TBK1 inhibitor for use in the treatment of vitiligo

Abstract

Vitiligo is a complex auto-immune skin disease resulting in melanocytes loss and skin depigmentation. Following-up on their previous work identifying mitochondrial DNA (mtDNA) in skin biopsies from some vitiligo patients, the inventors demonstrate here that melanocytes from non-lesional skin present elevated numbers of mtDNA variants associated with mtDNA release and can be classified as function of their number of mtDNA variants in low-variant (LV) and high-variant (HV) load. Vitiligo HV melanocytes display increased ROS production, reduced antioxidant capacity, elevated mitochondrial function and mtDNA release as compared to Vitiligo LV and healthy melanocytes. Sensing of mtDNA by the cGAS-STING pathway results in pro-inflammatory cytokine and chemokines production promoting the recruitment of cytotoxic CD8+ T cells. This cascade of events can be blocked with TBK1 inhibitors suggesting new therapeutic approaches for sub-population of vitiligo patients displaying HV mtDNA phenotype. The present invention relates to method for the treatment of vitiligo in a subject in need thereof comprising a step of administrating to the subject a therapeutically effective amount of TANK-binding kinase 1 (TBK1) inhibitor.

Description

[0001] TBK1 INHIBITOR FOR USE IN THE TREATMENT OF VITILIGO

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of dermatology. More particularly, the invention relates to methods and compositions for treating vitiligo in a subject in need thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] Vitiligo is a multifactorial autoimmune disease that causes melanocyte loss, leading to skin and hair depigmentation [1], The Thl response against melanocytes plays a key role in the adaptative immune response in vitiligo [2], [3], The importance of this pathway has been underlined by the efficacy of topical and systemic use of JAK inhibitors in treating vitiligo [4],

[0006] [5], These treatments represent a great advance in the care of this challenging disease. Despite these advancements, skin repigmentation still takes more than 1-2 years, and most patients do not attain a complete response. Thus, additional therapeutic options are needed and this requires a better understanding of the physiopathology of the disease resulting in the initiation of the autoimmune response against melanocytes. Exposomal factors occurring in genetically predisposed patients are well -demonstrated triggering factors of vitiligo flares. The innate immune response is the bridge between exposome factors and activation of the adaptative immunity [1],

[0007] In vitiligo non-lesional skin (NLS), the number of natural killer (NK) cells and other innate lymphoid cells group 1 (ILC1) are significantly increased compared to normal skin, making it more prone to respond to exposomal factors with pathogen-associated molecular patterns (PAMPs) and / or damage-associated molecular patterns (DAMPs). This activation leads to increased production of IFNy, which is associated with the release of chemokines such as CXCL9, CXCL10, CXCL11 and CXCL16 not only from keratinocytes but also from melanocytes and fibroblasts [6]- [8] . Those chemokines induce the activation of CD8+ T-cells and bind to CXCR3B receptors in melanocytes inducing their death and release of melanocytic antigens, establishing a positive feedback loop of autoimmune response against melanocytes

[0008] [6], The inventors previously showed that the inhibition of CXCR3B avoids melanocytic death and presentation of melanocytic agents in both non-segmental and segmental vitiligo patients [6], Nonetheless, the initial triggers preceding this cascade of events are yet to be unravelled. Vitiligo has long been described as an oxidative stress-oriented disorder [9]—

[0012] , linked to downregulation of reactive oxygen species (ROS) scavenger enzymes such as catalase (CAT)

[0009]

[0013] , The increase in ROS levels might drive the pathogenesis, initiating recruitment of innate immune cells at early stages.

[0010] Recently, a prospective randomized double-blinded, placebo-controlled study demonstrated that the use of a potent oral antioxidant in addition to narrowband UVB provided significantly higher repigmentation after 6 months of treatment compared to narrowband UVB and placebo

[0011]

[0014] , Furthermore, previous studies have demonstrated a strong correlation between the skin microbiome and its impact on autoimmune diseases

[0015] —

[0017] .The inventors’ group has previously shown gut and skin dysbiosis in vitiligo patients and that lesional skin (LS) from some vitiligo patients contained higher levels of mitochondrial DNA (mtDNA) compared to non-lesional skin (NLS) or healthy controls

[0018] , This phenotype was strongly correlated with the increase in opportunistic / pathogenic bacteria, decrease of commensals present at the same site, and with concomitant increase in blood levels of CXCL9, CXCL10, CXCL11 and CXCL16

[0018] , Therefore, since the presence of mtDNA was not observed in the LS of all vitiligo patients, the inventors wondered whether instead of being a byproduct of melanocyte destruction, it may be involved in vitiligo pathogenesis.

[0012] In this study, the inventors show that melanocytes from some vitiligo patients present elevated numbers of mtDNA variants associated with mtDNA release. Melanocytes presenting with this phenotype demonstrate increased mitochondrial function and ROS levels associated with reduced catalase activity. Further, sensing of mtDNA by the cGAS-STING pathway leads to the release of pro-inflammatory cytokines resulting in the recruitment of CD8+ T cells responsible for the initiation of an autoimmune response. Finally, they show that this cascade of events can be blocked by treatment with TBK1 inhibitors, suggesting new therapeutic approaches for this sub-population of vitiligo patients.

[0013] SUMMARY OF THE INVENTION:

[0014] The present invention relates to a method for the treatment of vitiligo in a subject in need thereof comprising a step of administrating to the subject a therapeutically effective amount of TANK- binding kinase 1 (TBK1) inhibitor. In particular, the present invention is defined by claims.

