Compositions containing MIR-335

Abstract

We describe a method for diagnosing a skin condition such as atopic dermatitis (AD) in an individual. The method comprises detecting the activity or expression level of miR-335 in a sample from the individual or in a sample from the individual. We also disclose the use of a histone deacetylase (HDAC) inhibitor, such as belinostat (PubChem CID: 6918638), in the preparation of a medicament for restoring barrier function or treating a skin condition such as atopic dermatitis.

Description

Technical Field

[0001] The present invention relates to the fields of medicine, cell biology, molecular biology and genetics. The present invention also relates to the field of medicine. Background Art

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease[1,2] that affects 15% to 30% of children and 2% to 10% of adults. AD, commonly referred to as "eczema," is a highly prevalent, chronic, relapsing inflammatory skin condition or disease that significantly impacts patients' quality of life.

[0003] Symptoms include recurrent episodes and remissions of skin inflammation, pruritus, scaling, and susceptibility to infection, which significantly reduce the patient's quality of life. The main characteristics of lesional skin in AD patients include skin barrier defects, impaired cornified envelope formation, and abnormal keratinocyte differentiation [3,4]. These skin lesions can occur anywhere on the body, and their morphology and distribution depend on age and disease severity [5]. In infants, AD lesions are commonly found on the face, scalp, and extensor surfaces. Children aged 2 to 12 years often have polymorphic manifestations, with different types of skin lesions on the flexural areas, neck, dorsum of the feet, and hands [6]. In adolescents and adults, lesions are often lichenified and present on the flexural surfaces of the extremities [7].

[0004] Although skin lesions represent the most obvious symptom of atopic dermatitis, many symptoms lie beneath the surface. AD is a complex systemic disease that is often the first indicator of “atopic progression” (the progression of affected individuals from atopic dermatitis to asthma and allergic rhinitis) [8,9]. In this model, it is hypothesized that epidermal barrier defects in AD lead to overexposure to environmental aeroallergens, and that this allergen sensitization triggers atopic progression

[10] . The critical role of epidermal barrier dysfunction in the genesis of AD is a relatively recent discovery. AD was initially thought to be caused by immune dysregulation, with skin barrier defects resulting from local inflammation

[11] . A strong genetic association is now known between AD and loss-of-function mutations in filaggrin (FLG)

[12] . FLG is a protein involved in corneal envelope formation and barrier function. Skin with AD lesions also shows reduced expression of other barrier proteins, such as loricrin and involucrin

[13] . The evidence that skin barrier defects predispose to AD provides an interesting opportunity for new therapeutic approaches to AD and atopy, as existing treatments are strictly symptomatic. Summary of the Invention

[0005] The development and maintenance of a healthy skin barrier depends on many factors, including translational control of microRNAs.

[0006] MicroRNAs (miRNAs) are small, noncoding RNAs that mediate post-transcriptional gene regulation by targeting mRNAs for degradation and / or inhibiting translation. The critical role of miRNAs in mammalian skin development has been revealed by the defective skin phenotypes exhibited by mice with skin-specific knockout of Dicer and Dgcr8, two key components of the miRNA biogenesis pathway. Epidermal-specific deletion of Dicer and Dgcr8 results in similar but abnormal skin phenotypes, such as reduced barrier function, defective hair follicle (HF) morphogenesis, and keratinocyte hyperproliferation [14-16].

[0007] These findings provide strong evidence for the involvement of miRNAs in skin differentiation but fail to identify key miRNAs required for effective skin barrier function.

[0008] According to a first aspect of the present invention, we provide the use of miR-335 for use in a method of diagnosing, treating, preventing or alleviating a dermatological condition such as atopic dermatitis.

[0009] miR-335 may comprise a polynucleotide sequence having miRBase Accession No. MI0000816. It may comprise a variant, homolog, derivative, or fragment of the polynucleotide sequence. Such a variant, homolog, derivative, or fragment of the polynucleotide sequence may comprise a sequence having 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence having miRBase Accession No. MI0000816. Such a variant, homolog, derivative, or fragment of the polynucleotide sequence may comprise miR-335 activity.

[0010] According to a second aspect of the present invention, there is provided an agent capable of upregulating the expression or activity of miR-335 for use in a method of treating, preventing or alleviating a skin condition such as atopic dermatitis. Such an agent may comprise a miR-335 agonist.

[0011] The agent may comprise a histone deacetylase (HDAC) inhibitor.

[0012] The agent may comprise belinostat (PubChem CID: 6918638).

[0013] According to a third aspect of the present invention, we provide a pharmaceutical composition comprising miR-335 as described above or an agent as described above and a pharmaceutically acceptable excipient, carrier or diluent.

[0014] The pharmaceutical composition may be formulated for topical administration.

[0015] As a fourth aspect of the present invention, provided is the use of a histone deacetylase (HDAC) inhibitor, such as belinostat (PubChem CID: 6918638), in the preparation of a medicament for restoring barrier function.

[0016] According to a fifth aspect of the present invention, we provide the use of a histone deacetylase (HDAC) inhibitor, such as belinostat (PubChem CID: 6918638), in the preparation of a medicament for treating, preventing or ameliorating a skin condition such as atopic dermatitis.

[0017] In a sixth aspect, the present invention provides a method for upregulating miR-335 expression in a cell. The method may comprise exposing the cell to a histone deacetylase (HDAC) inhibitor such as belinostat (PubChem CID: 6918638).

[0018] In a seventh aspect of the present invention, a method for treating, preventing or alleviating a skin condition such as atopic dermatitis in an individual is provided. The method may comprise upregulating the activity or expression level of miR-335 in the individual.

[0019] According to an eighth aspect of the present invention, we provide a method for diagnosing a skin condition such as atopic dermatitis (AD) in an individual. The method may comprise detecting the activity or expression level of miR-335 in a sample from the individual or detecting the activity or expression level of miR-335 in a sample from the individual.

[0020] A decrease in the activity or expression level of miR-335 compared to the activity or expression level of miR-335 in an individual known not to have a skin condition such as atopic dermatitis can indicate that the individual has or may have a skin condition such as atopic dermatitis.

[0021] miR-335 expression levels of 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less compared to the expression levels of miR-335 in individuals known not to have a skin condition such as atopic dermatitis or compared to the expression levels of miR-335 in individuals known not to have a skin condition such as atopic dermatitis can be indicative of a skin condition such as atopic dermatitis.

[0022] The sample may comprise a skin sample.

[0023] According to a ninth aspect of the present invention, there is provided a method of treating a skin condition such as atopic dermatitis in an individual. The method may comprise performing a method as described above. Upon determining that the individual has or may have a skin condition such as atopic dermatitis, the method may comprise administering to the individual a treatment for the skin condition such as atopic dermatitis.

[0024] According to a tenth aspect of the present invention, a method for treating a skin condition such as atopic dermatitis in an individual is provided. The method may comprise obtaining, from a sample of the individual or a sample derived from the individual, analysis of the expression level of miR-335 or a variant, homolog, derivative, or fragment thereof, such as a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to miR-335.

[0025] If the expression level of miR-335 is lower than a reference expression level, the method may further include treating the individual for a skin condition such as atopic dermatitis, wherein the reference expression level is the expression level of miR-335 in a sample from an individual known not to have a skin condition such as atopic dermatitis or the expression level of miR-335 in a sample from an individual known not to have a skin condition such as atopic dermatitis.

[0026] As an eleventh aspect of the present invention, we provide a kit for detecting a skin condition such as atopic dermatitis in an individual or detecting the individual's susceptibility to a skin condition such as atopic dermatitis. The kit may include means for detecting the activity or expression level of miR-335 in the individual or in a sample taken from him or her. The means may include a miR-335 polynucleotide or a fragment thereof or a nucleotide complementary to a miR-335 polynucleotide or a fragment thereof.

[0027] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of those skilled in the art and are explained in the literature. See, for example, J. Sambrook, EF Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F Met al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; JMPolak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; MJ Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; DMJ Lilley andJ.E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO.I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3. Each of these general texts is incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A to Figure 1 G is a graph showing miRNA screening in AD targeting miR-335 as an epithelial differentiation factor.

[0029] Figure 1 A is a graph showing scatter plots representing relative miR-335 levels in healthy unaffected skin samples (control, n=7) and lesional skin from AD patients (AD-LS, n=10).

[0030] Figure 1B is a diagram showing the expression of miR-335 detected in healthy unaffected skin and AD lesion skin by in situ hybridization using a specific probe for mature miR-335 or a control probe. The inset of the middle figure of healthy skin shows an enlarged view of the epidermis, showing the supra-basal specific expression of miR-335. The middle figure of unaffected skin shows the supra-basal expression of miR-335 with a higher magnification inset. The basal layer is divided by a dotted line. The basal layer is divided by a dotted line. MiR-335 is significantly downregulated in AD lesion skin (right figure). Scale bar, 100 μm.

[0031] Figure 1 C is a graph showing the gene ontology analysis of differentially regulated genes in N / TERT-1 cells expressing miR-335 compared with control cells.

[0032] Figure 1 D is a graph showing a heat map of selected genes generated using microarrays of RNA from N / TERT-1 cells transfected with miR-335 mimics or control RNA. Expression values ​​are shown as red (high) or blue (low) shades relative to the individual mean of the gene in a linear scale.

[0033] Figure 1 E is a graph showing the relative transcript abundance of IVL, TGM1, and SPRR, all of which are involved in keratinocyte differentiation, in N / TERT-1 cells transfected with miR-335 mimic or control RNA, as shown in bar graphs.

[0034] Figure 1 F is a bar graph showing the frequency of cornified envelopes per field in N / TERT-1 cell cornified envelope analysis on N / TERT-1 cells transfected with miR-335 mimic or control RNA.

[0035] Figure 1 G is a representative phase contrast micrograph of a mature cornified envelope. P values ​​were calculated using Student's t-test, with error bars representing ± sem. *P < 0.05 **P < 0.01. NS - not significant.

[0036] Figure 2 A to Figure 2 D is a graph showing that SOX6 is a direct target of miR-335.

[0037] Figure 2A is a graph showing heat maps of selected genes generated using microarrays of RNA from N / TERT-1 cells transfected with miR-335 mimics or control RNA. Expression values ​​are shown as red (high) or blue (low) shades relative to the individual mean of the gene in a linear scale.

