Methods of treatment of patients suffering from hypomelanosis of ITO

Abstract

Hypomelanosis of Ito is a clinical term for patients with mosaic syndromes characterized by skin hypopigmentation and developmental disorders. The genetic causes of these rare diseases remain largely unclear. Here, we report that GNA13 is a new gene that causes Hypomelanosis of Ito. We identified an identical mutation in this gene in four unrelated patients exhibiting pigmentary mosaicism. In depth functional investigations revealed that this is an activatory mutation that alters the cytoskeleton and morphology of melanocytes via a hyperactivation of the RHOA / ROCK signalling pathway. Our results also indicate that this pathology does not necessarily originate from a decreased production of melanin, but can originate from a defect in melanosome transfer to keratinocytes due to cell shape alterations. Thus, our findings suggest for the first time a mechanism by which the clinical symptoms of patients with Hypomelanosis of Ito appear, and pave the path for new therapeutic approaches. Altogether, the present invention relates to a method for treating a patient suffering from hypomelanosis of Ito by administering a ROCK inhibitor and / or RHOA inhibitor.

Description

[0001] METHODS OF TREATMENT OF PATIENTS SUFFERING FROM

[0002] HYPOMELANOSIS OF ITO

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular rare neurocutaneous disease.

[0005] BACKGROUND OF THE INVENTION:

[0006] Somatic mosaicism anomalies are rare diseases that manifest on the skin as linear hypopigmentation zones along the Blaschko lines and are classified using broad, undefined terms such as "Hypomelanosis of Ito"1. Aside from cutaneous abnormalities, developmental and neurological disorders have also been reported. Despite the importance of this medical field in terms of paving the way for novel treatments for so-called orphan diseases, i.e. those with no known therapeutic resources to date, the genetic origins of these rare diseases remain unclear, hindering diagnosis and patient care. The study of these genetic bases has only recently become possible, thanks to the development of high throughput sequencing. This approach led us to the discovery of mutations in the MTOR and RHOA genes that cause the "Hypomelanosis of Ito" syndrome2,3.

[0007] Here, we have discovered a novel mutation in the GNA13 gene that encodes for Gal3 in four unrelated patients with Hypomelanosis of Ito who all have cutaneous and developmental anomalies but no major neurological disorders. Gal3 is a subunit of a heterotrimeric G protein (aPy) coupled to specific transmembrane receptors known as G-protein coupled receptors (GPCR)4,5. Upon GPCR stimulation, the Gal3 subunit loads with GTP in exchange for GDP, causing it to change conformation and dissociate from both the GPCR and the P and y subunits. The Gal3-GTP active form can then interact with downstream effectors and kinases to regulate a variety of essential cellular functions such as cytoskeletal modifications, gene transcription, cell migration, and cell cycle division. The signalling process ends with the hydrolysis of GTP, which is mediated by the intrinsic GTPase activity of the Ga subunit.

[0008] Here, the inventors have identified the functional consequences of this mutation and propose a molecular and cellular mechanism by which the clinical characteristics of those patients manifest and pave the path for new therapeutic approaches.

[0009] SUMMARY OF THE INVENTION: The present invention is defined by the claims. The present invention relates to methods of treatment of patients suffering from hypomelanosis of Ito, and in particular suffering from hypomelanosis of Ito caused by a mutation in a gene coding for a G protein alpha subunit.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] Hypomelanosis of Ito is a clinical term for patients with mosaic syndromes characterized by skin hypopigmentation and developmental disorders. The genetic causes of these rare diseases remain largely unclear. Here, we report that GNA13 is a new gene that causes Hypomelanosis of Ito. They identified an identical mutation in this gene in four unrelated patients exhibiting pigmentary mosaicism. In depth functional investigations revealed that this is an activatory mutation that alters the cytoskeleton and morphology of melanocytes via a hyperactivation of the RHOA / ROCK signalling pathway.

[0012] Their results also indicate that this pathology does not necessarily originate from a decreased production of melanin, but can originate from a defect in melanosome transfer to keratinocytes due to cell shape alterations. Thus, their findings suggest for the first time a mechanism by which the clinical symptoms of patients with Hypomelanosis of Ito appear, and pave the path for new therapeutic approaches.

[0013] Methods for treating hypomelanosis of Ito

[0014] Accordingly, the invention refers to a method for treating hypomelanosis of Ito in a subject in need thereof comprising administering a therapeutically effective amount of a ROCK inhibitor and / or RHOA inhibitor.

[0015] As used herein, the term “subject” or “patient” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Notably, in the present invention, the subject is a human. In some embodiments, the subject according to the invention has or is susceptible to have hypomelanosis of Ito, and particularly an hypomelanosis of Ito caused by at least one mutation in a gene coding for a G-alpha subunits, and more particularly an hypomelanosis of Ito caused by at least one mutation in the gene coding for the G subunits alpha 13 (GNA13 gene). In some embodiments, the subject according to the invention has or is susceptible to have a skin linear hypopigmentation along Blaschko lines, asymmetric facial dysmorphism, limb asymmetry and malformation, wound healing issues, teeth and ocular anomalies, gastroenterological and nephrological abnormalities, and neurological defects due to malformative hydrocephalus. As used herein, the term “Hypomelanosis of Ito” (HI) also known as “pigmentary mosaicism” refers to a group of very rare neurocutaneous orphan-disease that causes unusual patches of light-colored (hypopigmented) skin and may be associated with eye, nervous system, and skeletal problems. Hypomelanosis of Ito can be caused by mutations in the MTOR and RHOA genes as previously disclosed2'3.

[0016] Herein, the inventors discovered a novel mutation in the GNA13 gene that encodes for Gal 3 in four unrelated patients with Hypomelanosis of Ito who all have cutaneous and developmental anomalies but no major neurological disorders.

[0017] In particular embodiment, the hypomelanosis of Ito is caused by at least one mutation in a gene coding for a G alpha subunit.

