in samples of skin from said spot and adjacent uninjured skin, of at least a gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C , SVIP, LRMP, NAV3 and PCLO, and possibly the MREG gene. Method according to claim 1, comprising comparing the expression levels of at least two distinct genes, preferably of at least five distinct genes chosen from said list. Method according to claim 1, where said gene is chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP and PCLO. Method according to claim 1, where said spot is confirmed as a hyperpigmentary spot when the measured expression level is:

• higher in the sample of skin from the spot compared to the level in the sample of adjacent uninjured skin, if the gene is chosen from ARC, NRN1, SYNPO2, RASIP1, and LRMP, and/or
• lower in the skin sample from the spot compared to the level in the adjacent uninjured skin sample, if the gene is chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1 , CHMP4C, SVIP, NAV3 and PCLO, and possibly MREG.

Method according to claim 1, wherein said pigmentary spot is an actinic, senile or solar lentigo. Method for evaluating the effectiveness of a treatment for actinic lentigo, comprising measuring and then comparing the expression levels measured in a sample of skin from actinic lentigo, before and after treatment, of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, NAV3 and PCLO, and possibly the MREG gene. Method according to claim 6, wherein said treatment is considered effective for the treatment of actinic lentigo when the level of expression measured is:

1. lower after treatment compared to the expression level before treatment, if the gene is chosen from ARC, NRN1, SYNPO2, RASIP1, and LRMP, and/or
2. higher after treatment compared to the expression level before treatment, if the gene is chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, NAV3 and PCLO and possibly MREG.

Methodin vitrofor screening active agents to prevent and/or treat skin pigment spots, said method comprising the following steps:

1. bring a cellular model representative of the skin into contact with at least one active agent to be tested, or expose said cellular model to a physical treatment to be tested;
2. carry out a measurement of the expression level of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1 , SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG, or the level of expression or activity of the expression product of said chosen gene, and
3. compare said measurement to a reference measurement.

Cosmetic method for treating or preventing actinic lentigo in human skin, comprising the application to the skin of a composition comprising at least one active agent modulating the level of expression or activity of at least one gene involved in the management of vesicles, preferably melanosomes, where said gene is chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C , SVIP, LRMP, PCLO, NAV3 and MREG. Method according to claim 9, wherein said method comprises the modulation of at least two genes, preferably at least five distinct genes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG. Method according to claim 9, wherein said modulation is an inhibition if the gene is chosen from ARC, NRN1, SYNPO2, RASIP1, and LRMP; and/or an increase in the level of expression or activity if the gene is chosen from JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG . Cosmetic use of a modulator of the expression level or activity of the expression product of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG in the treatment of actinic lentigo. Use according to claim 12, wherein said modulator is caffeine, calcitriol, folic acid, hyaluronic acid, palm oil, sesame oil, curcumin, and mixtures thereof. Method for preparing a cellular model capable of screening active agents to prevent and/or treat skin pigment spots, comprising the following steps:

1. transfect the keratinocytes collected using siRNA capable of repressing the level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN , TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG; and/or transfect said keratinocytes taken using an expression vector, preferably a plasmid, of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes ARC, NRN1, SYNPO2, RASIP1, and LRMP, then
2. cultivate the keratinocytes transfected in step i. or co-culture the transfected keratinocytes with the melanocytes previously collected.

Cellular model capable of screening active agents to prevent and/or treat skin pigment spots, capable of being obtained or directly obtained according to the preparation method as defined in claim 14. Cellular model capable of screening active agents to prevent and/or treat skin pigment spots, comprising keratinocytes and possibly melanocytes, in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG, repressed and/or in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes, overexpressed. In vitro method for screening active agent for preventing and/or treating skin pigment spots, said method comprising the following steps:

1. bring into contact a cellular model comprising keratinocytes and possibly melanocytes, in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of JAKMIP2 genes , KIF21A, SLITRK6, SCIN, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, ASGR1, CHMP4C, SVIP, PCLO, NAV3 and MREG, repressed and/or in which said keratinocytes present a level of expression of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the ARC, NRN1, SYNPO2, RASIP1, and LRMP genes, overexpressed with at least one active agent to be tested, or exposing said cellular model to a physical treatment to be tested ;
2. carry out a quantitative or qualitative measurement of melanin and/or a measurement of the expression level of at least one gene involved in the management of vesicles, preferably melanosomes, chosen from the list consisting of the genes JAKMIP2, ARC, KIF21A, SLITRK6, NRN1, SCIN, SYNPO2, TNNT1, SNTB1, ARHGAP29, RAB3IP, RHOBTB3, RASIP1, ASGR1, CHMP4C, SVIP, LRMP, PCLO, NAV3 and MREG, or the level of expression or activity of the product expression of said chosen gene, and compare said measurement to a reference measurement.