[0038] Figure 2 B shows the sequence of the miR-335 binding site in the 3'UTR of SOX6. To generate a mutant 3'UTR lacking the miR-335 binding site, the seed sequence was modified to the sequence shown in red.

[0039] Figure 2 C is a diagram showing the direct target of miR-335 verified by luciferase reporter gene analysis. Wild-type (WT) or mutant (Mut) 3'-UTR luciferase reporter gene constructs were used to co-transfect cells as indicated, or miR-335 or non-targeted scrambled control (scrambled control) were used to co-transfect cells. Standardized relative luciferase activity is shown as a bar graph. Luciferase activity is expressed as the mean value relative to the control (n=3). **p<0.001. P values ​​were calculated using Student's t test, and error bars represent ± sem.

[0040] Figure 2 D is a graph showing miR-335 expression (left figure) detected by in situ hybridization on a section of normal healthy (unaffected by AD) human skin (upper figure) and a section of AD lesion skin (lower figure) (n=5). Immunohistochemical analysis (right figure) of SOX6 from a section of normal healthy human skin (top) and a section of AD lesion skin (bottom). In unaffected skin, the expression of the target gene remains relatively low (n=5). However, in AD lesion skin (lower right) (n=5), it is clearly observed that the protein expression of the target gene Sox6 increases significantly. In the upper figure, the basal layer is divided by a dotted line. Scale bar = 100 μm.

[0041] Figure 3 A to Figure 3 I is a graph showing the transcriptomic landscape characteristics of miR-335 inducing epidermal differentiation by targeting SOX6.

[0042] Figure 3 A is a bar graph showing the relative transcript abundance of SOX6 in N / TERT-1 cells transfected with miR-335 mimic or control cells.

[0043] Figure 3B is a Western blot showing the relative levels of SOX6 in cells transfected with miR-335 mimics or control cells. β-actin levels in the same samples indicate equal loading.

[0044] Figure 3 C is a graph showing representative phase contrast images of N / TERT-1 cells expressing shRNA against SOX6 or a scrambled control. Scale bar = 400 μm.

[0045] Figure 3 D is a graph showing relative cell numbers in N / TERT-1 cells transduced with control shRNA and shSOX6 assessed by cell-titerglo analysis.

[0046] Figure 3 E is a graph showing gene ontology analysis of differentially regulated genes after SOX6 knockdown in N / TERT-1 keratinocytes.

[0047] Figure 3 F is a graph showing the relative transcript abundance of factors associated with keratinocyte differentiation, including IVL, TGM1, and SPRR, in N / TERT-1 keratinocytes transduced with control shRNA or shSOX6.

[0048] Figure 3 G is a graph showing a bar graph representing the number of cornified envelopes per field in N / TERT-1 keratinocytes transduced with shcontrol or shSOX6. A significant increase in the number of CEs was observed after SOX6 knockdown. Values ​​represent the mean of three independent experiments performed in quadruplicate and are expressed as mean ± SD. **p value < 0.01.

[0049] Figure 3 H is a graph showing chromatin immunoprecipitation analysis of N / TERT-1 keratinocytes using a SOX6-specific antibody or control IgG. SOX6 occupancy in the promoter regions of IVL, TGM1, and SPRR2F is shown as fold enrichment in response to IgG. No SOX6 enrichment was observed in the RPLP0 or KRT14 promoters.

[0050] Figure 3 Figure 1 shows a schematic diagram depicting SOX6-mediated transcriptional repression of IVL, TGM1, and SPRR2F via SOX6 binding motifs in their promoters under basal conditions. P values ​​were calculated using Student's t-test, and error bars represent ± sem. *P < 0.05 **P < 0.01. NS - not significant.

[0051] Figure 4A to Figure 4 D is a diagram showing that SOX6 interacts with the SMARCA chromatin remodeling complex and blocks epidermal cell differentiation:

[0052] Figure 4 A is a graph showing a list of selected potential SOX6 interacting partners (including SWI / SNF complex subunits) detected by immunoprecipitation coupled with mass spectrometry analysis.

[0053] Figure 4 B is a graph showing colocalization of SOX6 with SWI / SNF complex subunits. Immunocytochemistry of N / TERT-1 cells shows co-staining of SOX6 (green) with the indicated subunit complexes (red), namely SMARCA6, SMARCA4, and SMARCC1. Z-stack images were acquired, and representative images from a single plane are shown. The right panel depicts the relative fluorescence intensity along the white line. Pinin 1 (PNN1), a nuclear protein, did not show significant colocalization with SOX6.

[0054] Figure 4 C is a graph showing the Pearson colocalization coefficient calculated using Z-stack images from Olympus FloView software to quantify the degree of colocalization. The scatter plot shows the Pearson colocalization coefficient for each gene pair (SOX6 vs. SMARCA6 / SMARCA4 / SMARCC1 / PNN1) obtained from approximately 15 nuclei from two independent experiments.

[0055] Figure 4 D is a graph showing the relative transcript abundance of IVL, SPRR2F, and TGM1 mRNA levels in N / TERT-1 keratinocytes transiently transfected with control siRNA or siRNA targeting SMARCC1. P values ​​were calculated using the Student's t-test, and error bars represent ± sem. *P < 0.05 **P < 0.01.

[0056] Figure 5 A to Figure 5 F is a graph showing epigenetic regulation of miR-335 by histone deacetylase 2 (HDAC2).

[0057] Figure 5 A is a diagram showing the chromosomal location and genomic sequence of miR-335. MiR-335 is located in the second intron of the mesoderm-specific transcript (MEST) of chromosome 7q32.2 locus. The stem-loop sequence below shows the precursor sequence of miR-335, and sequence (green) represents mature miR-335 sequence.

[0058] Figure 5 B is a graph showing a bar graph representing the relative expression of miR-335 in N / TERT-1 cells treated with sodium butyrate (NaB) analyzed by qRT-PCR. Ct values ​​were normalized to the U6 probe.

[0059] Figure 5 C is a graph showing the relative expression of MEST in N / TERT-1 cells treated with sodium butyrate (NaB) analyzed by qRT-PCR. Ct values ​​were normalized to the U6 probe.

[0060] Figure 5 D is a graph showing chromatin immunoprecipitation (ChIP)-qPCR of HDAC1 and HDAC2 occupancy in the promoter regions of miR-335 and MEST in N / TERT-1 in the presence of NaB. Precipitated DNA was analyzed by qRT-PCR. Data are presented as mean ± SD (n = 2).

[0061] Figure 5 E is a graph showing the relative transcript abundance of keratinocyte differentiation markers KRT1, IVL, and TGM1 in N / TERT-1 cells after MEST knockdown, represented as a bar graph.

[0062] Figure 5 F is a graph showing the relative transcript abundance of IVL, SPRR2F, and TGM1 in NaB-treated N / TERT-1 cells measured by qRT-PCR, with Ct values ​​normalized to the RPLP0 probe. Data are representative of three independent experiments and plotted as mean ± SD. *p-value < 0.05, **p-value < 0.001 were calculated using Student's t-test.

[0063] Figure 6 A to Figure 6 H is a graph showing that belinostat restores barrier function and epidermal homeostasis through the miR-335 network.

[0064] Figure 6 A is a graph showing qRT-PCR analysis of miR-335 induced expression in N / TERT-1 cells treated with HDAC inhibitors, represented as a bar graph.

[0065] Figure 6 B is a graph showing the induced expression of miR-335 upon belinostat treatment, detected by in situ hybridization in N / TERT-1 cells using a probe specific for mature miR-335 (red).

[0066] Figure 6C is a graph showing a bar graph representing the number of cornified capsules per field of belinostat-treated N / TERT-1 keratinocytes.

[0067] Figure 6 D is a graph showing a representative phase contrast microscopic image of a mature cornified envelope.

[0068] Figure 6 E is a graph showing qRT-PCR analysis of miR-335 induced expression on human skin biopsies topically treated with acetone alone or with HDACi dissolved in acetone, represented as a bar graph.

[0069] Figure 6 F is a graph showing the expression of miR-335 in human skin biopsies topically treated with acetone alone or with belinostat dissolved in acetone, as detected by in situ hybridization using a probe specific for mature miR-335 or a control probe.

[0070] Figure 6 G is a graph showing immunohistochemical analysis of IVL from human skin biopsies topically treated with acetone alone or with belinostat dissolved in acetone. P values ​​were calculated using Student's t-test. *p-value < 0.05, **p-value < 0.001. Scale bar, 100 μm.

[0071] Figure 6 H shows a diagram depicting a model of how loss of miR-335 leads to dysregulation of molecular pathways in AD. Schematic diagram of restoration of epidermal homeostasis after belinostat treatment.

[0072] Figure 7 A to Figure 7 C is a graph showing the differential expression of miR-335 in AD lesional skin.

[0073] Figure 7 A is a graph showing heat maps of selected genes generated by microRNA microarray using total RNA from AD lesion skin samples compared to RNA from normal human skin samples. Expression values ​​are shown as red (high) or blue (low) shades relative to the individual mean of the gene in a linear scale.

[0074] Figure 7 B is a graph showing the relative transcript abundance of miR-335 in N / TERT-1 keratinocytes and normal human skin, represented as a bar graph.

[0075] Figure 7C is a graph showing the relative transcript abundance of miR-335 in N / TERT-1 cells transfected with miR-335 mimic compared to control cells, represented as a bar graph.

[0076] Figure 8 A and Figure 8 B is a diagram showing that SOX6 targets genes essential for keratinocyte differentiation and cornification.

[0077] Figure 8 A is a graph showing the relative transcript abundance of SOX6 in N / TERT-1 cells transduced with shSOX6 and shControl, represented as a bar graph.

[0078] Figure 8 B is a Western blot showing the relative levels of SOX6 in shSOX6 and shControl. β-actin levels in the same samples indicate equal loading. C) Inducible expression of SOX6 leads to a significant downregulation of IVL, which is essential for keratinocyte differentiation and cornification as shown in organotypic analyses.

[0079] Figure 9 A and Figure 9 B is a diagram showing the interaction between SOX6 and the SMARCA complex.