[0018] As used the term “G alpha subunits” (Ga) has its general meaning in the art and refers to one of the three types of subunit of guanine nucleotide binding proteins, which are membrane-associated heterotrimeric G proteins. Guanine nucleotide binding proteins (G proteins) are membrane-associated, heterotrimeric proteins composed of three subunits: alpha, beta and gamma (IPR001770). G proteins act as signal transducers, relaying a signal from a ligand-activated GPCR (G protein-coupled receptor) to an enzyme or ion channel effector. The heterotrimeric G protein alpha subunit is composed of two domains: a GTP -binding domain and a helical insertion domain. The GTP -binding domain is homologous to Ras-like small GTPases, and includes switch regions I and II, which change conformation during activation. There are several isoforms of G protein alpha subunit, many of which have splice variants. G alpha subunits includes but are not limited to : Gs alpha subunit (or Gas) which is encoded by the gene GNAS gene (Its Entrez reference is 2778, and its Uniprot reference is P63092); G12 alpha subunits (or Gal2) which is encoded by the gene GNA12 gene (Its Entrez reference is 2768 and its Uniprot reference is Q03113); G13 alpha subunits (or Gal3) which is encoded by the gene GNA13 gene (Its Entrez reference is 10672 and its Uniprot reference is Q14344); Gqalpha subunits (also known as Gaq) which is encoded by the gene GNAQ gene (Its Entrez reference is 2776 and its Uniprot reference is P50148); Gn alpha subunits (also known as Gal 1) which is encoded by the gene GNA11 gene (Its Entrez reference is 2767 and its Uniprot reference is P29992); G14 alpha subunits (also known as Gal4) which is encoded by the gene GNA14 gene (Its Entrez reference is 9630 and its Uniprot reference is P095837); G15 alpha subunits (also known as Gal 5) which is encoded by the gene GNA15 gene (Its Entrez reference is 2769 and its Uniprot reference is P30679); Gii alpha subunits (also known as Gail) which is encoded by the gene GN All gene (Its Entrez reference is 2770 and its Uniprot reference is P63096); Gi2 alpha subunits (also known as Gai2) which is encoded by the gene GNAI2 gene (Its Entrez reference is 2771 and its Uniprot reference is P04899); Gi3 alpha subunits (also known as Gai 3) which is encoded by the gene GN Al 3 gene (Its Entrez reference is 2773 and its Uniprot reference is P08754); Goialpha subunits (also known as Gaol) which is encoded by the gene GNAO1 gene (Its Entrez reference is 2775 and its Uniprot reference is P09471); Gz alpha subunits (also known as Gaz) which is encoded by the gene GN AZ gene (Its Entrez reference is 2781 and its Uniprot reference is P19086); Gti alpha subunits (also known as Gatl or Transducin) which is encoded by the gene GNAT1 gene (Its Entrez reference is 2779 and its Uniprot reference is Pl 1488); Gt2 alpha subunits (also known as Gat2 or Transducin 2) which is encoded by the gene GNAT2 gene (Its Entrez reference is 2780 and its Uniprot reference is Pl 9087); and Gt3 alpha subunits (also known as Gat3 or Gustducin) which is encoded by the gene GNAT3 gene (Its Entrez reference is 346562 and its Uniprot reference is A8MTJ3).In particular embodiment, the hypomelanosis of Ito is caused by at least one mutation in GNAQ gene, GNA11 gene, GNAS gene or GNA13 gene.

[0019] In particular embodiment, the hypomelanosis of Ito is caused by at least one mutation in a gene coding a G alpha subunit, said mutation causes a substitution of an arginine residue (R) by another amino acid residue in the Gal3, Gas, Gaq and / or Gal 1 subunits.

[0020] In particular embodiment, the Gal 3 subunits comprises or consists of the amino acid sequence of SEQ ID NO: 1

[0021] SEQ ID NO : 1 >sp | Q14344 | Guanine nucleotide-binding protein subunit alpha- 13 ( Gal3 subunit ) MADFLPSRSVLSVCFPGCLLTSGEAEQQRKSKEIDKCLSREKTYVKRLVKILLLGAGESGKSTFLKQMR I IHGQDFDQRAREEFRPTIYSNVIKGMRVLVDAREKLHI PWGDNSNQQHGDKMMSFDTRAPMAAQGMVETRVFLQ YLPAIRALWADSGIQNAYDRRREFQLGESVKYFLDNLDKLGEPDYI PSQQDILLARRPTKGIHEYDFEIKNVPFK MVDVGGQRSERKRWFECFDSVTSILFLVSSSEFDQVLMEDRLTNRLTESLNI FETIVNNRVFSNVSI ILFLNKTD LLEEKVQIVSIKDYFLEFEGDPHCLRDVQKFLVECFRNKRRDQQQKPLYHHFTTAINTENIRLVFRDVKDTILHD NLKQLMLQ

[0022] In particular embodiment, the Gas subunits comprises or consists of the amino acid sequence of SEQ ID NO: 2

[0023] SEQ ID NO : 2 >sp | P63092 | Neuroendocrine secretory protein 55 ( Gas subunit ) MGCLGNSKTEDQRNEEKAQREANKKIEKQLQKDKQVYRATHRLLLLGAGESGKSTIVKQMRILHVNGFN GEGGEEDPQAARSNSDGEKATKVQDIKNNLKEAIETIVAAMSNLVPPVELANPENQFRVDYILSVMNVPDFDFPP EFYEHAKALWEDEGVRACYERSNEYQLIDCAQYFLDKIDVIKQADYVPSDQDLLRCRVLTSGI FETKFQVDKVNF HMFDVGGQRDERRKWIQCFNDVTAI I FWASSSYNMVIREDNQTNRLQEALNLFKSIWNNRWLRTI SVILFLNKQ DLLAEKVLAGKSKIEDYFPEFARYTTPEDATPEPGEDPRVTRAKYFIRDEFLRI STASGDGRHYCYPHFTCAVDT

[0024] ENIRRVFNDCRDI IQRMHLRQYELL In particular embodiment, the Gaq subunits comprises or consists of the amino acid sequence of SEQ ID NO: 3

[0025] SEQ ID NO : 3 >sp | P50148 | Guanine nucleotide-binding protein G ( q) subunit alpha ( Gaq subunit )

[0026] MTLESIMACCLSEEAKEARRINDEIERQLRRDKRDARRELKLLLLGTGESGKSTFIKQMRI IHGSGYSD EDKRGFTKLVYQNI FTAMQAMIRAMDTLKI PYKYEHNKAHAQLVREVDVEKVSAFENPYVDAIKSLWNDPGIQEC YDRRREYQLSDSTKYYLNDLDRVADPAYLPTQQDVLRVRVPTTGI IEYPFDLQSVI FRMVDVGGQRSERRKWIHC FENVTSIMFLVALSEYDQVLVESDNENRMEESKALFRTI ITYPWFQNSSVILFLNKKDLLEEKIMYSHLVDYFPE YDGPQRDAQAAREFILKMFVDLNPDSDKI IYSHFTCATDTENIRFVFAAVKDTILQLNLKEYNLV

[0027] In particular embodiment, the Gal l subunits comprises or consists of the amino acid sequence of SEQ ID NO: 4

[0028] SEQ ID NO : 4 >sp | P 29992 | Guanine nucleotide-binding protein G ( q) subunit alpha- 11 ( Gal l subunit )

[0029] MTLESMMACCLSDEVKESKRINAEIEKQLRRDKRDARRELKLLLLGTGESGKSTFIKQMRI IHGAGYSE EDKRGFTKLVYQNI FTAMQAMIRAMETLKILYKYEQNKANALLIREVDVEKVTTFEHQYVSAIKTLWEDPGIQEC YDRRREYQLSDSAKYYLTDVDRIATLGYLPTQQDVLRVRVPTTGI IEYPFDLENI I FRMVDVGGQRSERRKWIHC FENVTSIMFLVALSEYDQVLVESDNENRMEESKALFRTI ITYPWFQNSSVILFLNKKDLLEDKILYSHLVDYFPE FDGPQRDAQAAREFILKMFVDLNPDSDKI IYSHFTCATDTENIRFVFAAVKDTILQLNLKEYNLV

[0030] In particular embodiment, the hypomelanosis of Ito is caused by at least one mutation in the Gal3, Gas, Gaq and / or Gal 1 subunits.

[0031] In particular embodiment, the at least one mutation consists of a substitution of an arginine residue (R) by another amino acid residue, such as a lysine residue (K), a cysteine residue (C), a histidine residue (H), a glutamine residue (Q).