[0015] DETAILED DESCRIPTION OF THE INVENTION: Vitiligo is a complex auto-immune skin disease resulting in melanocytes loss and skin depigmentation. Following-up on their previous work identifying mitochondrial DNA (mtDNA) in skin biopsies from some vitiligo patients, the inventors demonstrate here that melanocytes from non-lesional skin present elevated numbers of mtDNA variants associated with mtDNA release and can be classified as function of their number of mtDNA variants in low-variant (LV) and high-variant (HV) load. Vitiligo HV melanocytes display increased ROS production, reduced antioxidant capacity, elevated mitochondrial function and mtDNA release as compared to Vitiligo LV and healthy melanocytes. Sensing of mtDNA by the cGAS-STING pathway results in pro-inflammatory cytokine and chemokines production promoting the recruitment of cytotoxic CD8+ T cells. This cascade of events can be blocked with TBK1 inhibitors suggesting new therapeutic approaches for sub-population of vitiligo patients displaying HV mtDNA phenotype.

[0016] Mains definitions of the present invention

[0017] As used herein, the term “melanocyte” refers to melanin-producing neural crest-derived cells located in the bottom layer (the stratum basale) of the skin's epidermis, the middle layer of the eye (the uvea), the inner ear, vaginal epithelium, meninges, bones, and heart. Melanin is a dark pigment primarily responsible for skin color. Once synthesized, melanin is contained in special organelles called melanosomes which can be transported to nearby keratinocytes to induce pigmentation.

[0018] As used herein, the terms “pigmentation disorders” refer to disturbances of human skin color, either loss or reduction (depigmentation or hypopigmentation) which may be related to loss of melanocytes or to the inability of melanocytes to produce melanin or transport melanosomes correctly, or increase (hyperpigmentation) which is caused by an excessive production of melanin by melanocytes.

[0019] As used herein, the term “vitiligo” refers to an acquired depigmentation of the skin inducing a marked alteration of the quality of life of affected subjects. This disease is characterized by destruction of melanocytes that occurs mainly in the skin and results in the appearance of well circumscribed white macules. There are two types of vitiligo, i.e., segmental vitiligo located unilaterally on an area of the face, upper body, legs or arms, which in general does not change; and generalized vitiligo, which has more or less often bilateral symmetrical spots on areas of repeated friction or pressure and may become increasingly important over the years. The exact physiopathological mechanism that leads to the destruction of melanocytes is still elusive and involves autoimmunity (Passeron T; Ortonne JP 2005; Spritz 2007). Vitiligo is common and affects 1% to 2% of the general population. For many patients with vitiligo, the disfigurement caused by the disease has a great impact on their quality of life (Ongenae K et al. 2006).

[0020] As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with vitiligo.

[0021] As used herein, the term “biological sample” refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue biopsy. In a particular embodiment, the biological sample of the present invention is a skin sample.

[0022] Method of treatment of vitiligo

[0023] A first object of the present invention relates to a method for the treatment of vitiligo in a subject in need thereof comprising a step of administrating to the subject a therapeutically effective amount of TANK-binding kinase 1 (TBK1) inhibitor.

[0024] The inventors have particularly showed that melanocytes from some vitiligo patients present elevated numbers of mtDNA variants associated with mtDNA release. They also show that melanocytes presenting with this phenotype demonstrate increased mitochondrial function and ROS levels associated with reduced catalase activity. Further, sensing of mtDNA by the cGAS- STING pathway leads to the release of pro-inflammatory cytokines resulting in the recruitment of CD8+ T cells responsible for the initiation of an autoimmune response.

[0025] As used herein, the term “mitochondrial DNA” (mtDNA) refers to the DNA located in mitochondria, cellular organelles within eukaryotic cells that convert chemical energy from food into a form that cells can use, such as adenosine triphosphate (ATP). Mitochondrial DNA is only a small portion of the DNA in a eukaryotic cell; most of the DNA can be found in the cell nucleus. In some embodiment, the subject of the present invention is having a phenotype with a high- variant (HV) load of mitochondrial DNA (mtDNA).

[0026] As used herein the terms “high-variant (HV) load of mtDNA” refer to a melanocyte phenotype from some vitiligo patients that present elevated numbers of mtDNA variants associated with mtDNA release. Melanocytes presenting with this phenotype demonstrate increased mitochondrial function and ROS levels associated with reduced catalase activity.

[0027] In some embodiment, the subject of the present invention is having a phenotype with a low- variant (HV) load of mtDNA.

[0028] As used herein the terms “low-variant (LV) load of mtDNA” refer to a melanocyte phenotype from some vitiligo patients that present low numbers of mtDNA variants associated with mtDNA release.

[0029] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0030] The present invention relates to TANK-binding kinase 1 (TBK1) inhibitor for use in the treatment of vitiligo.

[0031] As used herein, the term “TANK-binding kinase 1” (TBK1) is an enzyme with kinase activity. Specifically, it is a serine / threonine protein kinase. It is encoded by the TBK1 gene in humans. This kinase is mainly known for its role in innate immunity antiviral response. However, TBK1 also regulates cell proliferation, apoptosis, autophagy, and anti-tumor immunity. Insufficient regulation of TBK1 activity leads to autoimmune, neurodegenerative diseases or tumorigenesis. TBK1 has the following Gene ID: 29110 and the following human UniProt number Q9UHD2.

[0032] As used herein, the term “TANK-binding kinase 1 inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of TBK1. In a particular embodiment, the antagonist of TBK1 is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

[0033] In a particular embodiment, the TBK1 inhibitor is a small organic molecule.

[0034] As used herein, the term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0035] Some examples of TBK1 inhibitor include but are not limited to BX795, GSK8612, Amlexanox, MRT67307, BAY-985, TBK1-IN-1, TBKl / IKKs-IN-4, TBKl / IKKs-IN-2, GSK319347A, MRT67307 hydrochloride, (Rac)-BAY-985, TBKl / IKKs-IN-1, SR8185

[0036] AZ 13102909, CYT387 and Domainex.

[0037] In a particular embodiment, the TBK1 inhibitor of the present invention is GSK8612.

[0038] As used herein, the term “GSK8612” refers to is a highly selective and potent Tank-binding Kinase-1 (TBK1) inhibitor. GSK8612 is having the following CAS Number : 2361659-62-1 and the following chemical structure :

[0039] In a particular embodiment, the inhibitor of TBK1 is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

[0040] As used herein, the term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of TBK1 is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

[0041] In some embodiments, the inhibitor of TBK1 is an antibody.