[0080] Figure 9 A is a graph showing SOX6 protein immunoprecipitated from nuclear extracts of HEK293T cells transfected with pTRIPZ-SOX6 in the presence of doxycycline, followed by Western blot analysis using the indicated antibodies. The extracts were immunoprecipitated with rabbit IgG, SOX6, or Myc antibodies.

[0081] Figure 9 B is a diagram showing a list of potential SOX6 interacting partners.

[0082] Figure 10 Figure 2 shows promoter analysis of the MEST / miR-335 locus. The CAGE tag is present only in the upstream region of MEST, but not in the upstream region of miR-335, indicating that miR-335 does not have an independent promoter.

[0083] Figure 11 Figure 1 shows that belinostat restores barrier function and FLG expression. Immunohistochemical analysis of FLG was performed on human skin biopsies topically treated with acetone alone or with belinostat dissolved in acetone. Scale bar, 100 μm. DETAILED DESCRIPTION

[0084] Here, we present data from a screen for miRNAs specifically involved in atopic dermatitis. We identify miR-335 as essential for keratinocyte differentiation and maintenance of epidermal homeostasis.

[0085] We disclose the identification of a role for miR-335 in skin and atopic dermatitis.

[0086] We also confirmed that miR-335 expression is lost in AD lesional skin. We demonstrated that in healthy skin, proliferating cells in the epidermis' basal layer express low levels of miR-335, while those in the suprabasal layer express high levels of miR-335. We also demonstrated that in AD lesional skin, miR-335 expression is lost in all layers, including the basal and suprabasal layers.

[0087] We identified SOX6 as a direct target of miR-335 and demonstrated that, in the absence of miR-335, SOX6 recruits components of the SMARCA complex and leads to epigenetic silencing of genes critical for epithelial differentiation, resulting in barrier defects.

[0088] Finally, we show that miR-335 is epigenetically regulated by histone deacetylases and that treatment with the HDAC inhibitor Belinostat effectively restores miR-335 expression and epidermal homeostasis.

[0089] We therefore disclose the use of Belinostat for the treatment of atopic dermatitis.

[0090] We proposed the development of a topical cream with Belinostat for the treatment of eczema.

[0091] Abbreviations

[0092] AD – atopic dermatitis, miR-335 – microRNA-335, 3′UTR – 3′-untranslated region, HDAC – histone deacetylase, LNA – locked nucleic acid, CE – cornified envelope, IVL – involucrin, SPRR – small proline-rich protein, TGM1 – transglutaminase-1, ChIP – chromatin immunoprecipitation, and NaB – sodium butyrate.

[0093] MIR-335

[0094] MICRORNA (MIRNA)

[0095] MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that participate in the post-transcriptional regulation of gene expression in multicellular organisms by affecting mRNA stability and translation. MiRNAs are transcribed by RNA polymerase II as part of a capped and polyadenylated primary transcript (pri-miRNA) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce a stem-loop precursor miRNA (pre-miRNA) of approximately 70 nt, which is further cleaved by the cytoplasmic Dicer ribonuclease to produce the mature miRNA and the antisense miRNA star (miRNA*) product. The mature miRNA is incorporated into the RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational repression or destabilization of the target mRNA. Coding miRNAs are dysregulated in a variety of cancers, including breast, colorectal, and prostate cancers.

[0096] MIR-335

[0097] References to miR-335 include references to sequences from any species, including cja-mir-335 (miRBASE accession number MI0031980); bta-mir-335 (miRBASE accession number MI0009804); ppy-mir-335 (miRBASE accession number MI0014900); efu-mir-335 (miRBASE accession number MI0028746); tch-mir-335 (miRBASE accession number MI0031291); dno-mir-335 (miRBASE accession number MI0039072); mmu-mir-335 (miRBASE accession number MI0000817); ssc-mir-335 (miRBASE accession number MI0013165); ocu-mir-335 (miRBASE accession number MI0039371); cfa-mir-335 (miRBASE accession number MI0008020); hsa-mir-335 (miRBASE accession number MI0000816); eca-mir-335 (miRBASE accession number MI0012696); chi-mir-335 (miRBASE accession number MI0030747); ptr-mir-335 (miRBASE accession number MI0008623); pal-mir r-335 (miRBASE accession number MI0032526); ggo-mir-335 (miRBASE accession number MI0020669); rno-mir-335 (miRBASE accession number MI0000612); mml-mir-335 (miRBASE accession number MI0007699); cpo-mir-335 (miRBASE accession number MI0038733); hsa-miR-335-3p (miRBASE accession number MIMAT0004703); hsa-miR-335-5p (miRBASE accession number MIMAT0000765); mmu-miR-335- 5p (miRBASE accession number MIMAT0000766); ggo-miR-335 (miRBASE accession number MIMAT0024122); dno-miR-335-5p (miRBASE accession number MIMAT0047815); mml-miR-335-3p (miRBASE accession number MIMAT0026853); eca-miR-335 (miRBASE accession number MIMAT0012942); cfa-miR-335 (miRBASE accession number MIMAT0006624); rno-miR-335 (miRBASE accession number MIMAT0000575);pal-miR-335-3p (miRBASE accession number MIMAT0040162); tch-miR-335-5p (miRBASE accession number MIMAT0036618); cja-miR-335-5p (miRBASE accession number MIMAT0039456); ocu-miR-335-3p (miRBASE accession number MIMAT0048383); bta-miR-335 (miRBASE accession number MIMAT0048384); T0009291); efu-miR-335 (miRBASE accession number MIMAT0035059); cja-miR-335-3p (miRBASE accession number MIMAT0039457); pal-miR-335-5p (miRBASE accession number MIMAT0040161); mml-miR-335-5p (miRBASE accession number MIMAT0006274); cpo-miR-335-3p (miRBASE accession number MIMAT0039457); ASE accession number MIMAT0047171); ocu-miR-335-5p (miRBASE accession number MIMAT0048382); ssc-miR-335 (miRBASE accession number MIMAT0013955); chi-miR-335-3p (miRBASE accession number MIMAT0036148); ppy-miR-335 (miRBASE accession number MIMAT0015837); cpo-miR-335- 5p (miRBASE accession number MIMAT0047170); ptr-miR-335 (miRBASE accession number MIMAT0008104); mmu-miR-335-3p (miRBASE accession number MIMAT0004704); chi-miR-335-5p (miRBASE accession number MIMAT0036147); and dno-miR-335-3p (miRBASE accession number MIMAT0047816).

[0098] Human MIR-335

[0099] Human miR-335 (hsa-mir-335) has miRBASE accession number MI0000816.

[0100] The sequence of miR-335 is as follows:

[0101]

[0102] Hsa-mir-335 has the following structure:

[0103]

[0104] The mature hsa-mir-335 may comprise hsa-miR-335-5p or hsa-miR-335-3p.

[0105] The mature hsa-miR-335-5p has miRBase accession number MIMAT0000765 and the following sequence:

[0106]

[0107] Unless the context indicates otherwise, the term miR-335 should be understood to include reference to any and all mature forms of miR-335.

[0108] Thus, with respect to hsa-mir-335, unless the context indicates otherwise, the term shall also refer to hsa-miR-335-5p and hsa-miR-335-3p, as appropriate.

[0109] References Hsa-mir-335:“Identification of many microRNAs thatcopurify with polyribosomes in mammalian neurons”Kim J,Krichevsky A,Grad Y,Hayes GD,Kosik KS,Church GM,Ruvkun G,Proc Natl Acad Sci USA.101:360-365(2004),“New human and mouse microRNA genes found by homologysearch”,Weber MJ,FEBS J.272:59-73(2005),“A mammalian microRNA expression atlas based on smallRNA library sequencing”,Landgraf P,Rusu M,Sheridan R,Sewer A,Iovino N,AravinA,Pfeffer S,Rice A,Kamphorst AO,Landthaler M,Lin C,Socci ND,Hermida L,FulciV,Chiaretti S,Foa R,Schliwka J,Fuchs U,Novosel A,Muller RU,Schermer B,BisselsU,Inman J,Phan Q,Chien M,Cell.129:1401-1414(2007)also'Patterns of known andnovel small RNAs in human cervical cancer”Lui WO,Pourmand N,Patterson BK,FireA,Cancer Res.67:6031-6043(2007)

[0110] The following publications also describe Hsa-mir-335 in detail: Yu Y, et al. Sci Rep (2016) 6:30185, Zhang JK, et al. Cancer Cell Int (2017) 17:28, Shu M, et al., Mol Cancer (2011) 10:59, Jebbawi F, et al. J Transl Med (2014) 12:218, Zhou XM, et al. Oncotarget (2016) 7:13634-13650, Ronchetti D, et al. BMC Med Genomics (2008) 1:37, Samaraweera L, et al. BMC Cancer (2014) 14:309, Wang S, et al. BMC Ophthalmol (2018) 18:93, Huang HY, et al. PLoS One (2012) 7:e48637, Zarfeshani A, et al. Clin Epigenetics (2014) 6:27, Garcia-Cruz R, et al. BMC Med Genet (2015) 16:46, Vojtechova Z, et al. BMC Cancer (2016) 16:382, Rajpathak SN, et al. Sci Rep (2017) 7:43235, Murdocca M, et al. Int J Mol Sci (2016) 17, Maciotta S, et al. PLoS One (2012) 7:e43464, McAlinden A, et al. PLoS One (2013) 8:e75012, Schade A, et al. Int J Mol Sci (2013) 14:10710-10726, Lopez-Camarillo C, et al. Int J Mol Sci (2012) 13:1347-1379, Gassling V, et al. PLoS One (2013) 8:e63015, Zammit V, et al. Genes (Basel) (2018) 9, Wang G, et al. BMC Cancer (2017) 17:805, Cossellu G, et al. PLoS One (2016) 11:e0161916, Tang J, et al. Int J Mol Sci (2012) 13:13414-13437, Mainieri A, et al.Evol Med Public Health(2018)2018:82-91,Allen-Rhoades W,et al.A,et al.Camcer Mead(2015)4:977-988,Lin CY,et al.Sci Rep(2018)8:4277,Chen YJ,et al.Oncotarget(2017)8:113598-113613,Shigunov P,et al.SciRep(2018)8:8411,Liu Z,et alSci Rep(2016)6:23709,Li R,et al.BMC Genomics(2015)16:884,Hass R,et al.Cell Commim Signal (2012)10:26,Tsai MM,et al.Int J Mol Sci(2016)17,Shi C,et al.Oncotarget(2016)7:40830-40845,Ekstrōm K,et al.PLoS One(2013)g:e75227,Melone MAB,et al.Cell Death Dis(2018)9:228,Cava C,et al.BMCSyst Biol(2015)9:62,Chan SH,et al.J Biomed Sci(2015)22:9,Hanieh H et al MolCancer(2015)14:172,Yeh CH,et al.Mol Cancer(2016)15:37,Mulero-Navarro S,etal.Front Cell Dev Biol(2016)4:45,Rodrigues CE,et al.L,et al.Stem Cell ResTher(2017)8:19,Jayavelu ND,et al.BMC Genomics(2015)16:1077,Papanagnou P,etal.Biomolecules(2016)6,Dahiya N,et al.PLoS One(2008)3:e2436,Gupta S,et al,JBiomed Semantics(2016)7:9,Gumerov V,et al.Biol Direct(2015)10:59,Karere GM,etal.BMC Genomics(2012)13:320,Wu HH,etal.Expert Rev Mol Med(2014)16:el,Panigrahi GK,et al.Oncotarget(2018)9,13894-13910,Hossain MM,et al.J OvarianRes(2013)6:36,Watahiki,APLoS et al. One(2011)6:e24950,McGregor RA,et al.CurrMolMed(2011)11:,304-316,Bettermann K,et al.Int J Mol Sci(2014)15:9924-94949494.BBShen Systhen. Biol(2016)10:18,Yuan F,et al.Sci Rep(2018)8:5674,Riester SM,et al.BMC Med Genomics(2015)8:59,Li J,et al.BMC Genomics(2016)7:15:15. J,et al.