[0032] In particular embodiment, the hypomelanosis of Ito is caused by a substitution of an arginine residue (R) in a G alpha subunit selected from the group consisting of Gal 3 subunit, Gaq subunit, Gal 1 subunit or Gas subunit.

[0033] In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 in the Gal 3 subunit.

[0034] In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 by a lysine residue (K) in the Gal 3 subunit.

[0035] In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 in the SEQ ID NO: 1.

[0036] In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 by a lysine residue (K) in the SEQ ID: 1.

[0037] The inventors identify the mutation R200K in the Gal 3 subunit in four unrelated patients exhibiting pigmentary mosaicism. This hypomelanosis of Ito is characterized by a skin linear hypopigmentation along Blaschko lines, asymmetric facial dysmorphism, limb asymmetry and malformation, wound healing issues, teeth and ocular anomalies, gastroenterological and nephrological abnormalities, and neurological defects due to malformative hydrocephalus

[0038] In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 in the Gaq and / or Gal l subunits. In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 in the SEQ ID NO:3 and / or SEQ ID NO:4.

[0039] In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 by a glutamine residue (Q) in the Gaq and / or Gal l subunits. In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 by a glutamine residue (Q) in the SEQ ID NO:3 and / or SEQ ID NO:4. In other words, in some embodiments, the hypomelanosis of Ito is associated with a Sturge-Weber syndrome.

[0040] As used herein, the term “Sturge-Weber syndrome” is a rare congenital neurological and skin disorder. It is often associated with port-wine stains of the face, glaucoma, seizures, intellectual disability, and ipsilateral leptomeningeal angioma (cerebral malformations and tumors). It is a mosaic disease arising from somatic activating mutations in GNAQ, which encodes the G protein subunit alpha q or from somatic activating mutations in GNA17, which encodes the G protein subunit alpha 11.

[0041] In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 in the Gas subunit. In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 by a cysteine residue (C) or a histidine residue (H) in the Gas subunit. In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 in SEQ ID NO:2. In particular embodiment, the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 201 by a cysteine residue (C) or a histidine residue (H) in SEQ ID NO:2. In other words, in some embodiments, the hypomelanosis of Ito is associated with a McCune- Albright syndrome. As used herein, the term “McCune-Albright syndrome” is a rare and complex genetic disorder, with estimated prevalence between 1 / 100,000 and 1 / 1,000,000, affecting the bone, skin and endocrine systems. It is a mosaic disease arising from somatic activating mutations in GNAS, which encodes the alpha-subunit of the Gs heterotrimeric G protein.

[0042] 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.]).

[0043] As used herein, the term “Rho-associated kinase”, also known as or “Rho-kinase” or “ROCK” or “ROK” has its general meaning in the art and refers to an effector of the small GTPases RhoA, RhoB and RhoC, and belongs to the serine-threonine protein kinase family. Rho kinases (ROCKs), which were the first downstream effectors of RhoA to be discovered were found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light chain phosphorylation. Rho-kinase has pleiotropic functions including the regulation of cellular contraction, motility, morphology, polarity, cell division, and gene expression. ROCK contains several domains including a kinase; a coiled-coil region which contains a RhoA- binding domain and a pleckstrin homology and cysteine-rich domain at the C-terminal. ROCK exists in two isoforms, ROCK1 and ROCK2 (T. Ishizaki et al, EMBO J., 1996, 15, 1885-1893). ROCK has been identified as an effector molecule of RhoA, a small GTP -binding protein (G protein) that plays a key role in multiple cellular signaling pathways.

[0044] As used herein, the term “ROCK inhibitor” refers to a natural or synthetic compound capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of ROCK including, for example, reduction or blocking the interaction between ROCK (i.e ROCK1 and / or ROCK2) and RhoA. In a particular embodiment the inhibitor is selective. The selective ROCK inhibiting compounds are not limited to a particular manner of selective ROCK inhibition. For example, in some embodiments, one or more of the selective ROCK inhibiting compounds selectively inhibit ROCK1 activity over ROCK2 activity. For example, in some embodiments, one or more of the selective ROCK inhibiting compounds selectively inhibit ROCK2 activity over ROCK1 activity. Moreover, in some embodiments, one or more of the selective ROCK inhibiting compounds selectively inhibit both ROCK1 activity and ROCK2 activity with similar capability.

[0045] By "biological activity" of ROCK is meant regulating actin organization by phosphorylation and activation of LIM kinase and / or myosin light chains (MLC) as well as regulating cell migration by promoting cellular contraction.

[0046] Tests for determining the capacity of a compound to be a ROCK inhibitor are well known to the person skilled in the art. In a preferred embodiment, the inhibitor specifically binds to ROCK (i.e. ROCK1 and / or ROCK2) in a sufficient manner to inhibit the biological activity of ROCK, i.e. the ROCK pathway. Binding to ROCK and inhibition of the biological activity of ROCK may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as ROCK inhibitor to bind to ROCK. The binding ability is reflected by the Kd measurement. The term "Kd", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd / Ka) and is expressed as a molar concentration (M). Kd values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an inhibitor that "specifically binds to ROCK is intended to refer to an inhibitor that binds to human R0CK1 and / or ROCK2 with a Kd of IpM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of ROCK. The functional assays may be envisaged such as evaluating the ability to inhibit a) F-actin polymerization and / or b) MLC phosphorylation and / or c) reverse the morphological effects (cell shape, motility) induced by Gal 3 R200K mutant as described in example (see Figure 4).

[0047] Accordingly, the ROCK inhibitor may be a compound selected from the group consisting of nucleic acid (e.g., a short interfering ribonucleic acid (siRNA)), antibodies, aptamers, polypeptides (including peptide or peptidometics) and small molecules.

[0048] In one embodiment, the ROCK inhibitor is a small organic molecule. As used herein, the term "small organic molecule" refers to a molecule of 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 2000 Da, and most preferably up to about 1000 Da.

[0049] Small organic ROCK inhibitors are well known in the art.

[0050] Example of ROCK inhibitors include but are not limited to; H-1152; AT-13148; 0- Elemene; Chroman 1; DJ4; GSK-576371; GSK429286A; LX-7101; RKI-1447; TCS-7001; isoquinoline derivatives such as Fasudil, Hydroxyfasudil, Belumosudil, Ripasudil, Verosudil, Netarsudil, Thiazovivin, Y-27632, Y-30141, Y-33075, Y-39983 and their derivatives.

[0051] Fasudil (hexahydro-l-(5-isoquinolylsulfonyl)-lH-l,4-di-azepime), also named as HA- 1077, is an isoquinoline sulfonamide derivative and a clinically available ROCK inhibitor codeveloped by Asahi Kasei of Japan and Department of Pharmacology of Nagoya University. Its Cas Number is 103745-39-7. A series of fasudil analogs were synthesized and their selectivity and inhibitory activity against ROCK were evaluated10'17.

[0052] Thiazovivin is a drug which acts as a potent and selective ROCK inhibitor. Its CAS Number is 1226056-71-8.

[0053] Rhosin is a cell-permeable compound that directly targets Rho GEF binding domain. Its CAS Number is 1173671-63-0.