[0042] As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93 / 1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06 / 030220 and WO 06 / 003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

[0043] In some embodiments, the antibody is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. In some embodiments, the inhibitor of TBK1 is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of TBK1. In a particular embodiment, the inhibitor of TBK1 expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide- long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0044] In some embodiments, the inhibitor of TBK1 is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

[0045] In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0046] In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 Bl and US 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534 / genetics.113.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0047] In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0048] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of TBK1) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0049] In a particular embodiment, the TBK1 inhibitor is administered topically.

[0050] A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0051] The inhibitor of TBK1 as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringeability or injectability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatine. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0052] In one embodiment, the TBK1 inhibitor is administered in combination with a classical treatment of vitiligo.

[0053] As used herein, the term “classical treatment” refers to any compound, natural or synthetic, and phototherapy used for the treatment of vitiligo.

[0054] In a particular embodiment, the classical treatment refers to phototherapy, such as narrow-band UVB (Nb-UVB), PUVA, excimer laser or lamp and topical treatments.

[0055] In another embodiment, the classical treatment refers to treatment acting on the auto immune response. Treatments include but are not limited to anti-IFNg, anti-IFN type 1, IL2 lutein to promote Treg response or BET inhibitor. According to the invention, compound used for the treatment of vitiligo may be selected in the group consisting in: topical corticosteroids (such as clobetasol propionate, betamethasone valerate, betamethasone diproprionate, prednisolone or hydrocortisone butyrate), topical calcineurin inhibitors (such as tacrolimus or pimecrolimus), topical JAK inhibitors, topical WNT agonists, topical GSK3b inhibitors, phenylalanine, psolarens (such as oxsoralen or trisoralen) and vitamin D analogues (such as calcipotriol or tacalcitol).

[0056] As used herein, the term “GSK3b inhibitors” refers to chemical compounds that are effective in inhibiting the activity of the glycogen synthase kinase 3p. Examples of GSK3b inhibitors include hymenialdisine, dibromocantharelline, debromohymenialdisine, thiadiazolidines, thiazoles, halomethylketones, aminopyrimidines, arylindolemaleimide, metal cation (such as beryllium, lithium chloride, copper, zinc), indirubin, manzamines, meridianin, tricantin and palinurin,

[0057] As used herein, the term “WNT agonists” refers to chemical compounds that are effective in activating the WNT signaling pathways.

[0058] As used herein, the term “JAK inhibitors” refers to chemical compounds that are effective in inhibiting the activity of one or more Janus kinase enzymes (JAK1, JAK2, JAK3 and TYK2). Examples of JAK inhibitors include tofacitinib, ruxolitinib, oclacitinib, baricitinib, filgotinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, pacritinib, upadacitinib, peficitinib, fedratinib, cucurbitacin I.

[0059] Method of screening

[0060] In another aspect, the present invention relates to a method of screening a drug suitable for the treatment of vitiligo comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity of TBK1.

[0061] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity of TBK1. In some embodiments, the assay first comprises determining the ability of the test compound to bind to TBK1. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of TBK1. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable to inhibit the activity of TBK1, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

[0062] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0063] FIGURES:

[0064] Figure 1: Some vitiligo melanocytes present elevated frequency of mtDNA variants associated with mitochondrial nucleic acids DNA release. (A) Number of mtDNA variants in melanocyte cultures from healthy donors and non-active vitiligo patients; (B) Measurement of double-stranded DNA (dsDNA) in the culture supernatant of melanocytes from healthy, Vitiligo low variants (LV) and Vitiligo high variants (HV). (C) Relative abundance of MT- ND1 or MT-CO1 transcript in melanocyte culture supernatant. (D) Correlation between the number of mtDNA variants present melanocytes and the amount of MT-ND1 or MT-CO1 transcripts detected in their culture supernatant. (E) Number of mtDNA variants found in melanocytes or PBMCs of various vitiligo melanocytes cultures. All data represent mean± SD. One-way ANOVA compared to healthy controls. *P < 0.05; ** P< 0.01; *** P < 0.001.

[0065] Figure 2: Vitiligo HV melanocytes display increased mitochondrial function. (A) Quantification of mitochondrial mass per micron and scoring of mitochondrial branching; (B) Correlation between mitochondrial mass and the number of mtDNA variants in melanocytes; (C) Total ATP production; (D) Quantification of basal respiration. (E) ATP-coupled respiration; All data represent mean ± SD. One-way ANOVA compared to healthy controls. *P

[0066] < 0.05; ** P< 0.01; *** P < 0.001; **** P < 0.0001.

[0067] Figure 3: Vitiligo HV melanocytes demonstrate elevated ROS levels and is associated with decreased peripheral catalase activity. (A) ROS measurement at baseline and (B) in response to oxidative stress induced by 50 pM menadione. (C) Catalase activity assay in matched PBMCs. All data represent mean ± SD. One-way ANOVA compared to healthy controls. *P < 0.05; ** P< 0.01; **** P < 0.0001.

[0068] Figure 4: Vitiligo HV triggers inflammatory response via the cGAS-STING pathway. (A) Melanocytes from healthy donors, Vitiligo LV orHV were immunostained for p-TBKl and the percentage of cells with p-TBKl foci was quantified. (B) Immunoblot analysis of melanocyte lysates for STING, TBK1, p-TBKl, NF-kB p65, NF-kB pl00 / p52, IRF3 and IRF7. Vinculin was used as a loading control.

[0069] Figure 5: Preventing mtDNA release or sensing blunts inflammatory response responsible from immune cells recruitment. (A) Measurement of dsDNA and a panel of cytokines (B-F) and chemokines (G-H) in the supernatant of melanocytes treated with NRF2 activator DMF (50 pM) or TBK1 inhibitor GSK8612 (10 pM). All data represent mean ± SD. n=3 melanocyte cultures per condition measured in technical triplicates. Two-way ANOVA compared to healthy controls. *P < 0.05; **P < 0.01; ***p < 0.001; ****p < 0.0001.