[0111] Front Mol Biosci(2015)2:31,Bhattacharya A,et al.PLoS One(2012)7:e46176,Xuan P,et al.PLoS One(2013)8:e70204,Harries LW,Genes(Basel)(2014)5:656-670,Hass R,et al.Cell Commun Signal(2011)9:12,Huang Z,et al.Sci Rep(2017)7:13673,Longati PBMC et al. Cancer(2013)13:95,Gurbuz I,et al.MolCancer(2014)13:22,Natarajan SK,et al.Biomolecules(2015)5:3309-3338,Pelosi L,etal.EBioMedicine(2015)2:285-293,Sandhu SK,et al.Adv Hematol(2011)2011:347137,Ren J,et al.J Transl Med(2018)16:65.

[0112] Koscianska E,et al.Cerebellum Ataxias(2014)1:7,Zhan Y,et al.J Ovarian Res(2015)8:48,Karere GM-et al.J Biomed Sci(2010)17:54,Burba l,etal.PLoS One(2011)6:e22158,Romania P,et al.Int J Mol Sci(2012)13:16554-16579,Lopez-Anton M,et al.Biomed Res Int(2015)2015:929806,Qin Z,et al.Viruses(2014)6:4571-4580,Erriquez D,et al.Int J Mol Sci(2013)14:19681-19704,Choi S,etal.Exp Mol Med(2017)49:e403,Santhanam AN,et al.PLoS One(2009)4:e4868,Ishii S,et al.Front Cell Neurosci(2015)9:207,Zhu Z,et al.Viruses(2014)6:1525-1539,Skalsky RL,et al.PLoS One(2011)6:e24248,Sana J,et al.JTransl Med(2012)10:103,Azuaje FJ,et al.BMC Med Genomics(2011)4:59,Dalan AB,etal.BMC Cancer(2017)17:207,Fiscon G,et al.SciRep(2018)8:7769,Di Leva G,et al.Ups J Med Sci(2012)117:202-216,Denk J,et al.PLoS One(2015)10:e0126423,Sui J,et al.Oncotarget(2017)8:65997-66018,Vuppalanchi R,et al.PLoS One(2013)8:e74471,Zhang C,et al.ParasitVectors(2016)9:278,Xie S,et al.Sci Rep(2017)7:2516,Koumangoye RB,et al.MolCancer(2015)14:24,Jiao DM,et al.PLoS One (2017) 12: e0172470, Stigliani S, et al. Oncotarget (2015) 6: 13295-13308, Chang L, et al. Oncotarget (2017) 8: 84384-84395, Wang CH, et al. Oncotarget (2015) 6: 42118-42129, Zhang ZJ, et al. Oncol Rep (2012) 27:903-910, Debeb BG, et al. Mol Cancer (2010) 9:180 and Valencia-Quintana R, et al. Front Microbiol (2014) 5:102.

[0113] Activity of MIR-335

[0114] The biological activities of miR-335 are known in the art.

[0115] The activity of miR-335 and its analysis are described in, for example, Kim et al (2015) miR-335 Targets SIAH2 and Confers Sensitivity to Anti-Cancer Drugs by Increasing the Expression of HDAC3. Mol Cells 38(6):562-72.

[0116] The activity of miR-335 may include an effect on HDAC3 and / or SIAH2 expression. The activity of miR-335 may include increasing sensitivity to anticancer drugs. The activity of miR-335 may include an apoptotic effect. The activity of miR-335 may include inhibiting the ubiquitination of HDAC3 in anticancer drug-resistant cancer cell lines. The activity of miR-335 may include negatively regulating the invasion, migration, and growth rate of cancer cells. The activity of miR-335 may include negatively regulating the tumorigenic potential of cancer cells.

[0117] Additional biological activities of miR-335 are described in the Examples.

[0118] The activity of miR-335 may include upregulating keratinocyte differentiation and / or cornification. Analysis of this activity is described in detail in the Examples.

[0119] The activity of miR-335 may include downregulating the expression of SOX6. This activity can be measured using a luciferase reporter gene construct comprising the 3'-UTR of SOX6.

[0120] MIR-335 MIRNA

[0121] The methods and compositions described herein can utilize miR-335, and any of its variants, homologs, derivatives, and fragments, for diagnosis, detection of susceptibility, treatment, alleviation, or prevention of a skin condition, such as atopic dermatitis, in an individual.

[0122] The terms "miR-335 miRNA" and "miR-335 nucleic acid" are used interchangeably.

[0123] These terms are also intended to include nucleic acid sequences that can encode miR-335 miRNAs and / or fragments, derivatives, homologs, or variants thereof. These terms are also intended to include nucleic acid sequences that are fragments, derivatives, homologs, or variants of miR-335 polynucleotides having a specific sequence disclosed herein.

[0124] When referring to miR-335 miRNA nucleic acid, it should be considered as a reference to the nucleic acid sequence capable of encoding such miRNA. Depending on the circumstances, such miRNA may contain one or more biological activities of native miR-335.

[0125] As described herein, miR-335 miRNA can be used in a variety of ways. For example, miR-335 miRNA can be used to treat individuals suffering from or suspected of having a skin condition such as atopic dermatitis, or to prevent such a condition or alleviate any symptoms caused by such a condition. Other uses will be apparent to those skilled in the art and are also included herein.

[0126] As used herein, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded DNA and double-stranded DNA, DNA with a mixture of single-stranded and double-stranded regions, single-stranded RNA and double-stranded RNA, and RNA with a mixture of single-stranded and double-stranded regions, including hybrid molecules of DNA and RNA that can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA with a main chain modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. DNA and RNA have been subjected to a variety of modifications; therefore, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA unique to viruses and cells. "Polynucleotide" also includes relatively short polynucleotides often referred to as oligonucleotides.

[0127] The skilled artisan will appreciate that due to the degeneracy of the genetic code, many nucleotide sequences can encode the same polypeptide.

[0128] As used herein, the term "nucleotide sequence" refers to a nucleotide sequence, an oligonucleotide sequence, a polynucleotide sequence, and variants, homologues, fragments and derivatives thereof (e.g., portions thereof). The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin, and may be double-stranded or single-stranded, representing either a sense strand or an antisense strand or a combination thereof. The term nucleotide sequence may be prepared using recombinant DNA technology (e.g., recombinant DNA).

[0129] The term "nucleotide sequence" may refer to DNA or RNA.

[0130] Other nucleic acids

[0131] We also provide nucleic acids that are fragments, homologs, variants or derivatives of the miR-335 miRNA.

[0132] The terms "variant," "homolog," "derivative," or "fragment" related to miR-335 include any substitution, variation, modification, replacement, deletion, or addition to one (or more) nucleic acid from the sequence of a miR-335 miRNA or to one (or more) nucleic acid of the sequence of a miR-335 miRNA. Unless the context dictates otherwise, references to "miR-335 miRNA," "miR-335 nucleic acid," "miR-335 nucleotide sequence," and the like include references to such variants, homologs, derivatives, and fragments of miR-335 miRNA.

[0133] The nucleotide sequence can encode a polypeptide having any one or more miR-335 miRNA activities. The term "homologue" may be intended to encompass consistency with respect to structure and / or function, such that the resulting nucleotide sequence encodes a polypeptide having miR-335 miRNA activity. For example, the expression level of a homologue of miR-335 miRNA, etc., may be increased or decreased in the cells of individuals suffering from skin conditions such as atopic dermatitis, compared to normal cells. With regard to sequence identity (i.e., similarity), there may be at least 70%, at least 75%, at least 85% or at least 90% sequence identity. It may have at least 95% sequence identity, for example, at least 98% sequence identity, to a related sequence (such as any nucleic acid sequence of miR-335 miRNA). These terms also include allelic variations of the sequence.

[0134] Variants of miR-335 can comprise variants having 95% or more, 95.1% or more, 95.2% or more, 95.3% or more, 95.4% or more, 95.5% or more, 95.6% or more, 95.7% or more, 95.8% or more, 95.9% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.6% or more, 96.7% or more, 96.8% or more, 96.9% or more, 97% or more, 97.1% or more, 97.2% or more, 97.3% or more, 97.4% or more In some embodiments, the present invention relates to a sequence having 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity.