[0054] Hydroxyfasudil (l-(l-Hydroxy-5-isoquinolinesulfonyl)homopiperazine hydrochloride hydrate) is an active metabolite of Fasudil in vivo, which has higher affinity to ROCK than Fasudil. Its Cas Number is 155558-32-0. Belumosudil, also known as SLx-2119 binds to and inhibits the serine / threonine kinase activity of ROCK2 and is indicated for the treatment of chronic graft-versus host disease (chronic GvHD). Its CAS Number is 911417-87-3.

[0055] Ripasudil, also known as K-l 15 is a derivative of fasudil which is used for the treatment of glaucoma and ocular hypertension. Its CAS Number is 223645-67-8.

[0056] Verosudil, also known as AR-12286 is another potent and selective ROCK Inhibitor investigated for the treatment of glaucoma. Its CAS Number is 1414854-42-4.

[0057] Netarsudil, also known as AR-13324 is another potent and selective ROCK Inhibitor clinically available for the treatment of glaucoma. Its CAS Number is 254032-66-0.

[0058] H-l 152 (4-methyl-5-[[(2S)-2-methyl-l,4-diazepan-l-yl]sulfonyl]isoquinoline), is another isoquinoline derivative which has been optimized on the basis of fasudil. Its CAS Number is 451462-58-1.

[0059] Y-27632 ((+)-(R)-trans-4-(l -Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide) is another type of ROCK inhibitor which inhibits both ROCK1 and ROCK2 through competitively binding to the ATP binding pocket. Its CAS Number is 146986-50-7.

[0060] Optimization of the compound Y-27632 leads to a more potent ROCK inhibitor, Y- 39983 ((R)-(+)-trans-4-(l-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride) , which is benefit for the treatment of the glaucoma (Kubo T, Yamaguchi A, Iwata N, The therapeutic effects of Rho-ROCK inhibitors on CNS disorders. Ther Clin Risk Manag 2008;4(3):605-15). Its Cas Number is 129830-38-2

[0061] AT13148 is a novel oral ROCK inhibitor which has been described in Rath N et al.18

[0062] Chroman l is a highly potent and selective ROCK inhibitor which is more potent against ROCK2 than ROCK1. Its Cas number is 1273579-40-0.

[0063] DJ4, also known as EX-A7863 ((2E,5Z)-5-((3a,7a-dihydro-lH-pyrrolo[2,3-b]pyridin- 3-yl)methylene)-2-(phenethylimino)thiazolidin-4-one), is a selective multi-specific ATP competitive inhibitor of activity of ROCK 1, ROCK2, MRCKa and MRCKP kinases.

[0064] GSK-576371 is a selective ROCK inhibitor which has been described in Phrommintikul A. et al19. GSK429286A (N-(6-fluoro-lH-indazol-5-yl)-6-methyl-2-oxo-4-[4-

[0065] (trifluoromethyl)phenyl]-3,4-dihydro-lH-pyridine-5-carboxamide)) is a selective ROCK inhibitor.

[0066] In particular embodiment, the ROCK inhibitor is Fasudil or Y-27632.

[0067] Other examples of ROCK inhibitors include those described in the international patent publications WO98 / 06433, WO00 / 09162, WO00 / 78351, WOOl / 17562, WO02 / 076976, EP1256574, W002 / 100833, W003 / 082808, W02004 / 009555, W02004 / 024717, W02004 / 108724, W02005 / 003101, W020Q5 / 035501, W02005 / 035503, W02005 / 035506, W02005 / 058891 , W02005 / 074642, W02005 / 074643, W02005 / Q80934, W02005 / 082367, W02005 / 082890, W02005 / 097790, W02005 / 100342, W02005 / 103050, W02005 / 105780, W02005 / 108397, W02006 / 044753, W02006 / 051311, W02006 / 057270, W02006 / 058120 , W02006 / 072792, WO201 1107608 Al, W02007026920A2, W02018108156,

[0068] WO / 2020211751, WO / 2021093795, W02022012409, and WO2023 / 134688.

[0069] In one embodiment, the ROCK inhibitor of the invention is a peptide or a peptidometic.

[0070] The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

[0071] In one embodiment, the ROCK inhibitor of the invention is an aptamer.

[0072] “Aptamers” are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consist of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two-hybrid methods (Colas et al., 1996). Then, after raising aptamers directed against ROCK as described above, the skilled man in the art can easily select those inhibiting ROCK.

[0073] In another embodiment, the ROCK inhibitor of the invention is an antibody (the term including “antibody portion”).

[0074] In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

[0075] As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

[0076] Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of ROCK. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

[0077] Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in ROCK. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hybridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

[0078] Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation. Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

[0079] It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitope specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and / or Fc / pFc' regions to produce a functional antibody. This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90 / 07861 also propose four possible criteria which may be used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization. In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and / or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., I. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans. In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference. Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies. The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.

[0080] In another embodiment, the antibody according to the invention 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. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH. The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in vitro maturation. VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

[0081] In another embodiment, the ROCK inhibitor of the invention is an antisense oligonucleotide, a small inhibitory RNAs (siRNAs) or short hairpin RNAs (shRNAs).

[0082] As used herein, “Anti-sense oligonucleotides”, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of mRNA of interest (here ROCK or RHOA mRNA) by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of proteins of interest (i.e. ROCK1, ROCK2 or RHOA), 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 encoding the protein of interest can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating 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).

[0083] As used herein, the term “Ras homolog family member A”, also known as “Transforming protein RhoA” or “Aplysia Ras-Related Homolog 12” or “RHOA” or “ARHA” has its general meaning in the art and refers to a small GTPase protein in the Rho family of GTPases that in humans is encoded by the RHOA gene. Rho proteins promote reorganization of the actin cytoskeleton and regulate cell shape, attachment, and motility. Overexpression of this gene is associated with tumour cell proliferation and metastasis.

[0084] As used herein, the term “RHOA inhibitor” refers to a natural or synthetic compound capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of RHOA including, for example, reduction or blocking the interaction between RhoA and its effectors. In some embodiments, the RhoA inhibitor is a direct inhibitor, preferably, a RhoA antagonist, such as rhosin. In other embodiments, the RhoA inhibitor is an indirect inhibitor.

[0085] By "biological activity" of RHOA is meant regulating actin organization by phosphorylation and activation of LIM kinase and / or myosin light chains (MLC) as well as regulating cell migration by promoting cellular contraction.

[0086] Tests for determining the capacity of a compound to be a RHOA inhibitor are well known to the person skilled in the art. In a preferred embodiment, the inhibitor specifically binds to RHOA in a sufficient manner to inhibit the biological activity of RHOA, i.e. the RHOA / ROCJ pathway. Binding to RHOA and inhibition of the biological activity of RHOA may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as RHOA inhibitor to bind to RHOA. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of RHOA. The functional assays may be envisaged such as evaluating the ability to inhibit a) F-actin polymerization and / or b) MLC phosphorylation and / or c) reverse the morphological effects (cell shape, motility) induced by Gal 3 R200K mutant as described in example (see Figure 4 or 5). Accordingly, the RHOA inhibitor may be a compound selected from the group consisting of nucleic acid (e.g., antisense oligonucleotide, siRNA or shRNA), antibodies, aptamers, polypeptides (including peptide or peptidomimetic) and small molecules.