[0070] Figure 6: Vitiligo HV triggers inflammatory response via the cGAS-STING pathway. (A) Melanocytes from healthy donors, Vitiligo LV orHV were immunostained for p-TBKl and the percentage of cells with p-TBKl foci was quantified (n=3 melanocyte cultures per condition and at least 45 cells per datapoint). (B) Immunoblot analysis of melanocyte lysates for STING, TBK1, p-TBKl, NF-kB p65, NF-kB pl00 / p52, IRF3 and IRF7 (n=3 melanocyte cultures per condition). Vinculin was used as a loading control. (C) ELISA assay from supernatants measuring IL-ip, IL- 18, IFNa, IFNP, CXCL9 and CXCL10 (n=3 melanocyte cultures per condition measured in technical triplicates). (D) Chemotaxis assay of CD8+ cells from healthy donors or vitiligo patients using culture supernatants from healthy melanocytes, Vitiligo LV or HV melanocytes (n=3 melanocyte cultures per condition measured in technical triplicates). All data represent mean ± SD. One-way ANOVA compared to healthy controls. * P < 0.05; ** P < 0 01; *** p < 0.001; **** P < 0.0001. EXAMPLE:

[0071] Material & Methods

[0072] Patients

[0073] Non-segmental vitiligo patients (n=16) were recruited from the Dermatology Department of the L’Archet Hospital, Nice CHU, and were enrolled in the study after informed, written consent was obtained. Healthy donors (n=14) surveyed for suspicion of skin cancer or familial history but negative on examination were recruited from the same clinic. The two groups were matched for age and gender, and vitiligo patients had no other auto-immune diseases (Data not shown . Sample collection consisted of a 4-mm skin punch biopsy from the non-lesional sites of vitiligo patients which was used for the isolation of melanocytes and 10 mL blood used for the isolation of peripheral blood mononuclear cells (PBMCs) by Ficoll gradient centrifugation (Lymphoprep®, Euromedex, France). The PBMCs were frozen in FCS 10 % DMSO and kept at -156 °C until subsequent analysis. The study was approved by the Regional Ethics Committee CPP Sud - Est VI, 1429 (N12.034) and conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

[0074] Isolation of primary human melanocytes

[0075] Upon arrival, skin biopsies were rinsed in 70 % ethanol followed by two times PBS 1 % Antibiotic-Antimycotic solution (Gibco) washes, prior to dissociation of dermal-epidermal junctions by overnight digestion in a Dispase solution (Life Technologies, Waltham, MA) at 4 °C. The next day, dermis was discarded and epidermis digested in a trypsin / EDTA solution for 20 min at 37 °C. Cellular suspension was passed through a 70 pm filter and melanocytes resuspended in MCDB 153 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2 % foetal bovine serum (FBS; Hyclone Perbio, Brevieres, France), 5 pg / ml insulin (Sigma- Aldrich), 0.5 pg / ml hydrocortisone (Sigma-Aldrich), 16 nM tetradecanoylphorbol- 13 -acetate (TP A) (Sigma-Aldrich), 1 ng / ml fibroblast growth factor (FGF; Promega, Madison, WI, USA), 15 pg / ml bovine pituitary extract (Invitrogen, Waltham, MA, USA) and 10 pM forskolin (Sigma- Aldrich). Melanocytes were maintained at 37 °C in a humidified 5 % CO2 atmosphere and supplemented with 0.08 % G418 geneticin (20 pg / ml, Invitrogen) for ~2 weeks to eliminate rapidly growing cells (i.e., keratinocytes hence selecting for melanocyte propagation). Once selected, melanocytes were grown in Cascade Biologies 254 medium (ThermoFisher Scientific, Waltham, MA) with Human Melanocyte Growth Supplement 1 (HMGS, Gibco).

[0076] Mitochondrial DNA sequencing mtDNA enrichment was performed through amplification of the mitochondrial genome using PCR, generating nine overlapping amplicons

[0040] ,

[0041] ,

[0042] Dobner et al., 2024b). These fragments were then sequenced on an Oxford Nanopore Technologies (ONT) MinlON device with a Flongle adaptor and flow cell, following a previously established protocol

[0043] , First, we pooled the PCR products and prepared a sequencing library using a ligation kit and barcodes (ONT). The library was then loaded onto a Flongle flow cell and sequenced for 24 h

[0044] , After sequencing, the fast5 files were basecalled using the guppy basecaller with specific parameters

[0045] , The resulting FASTQ files were concatenated, aligned to the mtDNA revised Cambridge Reference Sequence (rCRS) using minimap2, and variant calls were identified using Mutserve with a threshold of 0.05 or directly in Mitopore. Only mtDNA variants present in at least 12.5 % of reads were considered.

[0077] Immunofluorescence Microscopy

[0078] Cells were grown on glass coverslips, washed with PBS, fixed with a 3.7 % formaldehyde solution in PBS containing 250 nM HEPES for 15-min at room temperature, rinsed 3 times 5- min with ice-cold PBS, permeabilized for 3-min with 1% Triton X-100, rinsed another 5 times 5-min in room temperature PBS, blocked for 30-min with a 10% FBS solution in PBS and incubated with primary antibodies against dsDNA (PROGEN AC-30-10 at 1 / 500), TOM20 (BD Biosciences BD612278 at 1 / 500) or p-TBKl Serl72 (Cell Signalling Technologies #5483 at 1 / 150). This was followed by appropriate secondary antibodies including Alexa Fluor 594 goat anti-mouse IgG (H+L) (Al 1032), Texas-Red goat anti-rabbit IgG (H+L) (T6391) and Alexa Fluor 488 goat anti-Mouse IgM (SA5-10150) (Life Technologies). Nuclei were labelled with Hoechst 33342 (Sigma- Aldrich). The coverslips were mounted on glass slides using ProLong® Diamond Antifade Mountant (Life Technologies). The samples were visualized using Nikon AIR confocal microscope on an inverted Nikon Eclipse Ti stand (Nikon Instruments, Japan) using objectives Plan Apochromat 63x / 1.4 oil NA and Argon LASER 488 nm and / or DPSS 561 nm. The microscope was composed of 2 PMTs equipped with 450 / 50 and 700 / 75 filter set, 2 GaAsP equipped with 515 / 30 and 585 / 65 filter-set, and 1 external PMT for transmission. Z-acquisitions were performed using the microscope z-drive each 0.3 pm. Images were processed by Fiji software. Quantification of p-TBKl foci was performed on at least 45 cells per melanocyte culture. A p-TBKl focus was defined as a bright spot visible on at least two consecutive z-slices. A cell was considered positive if presenting at least three p-TBKl foci.