[0135] Variants, derivatives and homologues

[0136] MiR-335 miRNA nucleic acid variants, fragments, derivatives and homologs can comprise RNA. They may be single-stranded. They can also be polynucleotides containing synthetic or modified nucleotides. A variety of different types of oligonucleotide modifications are known in the art. These modifications include methylphosphonate and phosphorothioate backbones, with the addition of acridine or polylysine chains at the 3' end and / or 5' end of the molecule. For the purposes of this article, it will be understood that polynucleotides can be modified by any method available in the art. Such modifications can be performed to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0137] In the case of a double-stranded polynucleotide, both strands of the duplex, whether alone or in combination, are included in the methods and compositions described herein. In the case of a single-stranded polynucleotide, it is understood that the complementary sequence of the polynucleotide is also included.

[0138] The terms "variant," "homolog," or "derivative" with respect to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition to a nucleic acid (or nucleic acids) from or to the sequence (or nucleic acids). The variant, homolog, or derivative may encode a polypeptide having biological activity. As described above, such fragments, homologs, variants, and derivatives of miR-335 may comprise modulated activities.

[0139] As described above, with respect to sequence identity, a "homolog" may have at least 5% identity, at least 10% identity, at least 15% identity, at least 20% identity, at least 25% identity, at least 30% identity, at least 35% identity, at least 40% identity, at least 45% identity, at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to a related sequence (e.g., any nucleic acid sequence of a miR-335 miRNA).

[0140] There may be at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity.

[0141] A homologue, derivative or fragment of miR-335 may comprise a polypeptide having 95% or more, 95.1% or more, 95.2% or more, 95.3% or more, 95.4% or more, 95.5% or more, 95.6% or more, 95.7% or more, 95.8% or more, 95.9% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.6% or more, 96.7% or more, 96.8% or more, 96.9% or more, 97% or more, 97.1% or more, 97.2% or more, 97.3% or more, 97.4% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9 ... In some embodiments, the present invention relates to sequences having a sequence identity of at least 4%, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, sequences having a sequence identity of 99.7% or more, 99.8% or more, or 99.9% or more.

[0142] The comparison of nucleotide identity can be performed as described above. The sequence comparison program that can be used is the above-mentioned GCG Wisconsin Bestfit program. The default scoring matrix has a matching value of 10 for each identical nucleotide and a matching value of -9 for each mismatch. The default gap creation penalty for each nucleotide is -50, and the default gap extension penalty is -3.

[0143] hybridization

[0144] We also describe nucleotide sequences that are capable of selectively hybridizing to any of the sequences presented herein, or any variants, fragments, or derivatives thereof, or to the complement of any of the aforementioned sequences. The nucleotide sequence can be at least 5, 10, or 15 nucleotides in length, such as at least 20, 30, 40, or 50 nucleotides in length.

[0145] As used herein, the term "hybridization" shall include "the process by which a nucleic acid strand joins with a complementary strand through base pairing" as well as the process of amplification as performed in polymerase chain reaction techniques.

[0146] The polynucleotides capable of selectively hybridizing to the nucleotide sequences presented herein or their complements can be at least 40% homologous, at least 45% homologous, at least 50% homologous, at least 55% homologous, at least 60% homologous, at least 65% homologous, at least 70% homologous, at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous or at least 95% homologous to the corresponding nucleotide sequences presented herein (e.g., any nucleic acid sequence of miR-335 miRNA). Such polynucleotides can typically be at least 70%, at least 80% or 90% or at least 95% or 98% homologous to the corresponding nucleotide sequences over a region of at least 5, 10, 15 or 20, such as at least 25 or 30, such as at least 40, 60 or 100 or more consecutive nucleotides.

[0147] The polynucleotides may comprise 95% or more, 95.1% or more, 95.2% or more, 95.3% or more, 95.4% or more, 95.5% or more, 95.6% or more, 95.7% or more, 95.8% or more, 95.9% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.6% or more, 96.7% or more, 96.8% or more, 96.9% or more, 97% or more, 97.1% or more, 97.2% or more, 97.3% or more, 97.4% or more , 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity.

[0148] The term "selectively hybridizes" means that the polynucleotide used as a probe is used under conditions in which the target polynucleotide is found to hybridize to the probe at a level significantly above background. Background hybridization may occur due to the presence of other polynucleotides, for example in the cDNA or genomic DNA library being screened. In this context, background means a signal level resulting from the interaction between the probe and nonspecific DNA members in the library that is 10 times, for example, 100 times less intense than that observed for the specific interaction with the target DNA. This can be achieved, for example, by radiolabeling the probe, for example using 32 P or 33 Alternatively, the strength of the interaction can be measured using non-radioactive probes such as fluorescent dyes, biotin, or digoxigenin.

[0149] Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and are given the definition of "stringency" as explained elsewhere herein.

[0150] The highest stringency generally occurs at about Tm-5°C (5°C lower than the Tm of the probe); high stringency occurs at about 5°C to 10°C below Tm; medium stringency occurs at about 10°C to 20°C below Tm; and low stringency occurs at about 20°C to 25°C below Tm. As will be understood by those skilled in the art, the highest stringency hybridization can be used to identify or detect identical polynucleotide sequences, while medium (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.

[0151] We provide nucleotide sequences that may be capable of hybridizing to miR-335 nucleic acids, fragments, variants, homologs or derivatives under stringent conditions, such as 65°C and 0.1xSSC (1xSSC = 0.15 M NaCl, 0.015 M sodium citrate pH 7.0).

[0152] Generation of homologues, variants and derivatives

[0153] Polynucleotides that are not 100% identical to the sequence of interest (miR-335), but are also included, as well as homologues, variants and derivatives of miR-335 miRNA, can be obtained in a variety of ways. Other variants of the sequence can be obtained, for example, by probing RNA libraries made from a range of individuals (e.g., individuals from different populations).

[0154] For example, homologs of the miR-335 miRNA can be identified from other individuals or other species. Examples of these are listed above.

[0155] Further recombinant miR-335 miRNA nucleic acids and polypeptides can be generated by identifying corresponding positions in homologs and by synthesizing or producing molecules as described elsewhere herein.

[0156] In addition, other viral homologs / bacterial homologs or cell homologs of miR-335 miRNA can be obtained, particularly cell homologs found in mammalian cells (e.g., rat, mouse, cattle, and primate cells), and these homologs and fragments thereof are generally capable of selective hybridization with human miR-335 miRNA. These homologs can be used to design non-human miR-335 miRNA nucleic acids, fragments, variants, and homologs. Mutagenesis can be performed by methods known in the art to generate more variants.

[0157] Sequences of miR-335 miRNA homologs can be obtained by probing libraries made from other animal species and probing such libraries under conditions of moderate to high stringency with probes containing all or a portion of any miR-335 miRNA nucleic acid, fragment, variant, and homolog or other fragment of miR-335 miRNA.

[0158] Similar considerations apply to obtaining species homologs and allelic variants of the polypeptide or nucleotide sequences disclosed herein.

[0159] Variants and strain / species homologs can also be obtained using degenerate PCR, which uses primers designed to target sequences within variants and homologs encoding conserved amino acid sequences within the miR-335 miRNA nucleic acid sequence. Conserved sequences can be predicted, for example, by aligning amino acid sequences from several variants / homologs. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

[0160] Primers used in degenerate PCR will contain one or more degenerate positions and will be used under lower stringency conditions than those used to clone sequences using single sequence primers to known sequences. One skilled in the art will appreciate that the overall nucleotide homology between sequences from distantly related organisms may be very low, and therefore in these cases, degenerate PCR may be an option rather than screening the library with the miR-335 sequence having a tagged fragment.

[0161] Additionally, homologous sequences can be identified by searching nucleotide and / or protein databases using search algorithms such as the BLAST suite of programs.

[0162] Alternatively, such polynucleotides can be obtained by site-directed mutagenesis of characteristic sequences (such as miR-335 miRNA nucleic acids or variants, homologues, derivatives or fragments thereof). This may be useful, for example, in situations where it is necessary to change the silent codons of the sequence to optimize the codon preference of a specific host cell expressing the polynucleotide sequence. Other sequences may need to be changed to introduce restriction enzyme recognition sites, or to change the properties or functions of the polypeptides encoded by the polynucleotides.

[0163] The polynucleotides described herein can be used to generate primers (e.g., PCR primers, primers for alternative amplification reactions), probes (such as probes with revealing labels labeled by conventional methods using radioactive or non-radioactive labels), or the polynucleotides can be cloned into vectors. Such primers, probes, and other fragments will be at least 8, 9, 10, or 15, such as at least 20, such as at least 25, 30, or 40 nucleotides in length, and are also included in the term "polynucleotide" as used herein.

[0164] Polynucleotides such as DNA polynucleotides and probes can be produced recombinantly, synthetically, or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0165] Generally speaking, primers will be produced by synthetic means, which involves the stepwise preparation of the desired nucleic acid sequence one nucleotide at a time. The technology to achieve this using automated techniques is readily available in the art.

[0166] Primers comprising a miR-335 miRNA stretch are particularly useful in methods for detecting miR-335 miRNA expression, for example, upregulation or downregulation of miR-335 miRNA expression associated with skin conditions such as atopic dermatitis. Suitable primers for amplifying miR-335 miRNA can be generated from any suitable miR-335 miRNA stretch. Primers that can be used include those that are capable of amplifying a specific miR-335 miRNA sequence.

[0167] Although primers for miR-335 miRNA can be provided individually, they are most useful when provided as a primer pair comprising a forward primer and a reverse primer.

[0168] Recombinant methods are typically used, such as PCR (polymerase chain reaction) cloning techniques to produce longer polynucleotides. This will include preparing a pair of primers (e.g., about 15 to 30 nucleotides), contacting the primers with mRNA or cDNA obtained from animal or human cells, performing polymerase chain reaction under conditions that amplify the desired region, separating the amplified fragments (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. Primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

[0169] The polynucleotide or primer may be labeled with a visible marker. Suitable labels include radioisotopes such as 32 P or 35 S, digoxigenin, fluorescent dyes, enzyme labels or other protein labels such as biotin. Such labels can be added to polynucleotides or primers and can be detected using techniques known per se. Those skilled in the art can use labeled or unlabeled polynucleotides or primers or fragments thereof in nucleic acid-based tests to detect or sequence polynucleotides in humans or animals.