[0087] In one embodiment, the RHOA inhibitor is a small organic molecule.

[0088] Small organic RHOA inhibitors are well known in the art.

[0089] Example of RHOA inhibitors include but are not limited to Rhosin, G04, CT04, CCG- 1423 and ScafflO-8.

[0090] Rhosin ((2R)-2- Amino-3 -( 1 H-indol-3 -yl)-N'-(( 1 E)-quinoxalin-6- ylmethylidene)propanehydrazide), is a cell-permeable compound that directly targets Rho GEF binding domain and inhibits RhoA activity. Its Cas Number is 1173671-63-0.

[0091] G04 is an active metabolite of Rhosin (D-Tryptophan (2E)-2-(6- quinoxalinylmethylene)hydrazide hydrochloride). Its Cas Number is 1281870-42-5.

[0092] Scaffl0-8 bound to RhoA, inhibits the AKAP-Lbc-mediated RhoA activation. Its Cas Number is 777857-56-4.

[0093] CCG-1423, N-[2-[(4-Chlorophenyl)amino]-l-methyl-2-oxoethoxy]-3,5- bis(trifluoromethyl)-benzamide, is an inhibitor of RHOA. Its CAS number is 285986-88-1. Other examples of RHOA inhibitors include those described in the international patent publications WO / 2002 / 053143, WO / 2015 / 033565, WO / 2017 / 217796 and WO / 2022 / 102867.

[0094] Typically, the ROCK inhibitor and / or RHOA inhibitor of the present invention is 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 syringability exists. 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 gelatin. 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 vacuum-drying 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.

[0095] Thus, the present invention also relates to a pharmaceutical composition comprising a ROCK inhibitor and / or RHOA inhibitor according to the invention for use in the treatment of hypomelanosis of Ito.

[0096] Thus the present invention also relates to a pharmaceutical composition comprising a i) ROCK inhibitor and / or RHOA inhibitor according to the invention and ii) a pharmaceutically acceptable carrier for use in the treatment of hypomelanosis of Ito.

[0097] In particular embodiment, the hypomelanosis of Ito is associated with at least one mutation in a gene coding for a G alpha subunit.

[0098] In a particular embodiment, the hypomelanosis of Ito is associated with at least one mutation in GNAQ gene, GNA11 gene, GNAS gene or GNA13 gene.

[0099] In particular embodiment, the hypomelanosis of Ito is associated by at least one mutation in the Gal3, Gas, Gaq and / or Gal 1 subunits.

[0100] In a particular embodiment, the ROCK inhibitor is Y-27632 or Fasudil. 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.

[0101] FIGURE:

[0102] Figure 1. Effect of R200K mutation identified in patients on actin organization and cell morphology. Quantification of F-actin content (A.) and cell morphology parameters including perimeter (B.) circularity (C.) and solidity (D.) of Gal 3 WT-YFP, Gal 3 R200K-YFP and Gal 3 Q226L-YFP (an artificial mutant generated to mimic a constitutively active form of Gal3, used here as a positive control) overexpressed in B16-F0 cells. The data are represented as the mean + / - SEM. **: P < 0.01, ***: P < 0.001, ****: p < 0.0001.

[0103] Figure 2. Effect of R200K mutation identified in patients on cytoskeletal proteins. B16-F0 cells expressing Gal 3 WT-YFP, Gal 3 R200K-YFP and Gal 3 Q226L-YFP were fixed and labelled with anti-pMLC or anti-vinculin antibodies, and Hoechst for nuclei 48 h after being transfected. A. Quantification of the pMLC content. B. Quantification of the number of focal adhesions per cell + / - SEM. **: P < 0.01, ***: P < 0.001, ****: p < 0.0001.

[0104] Figure 3. Effect of R200K mutation identified in patients on RhoA signalling.

[0105] A. Quantification of RhoA GTP levels in Gal3 WT-YFP, Gal3 R200K-YFP and Gal3 Q226L-YFP overexpressing B16-F0 cells that correspond to the mean intensity of the signal generated from anti-GTP RhoA antibody labeling. Quantification of F-actin content using a (B.) phalloidin 647 staining and (C.) circularity in RhoA WT-GFP and RhoA Q63L - GFP overexpressing B16-F0 cells. Quantification of F-actin content using a (D.) phalloidin 647 staining and (E.) circularity in Gal 3 WT-YFP, Gal 3 R200K-YFP and Gal 3 Q226L-YFP overexpressing B16-F0 cells, upon RhoA inhibition treatment with CT04. Quantification of F- actin content using a (F.) phalloidin 647 staining and (G.) circularity in Gal3 WT-YFP, Gal3 R200K-YFP and Gal3 Q226L-YFP overexpressing B16-F0 cells, upon ROCK inhibition treatment with Y27632. The data are represented as the mean + / - SEM. ns: not significant. **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0106] Figure 4. Effect of Y27632, a ROCK inhibitor on pMLC content and cell morphology.

[0107] A. Quantification of pMLC content using anti-pMLC antibody staining in Gal3 WT- YFP, Gal3 R200K-YFP and Gal3 Q226L-YFP overexpressing B16-F0 cells, upon ROCK inhibition treatment with Y27632. Quantification of cell morphology parameters including (B.) perimeter and (C.) solidity. The data are represented as the mean + / - SEM. ns: not significant.

[0108] ****: P < 0.0001.

[0109] Figure 5: Effect of Fasudil, another ROCK inhibitor, on pMLC, actin content and cell morphology.

[0110] Quantification of (A.) F-actin content and (B.) pMLC content in Gal3 R200K-YFP overexpressing B16-F0 cells, upon ROCK inhibition treatment with Fasudil at 5, 10 or 20 pM. Quantification of cell morphology parameters including (C.) circularity (D.) perimeter and (E.) solidity. The data are represented as the mean + / - SEM. ns: not significant. *. P<0.05 **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0111] Figure 6. Effect of R200K mutation in patients on melanosome transfer to keratinocytes.

[0112] B16-F0 cells expressing Gal3 WT-YFP, Gal3 R200K-YFP or Gal3 Q226L-YFP were stimulated or not by MSH (Melanocyte Stimulating Hormone) for 48 hours. A. The number and length of cell extensions were quantified. B. The number of melanosomes transferred to HaCaT keratinocytes was quantified. The data are represented as the mean + / - SEM. ns: not significant. ****:p < 0.0001.

[0113] EXAMPLE:

[0114] Material and Methods:

[0115] Study participants

[0116] This example includes four unrelated affected children and their unaffected parents. Individuals were phenotyped and recruited by dermatologists and geneticists in Dijon and other French cities thanks to a nationwide collaborative effort to identify genes involved in skin mosaic syndromes.

[0117] Next generation sequencing

[0118] For all four patients, index case sequencing was performed as a skin / blood pair.

[0119] Constructs pcDNA3.1 and pLVX plasmids encoding for WT, R200K or Q226L Gal3 containing an internal YFP tag were previously described6. The pEGFP-C3-RHOA L63 construct was previously reported7.