[0079] Measurement of dsDNA and mitochondrial markers by QPCR in cultured supernatants dsDNA was measured using Nanodrop (Thermo Fisher Scientific) and results expressed as ng / pl. MT-ND1 mRNA and MT-CO1 mRNA were measured by qPCR. Initially, RNA was extracted using RNeasy kits (Qiagen, Dusseldorf, Germany). One pg RNA was used to synthesize cDNA using the Reverse Transcription System (Promega) which was then used as a template for amplification by real-time qPCR with SYBRTM Green reagent (Life Technologies, CA, USA) and specific primers directed against MT-ND1 and MT-CO1 (primer sequences are given in Table 1). All measurements were performed in triplicate and results normalized to the expression of the 18S rDNA housekeeping gene.

[0080] Electron Microscopy

[0081] Melanocytes were seeded in 12-well culture plates. Following confhiency, cells were washed twice in PBS and then fixed with 1.6 % glutaraldehyde in phosphate buffer (0.1 M pH 7.4) for 1 hr at room temperature. They were then rinsed in cacodylate buffer (0.1 M pH 7.4) and postfixed in 1% osmium tetroxide (reduced with 1% potassium ferrocyanide). After being rinsed in distilled water, cells were gradually dehydrated in ethanol, embedded in epoxy resin, and incubated at 60°C overnight for polymerization. Ultrathin sections (80 nm) were assembled on copper grids and contrasted with lanthanides salts (gadolinium and samarium) followed by lead citrate. Sections were observed under a JEOL JEM 1400 electron microscope equipped with a Morada SIS camera. Mitochondrial mass was assessed using Mitochondria Analyzer plugin in ImageJ software (version 2.1) and degree of mitochondrial branching was scored semi- quantitatively from EM images.

[0082] ATP Content

[0083] The steady-state intracellular ATP content was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega; Madison, WI, USA) following the manufacturer’s instructions. Mitochondrial ATP and glycolytic ATP were measured upon treatment with oligomycin (ATP synthase inhibitor, 5 pM) or 2-deoxy -glucose (hexokinase inhibitor, lOmM). Luminescence was monitored with a Thermo Scientific™ Multiskan™ FC Microplate Photometer and values were calculated based on an ATP standard curve. Data were normalized to cell count.

[0084] Oxygen consumption rate

[0085] The oxygen consumption rate (OCR) was measured using a Seahorse XF96 extracellular flux analyser (Agilent). Cells were seeded at an initial concentration of 1 x 104 cells / well of a XF96 plate, and they were allowed to adhere for 24 h. A measurement plate containing calibrant solution (100 pl per well) was placed in a CO2-free incubator at 37 °C overnight. The next day, this plate was run as calibration. Meanwhile, culture media were removed, cells washed twice with PBS and 100 pl of fresh media was added containing 5 mM glucose, 1 mM pyruvate and 4 mM glutamine. To eliminate CO2 residue in the medium, cells were incubated for 1 h prior to the experiment at 37 °C with CO2 in a non-humidified incubator. After calibration, OCR was assayed by sequential addition of mitochondrial inhibitors and uncoupler. Following stabilization of basal respiration, ATP-synthase inhibitor oligomycin (1 pM, port A) was initially added to evaluate the oxygen consumption independent of mitochondrial ATP production. Then, the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added, forcing the transport of H+ throughout the mitochondrial inner-membrane and reaching the maximum mitochondrial respiration (500 nM, port B and C). Finally, 0.5 pM rotenone (inhibitor of CI) plus antimycin A (inhibitor of CIII) were added (port D) to block mitochondrial respiration. At least 6-technical replicates were done in the same plate for each sample and 6- measurements were carried out at baseline and after each injection. Three independent experiments were performed. The OCR value was normalized to cell numbers per well.

[0086] Reactive oxygen species (ROS) production

[0087] At approximately 70 % confluency, primary human melanocytes were treated with 50 pM menadione A (Sigma-Aldrich) (or not treated as controls) for 1 h at 37 °C. Cells were then collected, washed with PBS, and stained with 5 pM dihydrorhodamine for 30 min at 37 °C. Finally, ROS-positive cells were analyzed using BD FACSCalibur (BD, Pont-de-Claix, France) and analysis quantified using FlowJo software (version 10.8.2).

[0088] DNA extraction

[0089] Genomic DNA isolation was performed using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's protocol. Whole exo me sequencing (WES)

[0090] WES was performed at BGI Genomics (Warszawa, Poland) using their standardized protocol. Briefly, upon arrival, DNA was quantified with the Qubit dsDNA BR Assay system (Life Technologies) and its quality was assessed by 1% agarose gel electrophoresis. After random fragmentation of genomic DNA using Covaris S-220, fragmented DNA was selected by Agencourt AMPure XP -Medium kit to an average size of 200-400 bp. The selected fragments were then run through end-repair, 3 ’-adenylation, adapters-ligation and PCR amplification followed by AxyPrep Mag PCR clean up. Hybrid capture was performed using SureSelect Human All Exon v.6 followed by another AxyPrep Mag PCR clean up. The double-stranded PCR products were heat-denatured and circularized by the splint oligo sequence forming the single strand circle DNA (ssCir DNA) as the final library. The library was amplified to make DNA nanoballs (DNBs) which have more than 300 copies of one molecular. The DNBs were loaded into the patterned nanoarray and paired 100 bases reads were generated in the way of sequenced by combinatorial Probe-Anchor Synthesis (cPAS) on DNBSEQ-G400 platform. After filtering with SOAPnuke, over 75 million clean reads were detected in each sample with an average Q30 of 95.26 %. Alignment on hg38 was performed with BWA v.0.7.17. On average, we obtained a 68.02x mean sequencing depth on the whole genome excluding gap regions. SNP and indel calling, and annotation were performed using GATK v.4.1.4.1. A special emphasis was placed on the analysis of genes involved in antioxidant mechanisms.