[0170] Such tests for detection generally include contacting a biological sample containing DNA or RNA with a probe comprising polynucleotides or primers under hybridization conditions, and detecting any duplex formed between the probe and the nucleic acid in the sample. Such detection can be achieved using techniques such as PCR or by fixing the probe on a solid support, removing nucleic acids that are not hybridized with the probe in the sample, and then detecting nucleic acids that hybridize with the probe. Alternatively, the sample nucleic acid can be fixed on a solid support, and the amount of the probe bound to this support can be detected. This and other forms of suitable assay methods can be found in, for example, WO89 / 03891 and WO90 / 13667.

[0171] Assays for sequencing nucleic acids such as miR-335 miRNA nucleic acids include contacting a biological sample containing target DNA or RNA with a probe comprising a polynucleotide or primer under hybridization conditions and determining the sequence by, for example, the Sanger dideoxy chain termination method (see Sambrook et al).

[0172] This method generally involves extending a primer by synthesizing a chain complementary to a target DNA or RNA in the presence of suitable reagents, and selectively terminating the extension reaction at one or more of A, C, G, or T / U residues; allowing chain extension and termination reactions to occur; and separating the extension products by size to identify the nucleotide sequence at which selective termination has occurred. Suitable reagents include a DNA polymerase, the deoxynucleotides dATP, dCTP, dGTP, and dTTP, a buffer, and ATP. Dideoxynucleotides are used for selective termination.

[0173] Isolation of miRNA

[0174] miRNA can be isolated from exosomes using any method known in the art.

[0175] Those skilled in the art will be aware of the various methods that have been developed for isolating miRNA from biological fluids. Commercially available miRNA isolation kits are available, such as the miRNeasy kit (Qiagen, CA), the mirVana PARIS kit (Ambion, TX), and the Total RNA Isolation Kit (Norgen Biotek, Canada). Any of these can be used to isolate miRNA from a sample.

[0176] The following exemplary protocol from the miRNeasy Serum / Plasma Handbook (QIAGEN, February 2012) can be used to isolate miRNA using the miRNeasy kit:

[0177] 1. Prepare serum or plasma or thaw frozen samples.

[0178] 2. Add 5 volumes of QIAzol Lysis Reagent (see Table 2 in the instructions). Mix by vortexing or pipetting up and down.

[0179]

[0180] Note: If the volume of plasma or serum is not limited, we recommend using 100 μl to 200 μl for each RNA preparation.

[0181] Note: After adding QIAzol Lysis Reagent, lysates can be stored at -70°C for several months.

[0182] 3. Place the tube containing the lysate on the bench at room temperature (15°C to 25°C) for 5 minutes.

[0183] 4. Add 3.5 μl of miRNeasy serum / plasma spike-in control (miRNeasy Serum / Plasma Spike-InControl, 1.6 x 10 8 copies / μl working solution) and mix thoroughly.

[0184] For details on preparing appropriate miRNeasy serum / plasma spiked control stock and working solutions, see Appendix B on page 25.

[0185] 5. Add an equal volume of chloroform to the tube containing the lysate and cap it tightly (see Table 2 in the guide). Vortex or shake vigorously for 15 seconds.

[0186] Adequate mixing is important for subsequent phase separation.

[0187] 6. Place the tube containing the lysate on the bench at room temperature (15°C to 25°C) for 2 to 3 minutes.

[0188] 7. Centrifuge at 12,000 × g for 15 minutes at 4°C. After centrifugation, warm the centrifuge to room temperature (15°C to 25°C) if the same centrifuge will be used for the next centrifugation step.

[0189] After centrifugation, the sample separates into three phases: an upper, colorless aqueous phase containing RNA; a white, intermediate phase; and a lower, red, organic phase. See Table 2 for approximate volumes of the aqueous phase.

[0190] 8. Transfer the upper aqueous phase to a new collection tube (not provided). Avoid transferring any interphase material. Add 1.5 volumes of 100% ethanol and mix thoroughly by pipetting back and forth several times. Do not centrifuge. Proceed immediately to step 9.

[0191] A precipitate may form upon addition of ethanol, but this will not affect the procedure.

[0192] 9. Use a pipette to transfer up to 700 μl of sample (including any precipitate that may have formed) into the RNeasy MinElute spin column in the 2 ml collection tube (provided). Gently close the cap and centrifuge at ≥8,000 × g (≥10,000 rpm) for 15 seconds at room temperature (15°C to 25°C). Discard the flow-through.

[0193] Reuse the collection tube in step 10.

[0194] 10. Repeat step 9 with the remaining sample. Discard the flow-through.*

[0195] Reuse the collection tube in step 11.

[0196] 11. Add 700 μl of Buffer RWT to the RNeasy MinElute spin column. Gently close the cap and centrifuge at ≥8,000 × g (≥10,000 rpm) for 15 seconds to wash the column. Discard the flow-through.

[0197] Reuse the collection tube in step 12.

[0198] 12. Use a pipette to transfer 500 μl of Buffer RPE to the RNeasy MinElute spin column. Gently close the cap and centrifuge at ≥8000 × g (≥10,000 rpm) for 15 seconds to wash the column. Discard the flow-through.

[0199] Reuse the collection tube in step 13.

[0200] 13. Use a pipette to transfer 500 μl of 80% ethanol to the RNeasy MinElute spin column. Gently close the cap and centrifuge at ≥8,000 × g (≥10,000 rpm) for 2 minutes to wash the spin column membrane. Discard the collection tube containing the flow-through.

[0201] Note: 80% ethanol should be prepared using ethanol (96% to 100%) and RNase-free water.

[0202] Note: After centrifugation, carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not come into contact with the flow-through. Otherwise, ethanol carryover will occur.

[0203] 14. Place the RNeasy MinElute spin column in a new 2 ml collection tube (provided). Open the spin column cap and centrifuge at full speed for 5 minutes to dry the membrane. Discard the collection tube containing the flow-through.

[0204] To avoid damaging their lids, place the columns in the centrifuge with at least one empty space between them. Orient the lids so that they point in the opposite direction of the rotor's rotation (e.g., if the rotor rotates clockwise, orient the lids counterclockwise).

[0205] It is important to allow the spin column membrane to dry as residual ethanol may interfere with downstream reactions. Centrifuging with the lid open ensures that no ethanol is retained during RNA elution.

[0206] 15. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube (provided). Add 14 μl of RNase-free water directly to the center of the spin column membrane. Gently close the cap and centrifuge at full speed for 1 minute to elute the RNA.

[0207] If a higher concentration of RNA is required, elution can be performed using as little as 10 μl of RNase-free water, but the yield will be reduced by approximately 20%. Do not use less than 10 μl of RNase-free water for elution, as this will not fully hydrate the spin column membrane.

[0208] The dead volume of the RNeasy MinElute spin column is 2 μl: elute with 14 μl of RNase-free water to obtain 12 μl of eluate.

[0209] Histone deacetylase inhibitors (HDAC inhibitors - HDIs)

[0210] We provide the use of histone deacetylase inhibitors (HDAC inhibitors) in methods of diagnosing, treating, preventing or ameliorating dermatological conditions such as atopic dermatitis. HDAC inhibitors may be known by various names, such as lysine deacetylase (KDAC).

[0211] HDAC inhibitor can be any one or more any substances that can reduce histone deacetylase activity.This activity can comprise histone deacetylase activity.HDAC inhibitor can suppress the removal of the acetyl group of the ε-N-acetyl lysine amino acid of histone by histone deacetylase.Analysis of deacetylase activity is known in the art.

[0212] HDAC proteins are divided into four classes based on function and DNA sequence similarity:

[0213] Class I, including HDAC1, HDAC2, HDAC3, and HDAC8, which are related to the yeast RPD3 gene;

[0214] Class IIA, including HDAC4, HDAC5, HDAC7, and HDAC9; Class IIB, HDAC6 and HDAC10, which are related to the yeast Hda1 gene;

[0215] Class III, also known as sirtuins, are associated with the Sir2 gene and include SIRT1-7

[0216] Class IV, which includes only HDAC11 that has characteristics of both classes I and II.

[0217] Classes I, II, and IV are considered "classical" HDACs, their activity inhibited by trichostatin A (TSA) and possessing a zinc-dependent active site, whereas class III enzymes are a family of NAD+-dependent proteins known as longevity proteins and are unaffected by TSA. Homologs of these three groups have been found in yeast and are named: reduced potassium dependency 3 (Rpd3), corresponding to class I; histone deacetylase 1 (hda1), corresponding to class II; and silent information regulator 2 (Sir2), corresponding to class III. Class IV contains only one isoform (HDAC11), which shows little homology to the yeast enzymes Rpd3 or hda1, thus assigning HDAC11 to its own class. Class III enzymes are considered a separate class of enzymes with distinct mechanisms of action; these enzymes are NAD+-dependent, whereas other classes of HDACs require Zn2+ as a cofactor.

[0218] The HDAC inhibitors described herein can inhibit the activity of any of the above-mentioned histone classes. HDAC inhibitors themselves can be divided into several groups, including:

[0219] Hydroxamic acids (or hydroxamates), such as trichostatin A,

[0220] Cyclic tetrapeptides (such as trapoxin B) and decapeptides,

[0221] Benzamides,

[0222] Electrophilic ketones, and

[0223] Fatty acid compounds, such as phenylbutyric acid and valproic acid.

[0224] "Second generation" HDIs include the hydroxamic acids: vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589); and the benzamides: entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103).

[0225] Any of these HDAC inhibitors may be used for the purposes described herein.

[0226] MOCETINOSTAT

[0227] Mocetinostat has a PubChem CID of 9865515 and is also known as 726169-73-9, MGCD0103, MGCD-0103, and N-(2-aminophenyl)-4-([[4-(pyridin-3-yl)pyrimidin-2-yl]amino]methyl)benzamide.

[0228] Mocetinostat is a rationally designed, orally available, selective, small-molecule 2-aminobenzamide HDAC inhibitor with potential antitumor activity. Mocetinostat binds to and inhibits class 1 HDAC isoforms, specifically HDAC1, HDAC2, and HDAC3. This can lead to epigenetic changes in tumor cells, leading to cell death. Although the precise mechanism has not yet been determined, tumor cell death may occur through induction of apoptosis, differentiation, cell cycle arrest, inhibition of DNA repair, upregulation of tumor suppressors, downregulation of growth factors, oxidative stress, and autophagy. Class 1 HDAC1, HDAC2, and HDAC3 have been found to be overexpressed in many tumors and are associated with poor prognosis.