[0120] Cell culture and transfection

[0121] Melanoma (B16-F0 or SK-mel28) and keratinocyte (HaCaT) cell lines were grown in DMEM (Thermo Fisher Scientific) with 10% FCS. Seventy percent confluent cultures were transfected according to the manufacturer’s protocol with the lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) and cultured for 48 h including an overnight cell starvation step. The cells were then fixed, labeled and examined under a fluorescence microscope.

[0122] RHOA and ROCK inhibition were performed in serum-free medium incubation for 4 hours and overnight using Rho Inhibitor I (CT04, Cytoskeleton Inc.) and Y27632 (Calbiochem) at final concentrations of 2 pg / ml and 5 pM, respectively.

[0123] For the annexin V staining, cells were harvested with Trypsin / EDTA three days after the transfection, washed with PBS, and resuspended in binding buffer containing the fluorochrome-conjugated Annexin V. After incubation for 15 min away from light, cell suspension was diluted with the binding buffer and centrifuged to pellet the cells. The cells were then resuspended with the binding buffer and analysed by FACS.

[0124] For co-culture experiments, HaCaT cells were seeded at 2.5 xl05 cells / well in six-well plates. The next day, B16-F0 cells were added to each well containing the keratinocytes at a keratinocytes to melanocytes seeding ratio of 2.5: 1. Following transfection, a serum-free medium containing 100 nM MSH was added and left for two days. Cultures were then fixed and labelled for immunofluorescence microscopy analysis.

[0125] Melanin quantification

[0126] Melanocyte Stimulating Hormone (MSH, Merck) was administered in serum-free medium for 48 hours following transfection at a final concentration of 100 nM. For the melanin content assay, cells were harvested with Trypsin / EDTA and the amount of melanin was quantified. A comparable number of cells were lysed with 100 pl 1 N NaOH, 10% DMSO, heated at 80 °C for lh30, and vortexed repeatedly to homogenize. Cell extracts were placed in 96-well plates in duplicate. The relative melanin content was determined by measuring absorbance at 490 nm with a Clariostar reader, and a wide range of pure synthetic melanin (Merck).

[0127] Immunocytochemistry

[0128] Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at room temperature (RT) and permeabilized with 0.1% Saponin (Fluka), 0.2% BSA (Sigma) for 15 min at RT. The latter buffer is used as a washing buffer all along the labeling. Cells were incubated with primary antibodies anti-pMLC (Cell Signaling Technology), anti- vinculin (Life Technologies) or anti-RHOA-GTP (CurieCoreTech) for Ih at RT. For co-culture experiment, the primary antibodies used were anti-TRPl and anti-cytokeratin (abeam). Cells were then washed and treated with appropriate secondary Alexa Fluor-conjugated antibodies anti-rabbit or anti-mouse (Invitrogen) for 30 min at RT, and washed again. To visualize the F- actin in parallel, Alexa Fluor 647-conjugated phalloidin was added along with the secondary antibody incubation. A hoechst staining in PBS was applied to the cells for 5 min at RT in darkness. Coverslips were finally washed with PBS, and treated with Fluorsave (Calbiochem) overnight to preserve the fluorescence. Image acquisitions were performed with a 100* oil immersion objective with a wide-field Nikon TE2000 microscope equipped with a CMOS camera (ORCA-flash4.0 LT, Hamamatsu). Images were acquired with the Metafluor software. Imaging analysis was performed using the Imaged software, where the fluorescence intensity was measured as a mean gray value, the perimeter and the circularity were calculated by the shape descriptors of the software.

[0129] Cell transduction and scratch wound assay

[0130] Lentivirus packaging was performed in HEK293T cells by co-transfection of the lentiviral plasmid encoding Gal3-pLVX with the packaging plasmids pVSVG, p8.9 and pREV. The media was changed the next day, and collected after 24 hours for centrifugation to collect the lentiviruses.

[0131] Sk-mel28 cells were seeded in flat bottom 96-well plates (Corning, Falcon, 80 000 cells / well) and incubated with the lentiviruses for 2 days until confluence.

[0132] The Essen Bioscience WoundMaker was used to create scratch-wounds of a standardized width (-600 pm) on the cell monolayer. Cells were tracked using the automated live-cell Essen IncuCyte Zoom live-cell microscopy system, taking an image every two hours. The Incucyte S3 software was used for image analysis and wound confluence determination.

[0133] Statistics:

[0134] ANOVA or Friedman tests were performed with GraphPad (Prism).

[0135] Results:

[0136] Four Hypomelanosis of Ito patients carry the same GNA13 variant

[0137] We evaluated four unrelated individuals with similar phenotypes as part of our mosaic skin disorders research program (MUSTARD cohort). The most common clinical features are skin linear hypopigmentation along Blaschko lines, asymmetric facial dysmorphism, limb asymmetry and malformation, all of which strongly suggest the presence of mosaicism (data not shown). These patients also have wound healing issues, teeth and ocular anomalies, gastroenterological and nephrological abnormalities, and neurological defects due to malformative hydrocephalus. However, no anomalies in growth or intelligence have been reported. We used genomic DNA derived from patients' affected skin and blood samples to perform whole-exome sequencing and / or targeted ultra-deep sequencing. We discovered the same de novo postzygotic mutation in the GNA13 gene (chrl7:g.63010910C>T, c.599G>A, p.(Arg200Lys)) that was supported by 21 to 36 % of reads in the hypo-pigmented skins of patients (data not shown). The mutation was not found in affected people's blood samples.

[0138] The mutated Arg200 is highly conserved

[0139] The Arg200 that is mutated into a Lys in the patients we study here, is found in the switch I region (data not shown), which is a flexible region that can bind to GTP or GDP, and change its conformation depending on the protein's activation state, allowing Gal3 to interact with various downstream signalling effectors.

[0140] In addition, the presence of an arginine at this position is completely conserved across species from Drosophila (data not shown) and among Ga subunits (data not shown).

[0141] Gal 3 R200K variant alters cellular morphology and increases actin polymerisation

[0142] To investigate the cellular impact of the p.(Arg200Lys) mutation found in patients, we expressed this variant in the B16-F0 melanoma cell line. The effects of the p.(Arg200Lys) mutation were systematically compared to those of a well-characterized artificial constitutively active Gal 3 mutant containing the p.(Glu226Leu) substitution.

[0143] We first wanted to determine if the mutation affected the cell's cytoskeletal organization. For this purpose, the network of filamentous actin (F-actin) was visualized and quantified in cells expressing YFP-tagged wild-type (WT) GNA13 or mutants. Cells expressing one of the mutants have higher F-actin content than WT expressing cells (Fig. 1A). Furthermore, actin labelling appears to be more localized in the cell periphery in most mutant cells. The shape of the cells was another intriguing observation made during the course of these experiments. Cells expressing the mutants exhibit a smaller perimeter, increased circularity and solidity (indicative of a more regular shape) when compared to WT cells (Fig. 1B-D). To ensure that these changes in shape induced by mutant Gal3 were not caused by dying cells, we performed an annexin labelling three days after the transfection. The findings confirmed that the two mutations have no effect on the percentage of dying cells (data not shown).