[0091] Catalase activity

[0092] Catalase activity was measured in lysates of 2 * 106 PBMCs using the Catalase Activity Assay Kit from Abeam according to the manufacturer's protocol.

[0093] Protein Extraction and Immunoblotting

[0094] Approximately 2.5 x 105 cells were seeded per 6-well plates. 24 h after incubation, cells were washed with PBS and 100 pl of IxRIPA buffer (Sigma-Aldrich) with protease and phosphatase inhibitors (Merck) were added in each well. Cells were scraped-off, collected and frozen at -80 °C until further use. For protein extraction and immunoblotting, cell lysates were thawed, and proteins were quantified by Pierce BCA protein assay kit. LDS Sample Buffer (Thermo Fisher Scientific) was added, and sample volume was adjusted to equal protein concentration. Samples were heated at 90 °C for 5 min and 15-20 pg of protein extract per lane was loaded on SDS-PAGE gel. After running at 100 V for 1.5 h, the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore) to a blotting membrane. The proteins were incubated with primary antibodies overnight at 4 °C using the following series of antibodies: total OXPHOS WB Antibody Cocktail (1 : 1,000 Abeam #abl 10413) containing the antibodies anti-NDUFB8, anti-SDHB, anti-UQCRC2, anti-COX II and anti-ATP5 as a premixed solution; vinculin (1: 1,000 Cell Signaling #4650); IRF7 (1 : 1,000 Cell Signaling #4920); IRF3 (1 : 1,000 Cell Signaling #4302); NF-KB p65 (1 : 1,000 Cell Signaling #8242); NF-KB pl00 / p52 (1 : 1,000 Cell Signaling #3017). The next day, membranes were incubated for Ih at 4 °C with the appropriate HRP-conjugated secondary antibodies: anti-rabbit HRP conjugated secondary (1 :3,000 Thermo Fisher Scientific) or anti-mouse HRP conjugated secondary (1:3,000 Thermo Fisher Scientific). Proteins were detected with an ECL System from Amersham.

[0095] Immune Function

[0096] Secretion of CXCL-9, CXCL-10, CCL-19, IL-ip, IL- 18, IFNa and IFNP were measured under basal condition or after pre-treatment of melanocytes with TBK1 inhibitor GSK8612 (10 pM, MedChem Express) using commercially available ELISA kits (PeproTech, USA and R&D Systems Quantikine ELISA, Lille France). After a 2-h pre-treatment with the indicated compound or recombinant protein, medium was changed to regular culture medium. Culture supernatants were collected for secretomes analysis 24 h later.

[0097] Migration Experiments

[0098] The ability of the secretomes of recombinant GSK8612-pretreated melanocytes from healthy individuals, Vitiligo LV or HV patients to attract PBMCs from either healthy or vitiligo subj ects was tested using a Cytoselect 96-well Cell Migration Assay (Cell Biolabs, San Diego, CA, USA). Migratory cells were quantified using CyQuant GR Fluorescent Dye and results were compared to secretomes from non-pretreated melanocytes from the same patients.

[0099] Statistical analyses

[0100] Statistical analyses were performed with GraphPad Prism® 6.0 software (La Jolla, CA, USA). Normality of data and homogeneity of variances was assessed using Shapiro-Wilk and Bartlett tests, respectively. Statistical significance was determined by unpaired Student’s t-test comparing two independent groups. Multiple groups comparison was performed using ordinary one-way or two-way ANOVA with appropriate post-hoc analysis (Mann-Whitney U to test unpaired differences between groups and Wilcoxon signed rank test for paired differences). Statistical significance was considered at a > 0.05. All experiments were performed with a minimum of 3 biological replicates, and whenever possible, 2 or more technical replicates. All data represent mean ± SD.

[0101] Results

[0102] Number of mtDNA variants in vitiligo melanocytes is associated with release of mitochondrial DNA

[0103] Previous study from our group showed that some skin biopsies from the lesion sites of vitiligo patients have increased levels of mitochondrial DNA (mtDNA) compared to the healthy controls

[0018] , Numerous reports indicate that mtDNA can be released upon accumulation of alterations in its sequence. Thus, we isolated primary melanocytes from 14 healthy donors and 16 patients with non-active vitiligo and subjected them to mtDNA amplicon sequencing (Data not shown . While 11 vitiligo samples presented less than 50 variants compared to the reference genome and were similar to heathy subjects, 5 samples exhibited supraphysiological number of variants, more than 50 (Figure LA). This allowed us to classify the vitiligo melanocytes with mtDNA high variant load (Vitiligo HV) from the vitiligo melanocytes with low variant load (Vitiligo LV). Melanocytes presenting with HV phenotype demonstrate increased mtDNA release, increased mitochondrial function and ROS production and ROS levels to be associated with reduced catalase activity. Noticeably, healthy, Vitiligo LV and HV groups were age- matched and there was no correlation between the number of mtDNA variants detected in the melanocytes and the age of the subjects suggesting that mtDNA variant were not randomly accumulating over time (Data not shown). This was further supported by the variant types mainly comprised of T>C and OT transitions (Data not shown). Vitiligo HV released more double-stranded (ds)DNA in their culture supernatant as compared to both melanocytes from Vitiligo LV group and healthy subjects (Figure IB). Moreover, we found a positive correlation between the load of mtDNA variants, and the amount of dsDNA measured from the culture supernatant (Data not shown). Consistent with this observation, fluorescence microscopy confirmed a decrease in DNA staining colocalizing with the mitochondrial marker TOM20, suggesting less mtDNA present in Vitiligo HV melanocytes (Data not shown). Furthermore, we found increased amounts of transcripts from the mitochondrial encoded genes MT-ND1 and MT-CO1 in Vitiligo HV culture supernatants (Figure 1C), positively correlating with the number of mtDNA variants (Figure ID). We sequenced matching PBMCs from the subpopulation of vitiligo patients and observed that they presented with significantly lower number of mtDNA variants than melanocytes indicating their somatic and not germline origin (Figure IE). Except for 1 patient, the mtDNA HV were predominantly found in melanocytes and not in the PBMCs.

