METHODS AND COMPOSITIONS FOR TREATING HYPERPIGMENTATION DISORDERS

Abstract

The present invention relates to a method for treating hyperpigmentary skin disorder. By using normal human melanocytes (NHMs) and normal human keratinocytes (NHKs), which are infected with CLEC12B siRNA/shRNA/RNAi lentiviral particles, inventors have showed that decreasing CLEC12B expression significantly reduce the transfer of melanin to the keratinocytes. These results demonstrate that CLEC12B is specifically expressed in the skin by melanocytes and plays a key role in the transfer or melanosomes to the keratinocytes. Accordingly, the invention relates to a method for treating hyperpigmentary skin disorder in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a CLEC12B antagonist, wherein CLEC12B antagonist is polypeptide, more particularly a decoy.

Claims

1. A method for treating hyperpigmentary skin disorder in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a CLEC12B antagonist.

2. The method according to claim 1, wherein the CLEC12B antagonist is an antibody.

3. The method according to claim 1, wherein the CLEC12B antagonist is a polypeptide.

4. The method according to claim 3, wherein the the polypeptide is a decoy.

5. The method according to claim 1, wherein the CLEC12B antagonist is a glycomimectic molecule.

6. A method of screening a drug suitable for the treatment of hyperpigmentary skin disorder comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity of CLEC12B.

Description

FIGURES

[0030] FIG. 1: The downregulation of Clec12B in melanocytes (NHM for normal human melanocyte and MNT1, a melanoma cell line commonly used for studying pigmentation) decreases the transfer of melanin to the keratinocytes in co-culture condition. A. Normalized quantity of melanin in keratinocyte cell lysates cultures with MNT1; B. Normalized quantity of melanin in keratinocyte cell lysates cultures with NHM.

[0031] FIG. 2: Silencing of CLEC12B using lenti-shRNA increases the production of melanin in Normal Human Melanocyte (NHM). Quantification of melanin in NHM transduced with control or CLEC12B lentiviral shRNA normalized to protein content.

[0032] FIG. 3: Silencing of CLEC12B using lenti-shRNA increases the activity of tyrosinase enzyme as shown by semi-quantification.

EXAMPLE

Material & Methods

Cell Culture

[0033] Normal human melanocytes (NHMs) and normal human keratinocytes (NHKs) were obtained from the foreskin of young children (skin type III or IV) undergoing circumcision. Tissue samples were kindly supplied by the Department of Pediatric Surgery, Lenval Hospital (Dr. Kurzenne, Nice, France). The samples were washed with phosphate buffered saline (PBS) containing 1% Antibiotic/Antimycotic (Gibco, Life Technologies, USA) 3 times for 5 minutes each. After removal of the subcutaneous tissue, tissue was cut into 22 mm.sup.2 pieces.

[0034] The foreskin samples were incubated within dispase enzyme (4 U/ml, Roche) for 12-16 h at 4 C. Once the dermis and epidermis of foreskins were separated with forceps, the epidermis was incubated within a trypsin/EDTA solution for 20 minutes at 37 C., the cells dispersed into cell suspensions and filtered by cell strainer (70 m, Falcon) before final wash with PBS.

[0035] NHMs were isolated in MCDB 153 medium (Sigma Aldrich) supplemented with 2% FBS (Fetal Bovin Serum, SV30160-03, Hyclone, USA), 5 g.ml.sup.1 insulin (Sigma Aldrich), 0.5 g.ml.sup.1 hydrocortisone (Sigma Aldrich), 16 nM TPA (phorbol 12-myristate 13-acetate , Sigma Aldrich), 1 ng.ml.sup.1 basic fibroblast growth factor (Promega; Madison, Wis.), 15 g.ml.sup.1 bovine pituitary extract (Gibco, Life Technologies, USA), 10 M forskolin (Sigma Aldrich), and 20 g.ml.sup.1 geneticin (Invitrogen) over 2 weeks. Melanocytes between passages 3 and 7 were used.

[0036] NHKs were isolated in keratinocyte basal medium 2 (C-20211, Promocell, Heidelberg, Germany) supplemented with human keratinocyte growth supplement (C-39011, HKGS, Cascade Biologics, Calif., USA). Keratinocytes between passages 2 and 5 were used.