[0229] QUISINOSTAT

[0230] Quisinostat has a PubChem CID of 11538455 and is also known as 875320-29-9, JNJ-26481585, N-hydroxy-2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl))piperidin-1-yl)pyrimidine-5-carboxamide, and UNII-9BJ85K1J8S.

[0231] Quisinostat is an orally bioavailable, second-generation, hydroxamic acid-based histone deacetylase (HDAC) inhibitor with potential anti-tumor activity. The HDAC inhibitor JNJ-26481585 inhibits HDAC, leading to the accumulation of hyperacetylated histones, which may lead to the induction of chromatin remodeling, inhibition of tumor suppressor gene transcription, inhibition of tumor cell division, and induction of tumor cell apoptosis. HDAC is an enzyme that is upregulated in multiple tumor types and deacetylates chromatin histones. Compared to some first-generation HDAC inhibitors, JNJ-26481585 induces greater HSP70 upregulation and Bcl-2 downregulation.

[0232] SCRIPTAID

[0233] Scriptaid has a PubChem CID of 5186 and is also known as 287383-59-9, Scriptide, 6-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-hydroxyhexanamide, and GCK 1026.

[0234] LMK-235

[0235] LMK-235 has a PubChem CID of 71520717 and is also known as 1418033-25-6, LMK 235, N-((6-(hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide, and CHEMBL 2312168.

[0236] BELINOSTAT

[0237] Belinostat has a PubChem CID of 6918638 and has the molecular formula C 15 H 14 N2O4S.

[0238] Belinostat is also known as CID 6918638, Belinostat, 414864-00-9, PXD101, Belinostat(PXD101), 866323-14-0, PXD-101, Beleodaq, (E)-N-hydroxy-3-(3-(N-phenylsulfamoyl)phenyl)acrylamide, NSC726630, PXD 101, N-Hydroxy-3-(3-phenylsulfonylphenyl)acrylamide, UNII-F4H96P17NZ, N-Hydroxy-3-[3-[(anilino)sulfonyl]phenyl]-2-acrylamide, PX-105684, 2-Acrylamide, N-Hydroxy-3-[3-[(anilino)sulfonyl]phenyl]-,(2E)-, F4H96P17NZ, (2E)-N-Hydroxy-3-[3-(phenylsulfamoyl)phenyl]propyl-2-enamine, CHEBI:61076, PX 105684, (2E)-N-Hydroxy-3-[3-(phenylsulfamoyl)phenyl]acrylamide, (E)-N-Hydroxy-3-[3-(phenylsulfamoyl)phenyl]propyl-2-enamine, E-Belinostat, (E)-N-Hydroxy-3-[3-(phenylsulfamoyl)phenyl]propyl-2-enamine, Belinostat [USAN:INN], Belinostat (random form), N-Hydroxy-3-(3-(phenylsulfamoyl)phenyl)propyl-2-enamine, PX105684, 2-Acrylamide, N-Hydroxy-3-(3-(anilino)sulfonyl)phenyl)-,(2E)-, Belinostat Ph3, Beleodaq (TN), PubChem 22405, Belinostat-PXD101, Belinostat (USAN / INN), N-hydroxy-3-(3-phenylsulfonylphenyl)acrylamide, cc-489, MLS006011091, CHEMBL 408513, GTPL 7496, Belinostat 866323-14-0, BDBM25150, CHEBI:94531, DTXSID60194378, EX-A180, QCR-181, (E)-3-[3-(phenylsulfamoyl)phenyl]propyl-2-enehydroxamic acid, BCPP000351, AOB87787, BCP01741, ZINC3818726, Belinostat, PXD101, PX105684, Belinostat / PXD101, PX105684 / , ​​ABP000140, s1085, AKOS025401741, BCP9000386, CCG-208758, DB05015, LS41098 、NSC-726630、SB16466、NCGC00263155-05、AC-25046、AS-17068、SC-71101、SMR004702879、AB0007889、SW219445-1、EC-000.2286、A25012、D08870、W-5363、J-523584、Q4882925、BRD-K17743125-001-01-9、N-Hydroxy-3-[(phenylamino)sulfonyl]-trans-cinnamamide、(E)-N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide、N-hydroxy-3-[3-[(phenylamino)sulfonyl]phenyl]-2-PR and 5OG. ,

[0239] Belinostat is a novel hydroxamic acid-type histone deacetylase (HDAC) inhibitor with antitumor activity. Belinostat targets HDAC enzymes, thereby inhibiting tumor cell proliferation, inducing apoptosis, promoting cell differentiation, and inhibiting angiogenesis. This agent can sensitize drug-resistant tumor cells to other antitumor agents, possibly through a mechanism that downregulates thymidylate synthase (National Cancer Institute).

[0240] Through drug screening, a broad-spectrum HDAC inhibitor "Belinostat" was identified as a candidate drug.

[0241] Belinostat can effectively restore miR-335 expression, inhibit pro-inflammatory factors and repair defective barrier, thereby alleviating difficult-to-treat skin conditions such as atopic dermatitis.

[0242] Belinostat can be used in its native form, or as a salt, hydrate, or solvate. Therefore, it may be convenient or desirable to prepare, purify, and / or process the corresponding belinostat salt, such as a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. ScL., Vol. 66, pp. 1-19.

[0243] Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ and other cations such as Al +3 Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4+ ) and substituted ammonium ions (e.g. NH3R + NH2R 2+ 、NHR 3+ NR 4+ Examples of some suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3) 4+ .

[0244] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

[0245] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose.

[0246] Preparation, purification and / or processing corresponding belinostat solvate may be convenient or desirable.Term " solvate " is used with conventional meaning in this article, refers to the complex of solute (for example, belinostat, belinostat salt) and solvent.If solvent is water, then can easily claim that solvate is hydrate, for example monohydrate, dihydrate, trihydrate etc.

[0247] Reference herein to belinostat should be understood as a reference to a salt, hydrate or solvate thereof.

[0248] Belinostat analogs

[0249] Analogs and derivatives of belinostat are known in the art. Preferably, such analogs and derivatives contain histone deacetylase (HDAC) inhibitor activity. They may also contain an N-hydroxycinnamic moiety.

[0250] For example, analogs and derivatives of belinostat are described in Zhang et al (2019) Design, synthesis and evaluation of belinostat analogs as histone deacetylase inhibitors. Future Medicinal Chemistry, 11 (21) and Li et al (2019) Design, synthesis, and biological evaluation of target water-soluble hydroxamic acid-based HDACi derivatives as prodrugs. Chemical Biology & Drug Design 94 (4), 1760-1767.

[0251] An example of a belinostat analog / derivative is compound 7e as described in Zhang et al (2019), which showed an IC of 1.5 nM in an HDAC inhibition assay. 50 value.

[0252] Unless the context requires otherwise, references to belinostat shall include references to analogs and derivatives of belinostat.

[0253] Skin conditions

[0254] We disclose the use of miR-335, an agent capable of upregulating the expression or activity of miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat, for treating, preventing, prophylactically, or ameliorating a skin disease or skin condition.

[0255] Examples of skin diseases and conditions are known in the art and include, for example, alopecia areata, atopic dermatitis, bullous pemphigoid, bullous systemic lupus erythematosus, dermatitis herpetiformis (DH), dermatomyositis, drug-induced pemphigus, eczema, epidermolysis bullosa acquisita (EBA), IgA pemphigus, lichen sclerosus, linear IgA bullous dermatosis, mucous membrane pemphigoid, paraneoplastic pemphigus, pemphigus gestationis, pemphigus, pemphigus erythematosus (PE), pemphigus foliaceus, pemphigus vegetative, pemphigus vulgaris, psoriasis, scleroderma, systemic sclerosis, and vitiligo.

[0256] Skin diseases and conditions may include, for example, atopic dermatitis (eczema).

[0257] Atopic dermatitis (AD) or eczema

[0258] Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin condition triggered by barrier defects and exposure to allergens.

[0259] A defective skin barrier and exposure to allergens contribute to the onset and progression of the disease.

[0260] Atopic dermatitis (eczema) is a condition that causes redness and itching of the skin. It often develops in children but can occur at any age. It is long-lasting (chronic) and tends to flare up in cycles. It may also occur with asthma or hay fever.

[0261] Signs and symptoms of atopic dermatitis (eczema) vary from person to person and include: dry, itchy skin (which may be severe, especially at night); red to brownish-gray patches (especially on the hands, feet, ankles, wrists, neck, upper chest, eyelids, the bends of the elbows and knees, and on the face and scalp in infants); small, raised bumps that may ooze fluid and form crusts when scratched; thickened, cracked, scaly skin; and rough, sensitive, swollen skin from scratching.

[0262] Atopic dermatitis most often begins before age 5 and may continue into adolescence and adulthood. In some people, it flares up periodically and then disappears for a while, even years.

[0263] Pharmaceutical composition

[0264] We disclose pharmaceutical compositions comprising miR-335, an agent capable of upregulating the expression or activity of miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat.

[0265] While a composition comprising miR-335, an agent capable of upregulating miR-335 expression or activity, a histone deacetylase (HDAC) inhibitor, or belinostat can be administered alone, it is preferred that the active ingredients be formulated as a pharmaceutical preparation.

[0266] The pharmaceutical formulations disclosed herein comprise an effective amount of miR-335, an agent capable of upregulating the expression or activity of miR-335, a histone deacetylase (HDAC) inhibitor or belinostat, and one or more pharmaceutically acceptable carriers.

[0267] An "effective amount" of miR-335, an agent capable of upregulating the expression or activity of miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat is an amount sufficient to restore, maintain, or enhance the skin barrier function of a subject.

[0268] The effective amount will vary depending on the specific disease or syndrome to be treated or alleviated, as well as other factors, including the age and weight of the patient, the state of progression of the disease, etc., the patient's general health, the severity of the symptoms, and whether miR-335, an agent capable of upregulating the expression or activity of miR-335, a histone deacetylase (HDAC) inhibitor or belinostat is administered alone or in combination with other therapies.