[0144] As a result, we can conclude that the p.(Arg200Lys) mutation causes an increase in actin polymerization and a rounder cell shape, independently of any cell surface receptor engagement. These findings are consistent with this particular variant being constitutively active, indicating that the mutation found in the patients is a gain-of-function mutation. Gal 3 R200K variant has a broad effect on the cytoskeleton

[0145] First, we investigated the status of two other major cytoskeletal proteins: myosin and vinculin. Non-muscle myosins are proteins that interact with actin to control cell morphology. To determine whether the mutation affects myosin activity, we performed immunocytochemistry labelling of the phosphorylation of the myosin light chains (pMLC), which represent their active form. The results show that both mutants' expression induces an increase in the pMLC cell content (Fig. 2A). This suggests that myosin / actin interaction is increased by the pathogenic R200K variant. This is expected to favour cell contractility, which may be responsible for the induction of the round shape of the cells.

[0146] Next, we focused our analysis on vinculin which is a component of focal adhesions that links the cytoskeleton to extracellular matrix proteins. Vinculin staining and quantification of the vinculin dots revealed that cells expressing the Gal 3 R200K mutant have fewer focal adhesions and are more likely to be localized at the cell periphery than cells expressing the WT form (Fig. 2B).

[0147] These findings indicate that the p.(Arg200Lys) mutation affects cell cytoskeletal organization and may disrupt cell adhesion.

[0148] Gal 3 R200K hyper activates the RHOA / ROCK signalling pathway

[0149] We tested the activation state of RHOA in cells transfected with the Gal 3 mutants by quantifying the levels of active GTP -bound RHOA. The results show that at the basal level, independently from any cell surface receptor engagement, both R200K and Q226L Gal 3 mutants hyperactivate RHOA (Fig. 3A). In parallel, we show that the cells expressing the constitutively active form of RHOA (RHOAL63) exhibit similar features than those expressing the R200K mutation, regarding the increase in actin polymerisation (Fig. 3B) and the round morphology (Fig. 3C).

[0150] ROCK1 and ROCK2 are RHOA effectors that have been shown to phosphorylate MLC. To determine whether the cytoskeletal and morphological consequences elicited by the Gal 3 R200K mutation occur via a hyperactivation of the RHOA / ROCK pathway, we pharmacologically inhibited either RHOA with CT04 (Fig. 3D-E) or ROCK with Y27632 (Fig. 3F, 3G) in cells expressing WT or mutant Gal3. The inhibition of both RHOA and ROCK had no effect on the F-actin content of cells expressing WT Gal3, but it largely blocked the increased F-actin polymerization caused by the Gal3 mutants (Fig. 3D, 3F). Furthermore, it also reduced the enhanced circularity caused by the mutations and partially restored their original shapes (Fig. 3E, 3G). Blocking ROCK also resulted in the loss of the mutant-induced enhanced pMLC (Fig. 4A), confirming that MLC was phosphorylated by ROCK. Interestingly, ROCK inhibition also reversed the morphological effects induced by the mutants as measured by the perimeter and solidity parameters (Fig. 4B, C). These results were confirmed by using Fasudil, a FDA-approved ROCK inhibitor (Fig. 5A-5E).

[0151] Thus, these findings indicate that the functional defects reported above occur via a hyperactivation of the RHOA / ROCK signalling pathway.

[0152] Gal 3 R200K inhibits cell migration

[0153] Next, we aimed to determine if the mutations had any effect on cell migration. We chose the SK-mel28 melanoma cell line because, unlike B16-F0, it can be transduced with about 90% efficiency with Gal 3. We first confirmed that the morphology changes and increased actin polymerization caused by the two R200K and Q226L mutations also occur in SK-mel28 cells (data not shown). We then used a scratch wound assay to track the ability of Gal 3 -expressing cells to migrate and close the wound within two days of the scratch. The results show that noninfected control cells and cells expressing WT Gal 3 have regular and comparable migratory abilities (data not shown). However, the migration of cells expressing either R200K or Q226L mutants was significantly hampered.

[0154] Altogether, these results indicate that the morphological alterations elicited by the pathogenic R200K Gal 3 variant are responsible for a defect in cell migration.

[0155] Gal 3 R200K inhibits melanosomes transfer to keratinocytes

[0156] Finally, to get as close as possible to the original skin syndromes exhibited by the patients carrying the R200K Gal 3 variant, we have studied the impact of this particular mutation on melanin expression and transfer.

[0157] The skin pigmentation process occurs through two important steps: (i) the production of melanin by the melanocytes in vesicles called melanosomes which undergo maturation steps, and (ii) the transfer of melanosomes from melanocytes to keratinocytes. Because the latter step is dependent on the cytoskeleton and the morphology of the melanocytes, we hypothesized that the R200K-induced changes in melanocytes shape could thus disrupt the transfer of melanosomes to keratinocytes. Melanin Stimulating Hormone (MSH) is a hormone that induces the maturation of melanosomes and their transfer to keratinocytes through some morphological changes in the melanocytes. We show that MSH stimulation of B16-F0 cells expressing WT Gal 3 allows them to acquire long and numerous cytoplasmic extensions (Fig. 6A). However, the formation of these extensions is very much perturbed in cells expressing mutant Gal3, as shown by the decrease in both numbers and lengths of these acquired extensions in cells expressing the Gal 3 mutants (Fig. 6A). Interestingly, these Gal 3 mutations do not affect the amount of melanin pigmentation of the melanocytes (data not shown). In order to test whether these extensions affect the transfer of melanosomes to the keratinocytes, the B16-F0 melanocytes expressing Gal 3 WT or mutants were co-cultured with HaCat keratinocytes, and stimulated with MSH. The results show that both Gal 3 mutations negatively affect the number of transferred melanosomes in keratinocytes, up to the level where these mutants exert a complete repression of the effect of MSH on melanosomes transfer (Fig. 6B).

[0158] These important results therefore favour an inhibition of melanosomes transfer as the explanation for the pathogenicity of the R200K Gal 3 variant.

[0159] Conclusion

[0160] Here, we have discovered a novel postzygotic mutation in the GNA13 gene encoding Gal 3 in patients with rare skin and developmental disorders. These findings highlight the importance of Gal3 in human development and pave the way for further research into the etiology of Hypomelanosis of Ito.

[0161] To investigate the impact of this mutation, we chose a melanocyte cell line model to express the mutation. Indeed, direct study of patient skin samples is limited by ethical and medical considerations - such as the wound healing issues observed at the skin biopsy site in the patients reported here - to justify the less invasive explorations. In addition, both the potential loss of the mutation in primary cell cultures derived from the biopsy in affected skin and the observed absence of Gal 3 variant in blood, as we already observed in the RHOA mutated patients2, confirms the importance of Gal3 / RHOA in hematopoietic stem cell and lymphocyte development, suggesting negative selection of mutant blood cells. As a result, as with other mosaic conditions, diagnosis of mutation testing in Gal3-related mosaic disorders should be performed on a biopsy from affected skin.