[0037] MNT-1 human melanoma cells were cultured in DMEM (Gibco, Life Technologies, USA) supplemented with 10% Aim-V medium (Gibco, Life Technologies, USA), 20% fetal bovine serum (Hyclone, Logan, USA), 1 mM Sodium pyruvate (Gibco, Life Technologies, USA) and 0.1 mM nonessential amino acids (Gibco, Life Technologies, USA).

[0038] All cells were maintained at 37 C. in a 5% CO2 atmosphere.

Lentiviral Infection for RNAi Gene Knockdown

[0039] CLEC12B siRNA/shRNA/RNAi lentiviral particles (iV004749) and scrambled siRNA GFP Lentiviral particles (LVP015G) were purchased from Applied Biological Materials (Canada).

[0040] NHMs were serially passaged and used at passage 3 for viral infection. Cells were plated on 24-well plates at a density of 610.sup.4 cells per well and infected with a multiplicity of infection of 10 in the presence of 8 g/ml polybrene (Sigma Aldrich). Cells were incubated with virus for 48 hours and polybrene was added during the last 4 hours.The infected cells were selected for stable expression using puromycin at 1 ug/ml. Infected NHMs were cultured in medium 254 supplemented with human melanocyte growth supplement (Cascade Biologics, Calif., USA).

Co-Culture Experiment

[0041] For our coculture model, melanocytes were plated on 6-well plates at a density of 610.sup.4 cells per well. 24 h later, keratinocytes were added to each well (310.sup.5 cells), with an initial seeding ratio of 5:1.

[0042] Cocultures were then maintained in in keratinocyte basal medium 2 (C-20211, Promocell, Heidelberg, Germany) supplemented with human keratinocyte growth supplement (C-39011, HKGS, Cascade Biologics, USA).

[0043] After 24 or 96 hours of coculture, melanocytes and keratinocytes were separated by differential trypsinization method.

[0044] Differential trypsinization was performed as follows: MHNs were initially digested with 0.05% trypsin/EDTA for 2-5 min at 37 C. When MHNs become round and withdraw their dendrites, MHN single-cell suspension was obtained by carefully pipetting the MHN as they are more easily digested out of the substratum than KCs. The KHNs single-cell suspensions were obtained following a second digestion of trypsin. Trypsin is neutralized by medium containing 10% FBS. MHNs and KHNs were pelleted by centrifugation and resuspended in PBS before determination of melanin content and protein content.

Determination of Melanin Content Melanin Assay

[0045] Cell suspensions were centrifuged and pellets photographed before cells were solubilized in 120 l of 0,5N NaOH at 80 C. for 1 hr to dissolve melanin. Melanin absorbance was measured spectrophotometrically at 405 nm using a plate reader. Melanin production was calculated by normalizing the total melanin values with protein content.

Results

[0046] We have demonstrated that CLEC12B is expressed specifically in the skin by melanocytes. CLEC12B is expressed at the membrane surface of the melanocytes and in the cytoplasm. CLEC12B colocalized with microtubules. It also colocalized with and actine fibers but only when melanocytes are stimulated with MSH or forskolin or ultraviolet. Using videomicroscopy we have followed the CLEC12B thanks to a lentivirus-GFP/CLEC12B construction and we have observed that after stimulation with MSH or forskolin, CLEC12B colocalizes with melanosome and interact with the membrane of the keratinocytes. Then, using co-culture experiment with melanocytes and keratinocytes we have showed that decreasing CLEC12B expression significantly reduce the transfer of melanin to the keratinocytes.

[0047] Normal Human Melanocyte (NHM) are transduced with control or CLEC12B lentiviral shRNA. Cells images (data not shown) and cells lysates (data not shown) shown that silencing of CLEC12B using lenti-shRNA increases the production of melanin in NHM. The quantification of melanin in NHM transduced with control or CLEC12B lentiviral shRNA normalized to protein content (FIG. 2).

[0048] Silencing of CLEC12B using lenti-shRNA increases the activity of tyrosinase enzyme as shown on FIG. 3 by semi quantification.

[0049] Silencing of CLEC12B using lenti-shRNA increases microphthalmia-associated transcription factor (MITF) and melanogenesis gene expression of DCT and Tyrosinase (data not shown).

[0050] Taken together these results demonstrate that CLEC12B is specifically expressed in the skin by melanocytes and plays a key role in the transfer of or melanosomes (and thus melanin) to the keratinocytes. Decreasing the expression of CLEC12B or preventing the interaction between CLEC12B with keratinocytes is a specific and effective way to decrease pigmentation in the skin.

REFERENCES

[0051] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.