[0104] High load of mtDNA variants is associated with enhanced mitochondrial function

[0105] Mitochondrial dysfunctions caused by oxidative stress have been extensively studied and connected to changes in organelle shape and dynamics

[0019] , To investigate whether mitochondrial impairment could be the cause for the observed mtDNA release in Vitiligo HV cultured melanocytes, we first performed electron microscopy to evaluate mitochondrial morphology (Data not shown). We observed that melanocytes with HV have greater mitochondrial mass and branching (Figure 2A). There was a strong positive correlation between the number of mtDNA variants and the mitochondrial mass (R2=0.8050) (Figure 2B).

[0106] We then set out to characterize their mitochondrial metabolism. Both Vitiligo LV and HV showed decreased mitochondrial complexes proteins subunits compared to healthy controls (Data not shown). To further investigate the impact on the overall cellular energetic state, we measured total ATP production. Vitiligo HV melanocytes exhibited higher total ATP levels compared to Vitiligo LV or healthy melanocytes (Figure 2C). Furthermore, oxygen consumption rate (OCR) showed that melanocytes from vitiligo samples display significantly increased basal respiration compared to healthy melanocytes, with the greatest increase seen in Vitiligo HV melanocytes (Figure 2D). The increase in mitochondrial oxygen consumption was associated with higher ATP levels produced from the electron transport system, named ATP- Coupled respiration (Figure 2E). Measurements of ATP contribution from glycolysis by using the 2-deoxy-glucose (2DG) inhibitor, showed that Vitiligo HV melanocytes rely on mitochondrial activity rather than glycolysis to produce their ATP (Data not shown). Together, these data showed that Vitiligo HV melanocytes have increased energy metabolism driven by upregulation of their mitochondrial function.

[0107] Vitiligo HV show increased ROS levels correlated with lower catalase activity

[0108] Upregulation of mitochondrial oxidative phosphorylation leads to increased ROS levels. Consistent with their high mitochondrial function, Vitiligo HV demonstrated elevated levels of mitochondrial ROS monitored by dihydrorhodamine 123 (Figure 3A). Furthermore, Vitiligo HV melanocytes produced much higher levels of ROS when treated with the pro-oxidant menadione (Figure 3B), suggesting an impaired antioxidant capacity. To identify germline predictive biomarkers of the Vitiligo HV phenotype, we looked for possible alterations in redox balance genes through whole exome sequencing (WES) from PBMCs of five Vitiligo HV and four Vitiligo LV patients. Taking into consideration the expected frequency of the variants, we found rs371396899 and rs 17268652, two uncommon SNPs in catalase (CAT), to be tightly associated with the Vitiligo HV phenotype (Data not shown . Functional assay confirmed lower catalase activity in Vitiligo HV melanocytes (Figure 3C), supporting lower ROS scavenging capacity leading to greater oxidative stress.

[0109] Cytosolic release of mitochondrial DNA triggers innate inflammatory response via the cGAS- STING pathway mtDNA release in the cytosol has been linked to activation of the cGAS-STING pathway which activates immune responses

[0020] , Interestingly, we observed an induction of phosphorylated TBK1 (p-TBKl) foci downstream of the cGAS-STING pathway in Vitiligo HV but not in Vitiligo LV or healthy melanocytes (Figure 4A}. The activation of the cGAS-STING pathway was further confirmed by the induction of its targets TANK-binding kinase (TBK)l resulting in the induction of interferon regulatory factor (IRF)3 and IRF7, as well as the NFKB pathway detected by western blot (Figure 4B}. These transcription factors are critical for chemokines and cytokines production that play crucial roles in the stimulation of immunity and inflammation. Indeed, we demonstrated increased presence of type I interferons (IFNa / p), IL1- P, IL- 18, CXCL9 and CXCL10 in the culture supernatants of Vitiligo HV melanocytes detected by ELISA (Data not shown . Supernatants from Vitiligo HV melanocytes, containing such pro- inflammatory secretome, were capable of inducing a much more pronounced recruitment of PBMCs, than the secretome from LV or healthy melanocytes (Data not shown}. These results indicate a link between the oxidative stress occurring in vitiligo melanocyte mitochondria and the initiation of the innate immune response.

[0110] Preventing mitochondrial oxidative stress and mtDNA release or sensing blunts inflammatory response responsible for recruitment of immune cells

[0111] Finally, we assessed the effect of compounds capable of reducing mitochondrial oxidative stress in preventing activation of the downstream targets associated with mtDNA release. Although the TBK1 inhibitor GSK8612 had no effect on dsDNA release more upstream in the pathway, it effectively prevented cytokine and chemokine release except for IL-ip and CCL19 (Figures 5A to 5H). These agents had no effect on the immune function of Vitiligo LV melanocytes, nor healthy melanocytes. Therefore, targeting mitochondrial ROS using specific mitochondrial ROS scavengers inhibits the release of dsDNA and pro-inflammatory mediators in Vitiligo HV melanocytes. Finally, we showed that the secretome from the HV melanocytes pre-treated with the TBK1 inhibitor GSK8612 (Data not shown)), significantly blunted the CD8+ T-cell recruitment compared to the recruitment seen using the secretome from non-treated HV melanocytes (Data not shown)). These results demonstrate that targeting mitochondrial oxidative ROS and cGAS-STING pathway in HV melanocytes, can prevent recruitment of cytotoxic CD8+ T cells (Data not shown). We also showed that Vitiligo HV triggers inflammatory response via the cGAS-STING pathway (Figures 6A-6D).