[0162] From a mechanistic point of view, we demonstrate that the R200K Gal 3 variant found in patients is an activating mutation that alters the cytoskeleton and the morphology of the cells via a hyperactivation of the RHOA / ROCK signalling pathway. These shape alterations inhibit cell adhesion and migration, and the transfer of melanosomes towards keratinocytes but not the amount of melanin produced per se. These observations likely explain the hypo-pigmented areas of the patients' skin and allow a shift in the paradigm regarding the cause of Hypomelanosis of Ito: more than melanin production, defects in intercellular contacts and communication can lead to pigmentation defects. Moreover, based on our results showing an effect of the mutation on cell migration, we cannot exclude that melanocytes are unable to migrate correctly from the neural crest to the epiderm during development in the patients.

[0163] Other genes encode other isoforms of the Ga protein subunit and are also involved in mosaic disorders in humans, for example GNAQ and GNA11 in Sturge-Weber syndrome8, or GNAS in McCune-Albright syndrome9. Interestingly, it is the same homologous arginine mutation conserved among these Ga isoforms that is found in the post-zygotic state in these patients, although no mechanistic explanation has been provided so far. Based on the results we present here, we propose that the signalling pathways downstream of these Ga could, by crosstalks between RHO GTPases, affect also the activation of RHOA.

[0164] Finally, we previously reported linear hypopigmentation in six A / ZGA-r elated hemimegalencephaly patients and five RHOA -related mosaic ectodermal dysplasia patients2,3. Interestingly, the RHOA mutations are located in the switch regions of RHOA and appear to be inactivating, resulting in a decrease in MLC phosphorylation. Although these observations could seem contradictory with the results reported here, they could rather highlight the significance of the cycle between active and inactive forms of RHOA depending on cell needs, and that deregulation of this balance towards either form, even if affecting the downstream signalling pathways in opposite ways, can result in similar phenotypes. Regarding mTOR, it can be activated by the PI3K / AKT pathway. Interestingly, Arg200 is close to the Thr203, both in switch I region, which is a phosphorylation site for AKT, affecting the RHOA signalling pathway. Thus, these observations could imply that all the Gal3, RHOA and mTOR proteins implicated in mosaic development diseases may be linked in a single disrupted pathway that is still undefined.

[0165] To summarize, the discovery of GNA13 as a new gene at the root of dermal mosaic syndromes, could reveal common pathogenic pathways, improving our understanding of their causes and, ultimately, opening up new therapeutic avenues such as the use of RHOA or ROCK inhibition that restored some of the cell alterations caused by the mutation, specifically the cell morphology and cytoskeleton.

[0166] REFERENCES:

[0167] 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. Sybert VP. Hypomelanosis of Ito: a description, not a diagnosis. J Invest Dermatol. 1994 Nov;103(5 Suppl): 141S-143S. Vabres P, Soriin A, Kholmanskikh SS, Demeer B, St-Onge J, Duffourd Y, et al. Postzygotic inactivating mutations of RHOA cause a mosaic neuroectodermal syndrome. Nat Genet. 2019 Oct;51(10): 1438-41. Carmignac V, Mignot C, Blanchard E, Kuentz P, Aubriot-Lorton M-H, Parker VER, et al. Clinical spectrum of MT OR-r elated hypomelanosis of Ito with neurodevelopmental abnormalities. Genet Med. 2021 Aug;23 (8): 1484-91. Oldham WM, Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol. 2008 Jan;9(l):60-71. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615-49. Maziarz M, Park J-C, Leyme A, Marivin A, Garcia-Lopez A, Patel PP, et al. Revealing the Activity of Trimeric G-proteins in Live Cells with a Versatile Biosensor Design. Cell. 2020 Aug 6;182(3):770-785.el6. Faure S, Salazar-Fontana LI, Semichon M, Tybulewicz VLJ, Bismuth G, Trautmann A, et al. ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation. Nat Immunol. 2004 Mar;5(3):272-9. Jordan M, Carmignac V, Soriin A, Kuentz P, Albuisson J, Borradori L, et al. Reverse Phenotyping in Patients with Skin Capillary Malformations and Mosaic GNAQ or GNA1 1 Mutations Defines a Clinical Spectrum with Genotype-Phenotype Correlation. J Invest Dermatol. 2020 May; 140(5): 1106-1110.e2. Roszko KL, Guthrie L, Li X, Collins MT, de Castro LF, Boyce AM. Identification of GNAS Variants in Circulating Cell-Free DNA from Patients with Fibrous Dysplasia / McCune Albright Syndrome. J Bone Miner Res. 2023 Mar;38(3):443-50. Satoh N, Toyohira Y, Itoh H, Stimulation of norepinephrine transporter function by fasudil, a Rho kinase inhibitor, in cultured bovine adrenal medullary cells. Naunyn Schmiedebergs Arch Pharmacol 2012;385(9):921-31. Nakabayashi S, Nagaoka T, Tani T, Retinal arteriolar responses to acute severe elevation in systemic blood pressure in cats: role of endothelium-derived factors. Exp Eye Res 2012;103:63-70. Sun X, Minohara M, Kikuchi H, The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune encephalomyelitis. J Neuroimmunol 2006; 180(1 -2): 126-34. 13. Yu JZ, Ding J, Ma CG, Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil, a Rho kinase inhibitor. J Neurosci Res 2010;88(8): 1664- 72; Hou SW, Liu CY, Li YH, Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through anti-inflammatory effect. CNS Neurosci Ther 2012;18(l l):909-17.

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Claims

CLAIMS:

1. A method for treating hypomelanosis of Ito in a subject in need thereof comprising administering a therapeutically effective amount of a ROCK inhibitor and / or RHOA inhibitor.

2. The method of claim 1, wherein the hypomelanosis of Ito is caused by a mutation in a gene coding for a G alpha subunit.

3. The method of claim 2, wherein the hypomelanosis of Ito is caused by at least one mutation in GNAQ gene, GNA11 gene, GNAS gene or GNA13 gene.

4. The method of claim 3, wherein the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 in the Gal 3 subunit.

5. The method of claim 3, wherein the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 200 by a lysine residue (K) in the G l 3 subunit.

6. The method of claim 3, wherein the hypomelanosis of Ito is caused by the substitution of the arginine residue (R) at position 183 in the Gaq and / or Gal 1 subunits.

7. The method of claim 3, wherein the hypomelanosis of Ito is caused with the substitution of the arginine residue (R) at position 201 in the Gas subunit.

8. The method of any one of claim 1 to 7, wherein the ROCK inhibitor is selected from the group consisting of antibodies, aptamers, polypeptides and small molecules.

9. The method of any one of claim 8, wherein the ROCK inhibitor is a small molecule selected from the group consisting ofH-1152, AT-13148, P-Elemene; Chroman 1, DJ4, GSK-576371; GSK429286A; LX-7101; RKL1447; TCS-7001; Fasudil,Hydroxyfasudil, Belumosudil, Ripasudil, Verosudil, and Netarsudil, Thiazovivin, Y- 27632, Y-30141, Y-33075, Y-39983 and their derivatives.

10. The method of claim 9, wherein the ROCK inhibitor is Y-27632 or Fasudil.

11. A pharmaceutical composition comprising a ROCK inhibitor for use in the treatment of hypomelanosis of Ito.

12. The pharmaceutical composition for use according to claim 11, wherein the hypomelanosis of Ito is caused by at least one mutation in GNAQ gene, GNA11 gene, GNAS gene or GNA13 gene.

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