[0112] Discussion

[0113] Many studies on vitiligo aim to decipher the mechanisms that ultimately lead to melanocytes death, including abnormal innate immunity response and inflammation [6], [7], increased reactive oxygen species (ROS) production [9],

[0012] ,

[0021] ,

[0022] , and changes in skin microbiome

[0015] ,

[0018] ,

[0023] , Mitochondrial (mt) DNA release, increased mtROS levels and altered ATP production are major mitochondrial damage-associated molecular patterns (DAMPs). Here we show, in accordance with our previous findings

[0018] , that some melanocytes extracted from the skin of some vitiligo patients release high amounts of mtDNA. This phenomenon, seen on proliferating monoculture of vitiligo melanocytes, indicates that mtDNA do not originate from the destruction of melanocytes by immune cells but rather from intact melanocytes.

[0114] The observed mtDNA release tightly correlated with the elevated number of mtDNA variants, mtROS, ATP-coupled respiration and catalase polymorphisms in matched PBMCs with reduced antioxidant capacities. Our observations are in line with previous reports demonstrating that mitochondrial oxidative stress can disrupt mtDNA integrity and facilitate its release in the cytoplasm and the extracellular space

[0024] ,

[0025] , We have shown that cytosolic mtDNA triggers the cGAS-STING pathway that activates the transcription factors IRF3 and IFR7 downstream of TBK1, and NF-KB resulting in proinflammatory cytokines and chemokines production and CD8+ T cells recruitment. Thus, our work introduces the concept that numerous mtDNA variants (Vitiligo HV) in the skin are biomarkers of DAMPs leading to activation of innate immune response.

[0115] Our results indicate that most of ATP produced in Vitiligo HV is generated by mitochondria, suggesting that due to oxidative damage and decreasing in mitochondrial subunits, mitochondria need to compensate their potentially “impaired” function with increased 02 consumption and ATP production. Impaired mitochondrial function has been reported in Alzheimer's disease, multiple sclerosis and systemic lupus erythematosus

[0026] —

[0028] , In accordance with our results in vitiligo patients, recent data demonstrated that mtDNA variants linked to oxidative damages are not correlated with age

[0029] , This observation is further supported by the type of variants mainly comprised of OT and T>C transitions previously reported to be associated with elevated mitochondrial hydroxyl radical levels

[0024] ,

[0030] , We have demonstrated mtDNA variants to have a somatic origin supported by a much lower frequency in matched PBMCs. These somatic mtDNA variants have been reported in aging and neurodegenerative disorders

[0031] , but there are very limited data of somatic variants in mtDNA in auto-immune diseases. Somatic mtDNA variants in synoviocytes have been found to be significantly increased in rheumatoid arthritis

[0032] ,

[0033] , The mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS) is due, in most cases, to a mutation at base pair (bp) 3243 in the mtDNA. Interestingly, vitiligo is found in 11% of cases of patients suffering from MELAS

[0034] , This represents a 10-fold increased risk compared to the general population. Similarly, mitochondrial oxidative stress has been suggested to play a key role in the pathophysiology of Vogt-Koyanagi-Harada syndrome, an autoimmune disease which specifically affects vision and hearing, and is associated with uveitis, meningitis and development of vitiligo

[0035] , Kabuki syndrome, a rare polymalformative genetic disorder caused by the loss of function of the histone methyltransferase KMT2D, leads to a dysregulation of mitochondrial respiration

[0036] , In this syndrome, mitochondrial dysregulation is associated with increased frequency of auto-immune disorders such as vitiligo, witnessed in over 5 % of all cases reported

[0037] , Thus, together with our results, there is strong evidence placing the dysregulation of mitochondrial homeostasis as a cornerstone in vitiligo initiation.

[0116] Building on this conclusion, the TBK1 inhibitor GW8612 was effective in inhibiting the melanocyte release of some cytokines but not IL-ip nor CCL19. This can be explained by the fact that part of STING activity is independent from TBK1 and can directly activate NF-KB

[0039] , Nevertheless, GW8612 efficiently decreased CD8+ T cell chemotaxis, indicating that targeting TBK1 represents a therapeutic option for Vitiligo HV patients. The impact of these mitochondrial targeted treatments is significantly higher in melanocytes with high mtDNA variant load compared to low variant load that appears, along with mtDNA release in the skin, as an interesting companion test for such therapeutic approaches (Data not shown . Future studies with larger cohorts will have to address what proportion of vitiligo patients presents with HV phenotype. This new understanding of the pathophysiology of vitiligo opens new avenues for blocking one of the leading causes of the establishment of the adaptive anti- melanocytic immunity and subsequent destruction of melanocytes.

[0117] REFERENCES:

[0118] 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.

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Claims

CLAIMS:

1. A method for the treatment of vitiligo in a subject in need thereof comprising a step of administrating to the subject a therapeutically effective amount of TANK-binding kinase 1 (TBK1) inhibitor.

2. The method for the treatment of vitiligo according to claim 1 wherein the subject is having a phenotype with a high-variant (HV) load of mitochondrial DNA (mtDNA).

3. The method for the treatment of vitiligo according to claim 1 wherein the TBK1 inhibitor is a small organic molecule.

4. The method for the treatment of vitiligo according to claim 3 wherein the a small organic molecule is GSK8612.

5. The for the treatment of vitiligo according to claim 1 wherein the TBK1 inhibitor is an antibody.

6. The for the treatment of vitiligo according to claim 1 wherein the TBK1 inhibitor is siRNA.

7. The method for the treatment of vitiligo according to claim 1 wherein the TBK1 inhibitor is administered topically.

8. A method of screening a drug suitable for the treatment of vitiligo comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity of TBK1.

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