[0269] Therefore, the term "treatment" includes conjoint therapy and therapy, wherein two or more treatments or the combination of therapy in sequence or combination simultaneously.For example, belinostat can also be used for conjoint therapy, for example, with other agents (such as dermatological agents etc.) in combination. The example of treatment and therapy includes but is not limited to chemotherapy (activating agent, including such as HDAC inhibitors, antibodies (for example, in immunotherapy), prodrugs (for example, in photodynamic therapy, GDEPT, ADEPT etc.) administration);Surgery;Radiotherapy;And gene therapy.

[0270] The miR-335, histone deacetylase (HDAC) inhibitor, or belinostat can be administered in any suitable amount. For example, a composition containing 10 μg or less, such as 5 μg or less, such as 2 μg or less, such as 1 μg or less, such as 0.5 μg or less, such as 0.3 μg of miR-335, histone deacetylase (HDAC) inhibitor, or belinostat can be administered to a subject.

[0271] The pharmaceutical composition may comprise 40 μg / ml or less, 20 μg / ml or less, 8 μg / ml or less, 4 μg / ml or less, 2 μg / ml or less, or 1.2 μg / ml or less of miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat.

[0272] The composition can be administered for any suitable length of time, e.g., at least 1 week to 12 weeks. The amount of miR-335, histone deacetylase (HDAC) inhibitor, or belinostat administered can include any suitable amount, e.g., from about 0.0001 mg to about 100 g per day.

[0273] An effective amount of the pharmaceutical composition described herein may include any amount effective to achieve its purpose. The effective amount, typically expressed in mg / kg, can be determined by those skilled in the art by routine methods during preclinical and clinical trials.

[0274] MiR-335, a histone deacetylase (HDAC) inhibitor, or belinostat can be administered to an animal in need thereof, such as a mammal. The animal can be any animal. Examples include laboratory animals, such as mice, rats, or guinea pigs; or primates, such as monkeys, orangutans, apes, chimpanzees, or humans. For example, the mammal can be a human.

[0275] Suitable pharmaceutically acceptable carriers are known in the art and vary with the desired form and mode of administration of the pharmaceutical preparation. For example, they can include diluents or excipients, such as fillers, adhesives, wetting agents, disintegrants, surfactants, lubricants, etc. Typically, the carrier is a solid carrier, a liquid carrier, or an evaporable carrier, or a combination thereof. Each carrier should be "acceptable," i.e., compatible with the other ingredients in the preparation and harmless to the patient. When administered to a host, the carrier should be biologically acceptable and should not cause adverse reactions (such as immune responses).

[0276] The pharmaceutical compositions include topical formulations, which are preferred in cases where the affected tissue is primarily the skin or epidermis (e.g., epidermal diseases such as atopic dermatitis, etc.). Topical formulations include those compositions in which the composition is applied externally by direct contact with the surface of the skin to be treated. Conventional pharmaceutical forms for topical application include soaks, ointments, creams, lotions, pastes, gels, sticks, sprays, aerosols, bath oils, solutions, and the like. Topical treatments are carried out using a variety of carriers, the choice of which can be important and is generally dependent on whether the condition is acute or chronic.

[0277] Lotions (powder suspended in water) and solutions (drug dissolved in a solvent) are ideal for hairy and intertriginous areas. Ointments or water-in-oil emulsions are the most effective moisturizers for dry, scaly rashes, but they are greasy and sometimes unsuitable depending on the location of the lesion.

[0278] When appropriate, they can be used in combination with bandage, particularly when needing to increase miR-335, can raise the expression of miR-33 or active agent, histone deacetylase (HDAC) inhibitor or belinostat compositions to the penetration of focus (lesion). Cream or oil-in-water emulsion and gel are absorbable, and are the most acceptable (Guzzo et al, in Goodman&Gilman's Pharmacological Basis of Therapeutics, 9thEd., p.1593-15950(1996)) to patients in terms of beauty. Cream preparations generally include components such as petroleum, lanolin, polyethylene glycol, mineral oil, glycerol, isopropyl palmitate, glyceryl stearate, cetearyl alcohol, tocopheryl acetate, isopropyl myristate, lanolin alcohol, dimethicone, carbomen, methylchloroisothiazolinone, methylisothiazolinone, cyclomethicone and hydroxypropyl methylcellulose, and their mixtures.

[0279] Other formulations for topical administration include shampoos, soaps, shakes, and the like, particularly those formulated to leave a residue on the underlying skin, such as the scalp (Arndt et al, in Dermatology In General Medicine 2:2838 (1993)).

[0280] Typically, the concentration of the composition of the miR-335, the expression or active agent that can raise miR-335, histone deacetylase (HDAC) inhibitor or belinostat in topical preparations is about 0.5% to 50% based on described composition weight, preferably about 1% to 30%, more preferably about 2% to 20%, most preferably about 5% to 10%. The concentration initially used can be in the top of this scope, along with the continuation for the treatment of, can reduce concentration or reduce the frequency of application of preparation. Topical application is typically applied twice a day. However, the application of a larger dose once a day or more frequently a smaller dose can be effective. The stratum corneum can serve as a reservoir, and allows medicine to gradually penetrate into the living skin layer over the time period of prolonged.

[0281] In topical application, a sufficient amount of the miR-335, an agent capable of upregulating the expression or activity of miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat must penetrate the patient's skin to obtain the desired pharmacological effect. It is generally believed that the absorption of drugs by the skin depends on the drug properties, carrier characteristics, and skin. Three major variables lead to the absorption rate or flux of different topical drugs or the same drug in different carriers: the difference in drug concentration in the carrier, the drug partition coefficient between the stratum corneum and the carrier, and the diffusion coefficient of the drug in the stratum corneum. In order to effectively treat, the drug must pass through the stratum corneum responsible for the skin barrier function. Generally, topical preparations with high in vitro skin permeability are effective in vivo. Ostrenga et al. (J. Pharm. Sci., 60: 1175-1179 (1971)) demonstrated that the in vivo efficacy of topical steroids is directly proportional to the permeability of steroids into dermatomed human skin from in vitro.

[0282] Skin penetration enhancers that are dermatologically acceptable and compatible with miR-335, agents that can up-regulate the expression or activity of miR-335, histone deacetylase (HDAC) inhibitors, or belinostat can be incorporated into formulations to increase the permeability of the active compound from the skin surface to epidermal keratinocytes. Skin enhancers that increase the absorption of the active compound by the skin can reduce the amount of miR-335, agents that can up-regulate the expression or activity of miR-335, histone deacetylase (HDAC) inhibitors, or belinostat necessary for effective treatment, and provide a more lasting effect for the formulation. Skin penetration enhancers are known in the art. Known surfactants include, for example, dimethyl sulfoxide (U.S. Pat. No. 3,711,602); oleic acid, 1,2-butanediol (Cooper, J. Pharm. Sci., 73: 1153-1156 (1984)); a combination of ethanol and oleic acid or oleyl alcohol (EP 267,617), 2-ethyl-1,3-hexanediol (WO 87 / 03490); decylmethyl sulfoxide and Azone.RTM. (Hadgraft, Eur. J. Drug. Metab. Pharmacokinet, 21: 165-173 (1996)); alcohols, sulfoxides, fatty acids, esters, Azone.RTM., pyrrolidone, urea, and polyols (Kalbitz et al. al, Pharmazie, 51:619-637 (1996)); terpenes, such as 1,8-cineole, menthone, limonene and nerolidol (Yamane, J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone.RTM. and diethylene glycol monoethyl ether (Transcutol) (Harrison et al, Pharmaceutical Res. 13:542-546 (1996)); and oleic acid, polyethylene glycol and propylene glycol (Singh et al, Pharmazie, 51:741-744 (1996)) can improve the skin penetration of active ingredients.

[0283] The permeation level of miR-335, an agent capable of upregulating the expression or activity of miR-335, a histone deacetylase (HDAC) inhibitor, or a belinostat composition can be determined by techniques known to those skilled in the art. For example, radiolabeling an active compound and subsequently measuring the amount of the radiolabeled compound absorbed by the skin enables one skilled in the art to determine the level of the composition absorbed using any of several methods for determining the skin permeability of a test compound. Publications related to skin permeation studies include Reinfenrath, WG and G SHawkins. The Weanling Yorkshire Pig as an Animal Model for Measuring Percutaneous Penetration. In: Swine in Biomedical Research (M E Tumbleson, Ed.) Plenum, New York, 1986, and Hawkins, GS Methodology for the Execution of In Vitro Skin Penetration Determinations. In: Methods for Skin Absorption, BW Kemppainen and WG Reifenrath, Eds., CRC Press, Boca Raton, 1990, pp. 67-80; and WG Reifenrath, Cosmetics & Toiletries, 110: 3-9 (1995).

[0284] For some applications, preferably, long-acting form of miR-335, expression or active agent of miR-335 can be raised using preparations known in the art (e.g., polymer), histone deacetylase (HDAC) inhibitor or belinostat compositions can be applied. According to methods known in the art, miR-335, expression or active agent of miR-335 can be raised, histone deacetylase (HDAC) inhibitor or belinostat can be incorporated into skin patch (Junginger, HE, in Acta Pharmaceutica Nordica 4:117 (1992); Thacharodi et al, in Biomaterials 16:145-148 (1995); Niedner R., in Hautarzt 39:761-766 (1988)) or bandage to improve the efficiency of drug delivery to the area to be treated.

[0285] Optionally, the topical formulations described herein may contain additional excipients: for example, preservatives (such as methylparaben, benzyl alcohol, sorbic acid or quaternary ammonium compounds), stabilizers (EDTA), antioxidants (such as butylated hydroxytoluene or butylated hydroxyanisole), buffers (such as citrates and phosphates).

[0286] Other therapeutic agents suitable for use in the present invention are any compatible drugs that are effective for the intended purpose, or drugs that complement the retinol formulations. For example, treatment with the formulations described herein can be combined with other therapies, such as topical treatments with corticosteroids, calcipotrine, coal tar formulations, and systemic treatments with methotrexate, retinoids, cyclosporine A, and photochemotherapy. Combination therapy is particularly important for treating acute or severe skin diseases. The formulations used in combination therapy can be administered simultaneously with the other treatments or sequentially to achieve a combined effect.

[0287] The modified-release dosage form of miR-335, histone deacetylase (HDAC) inhibitor or belinostat can also be used.This is contrary to the quick-release dosage form of releasing medicine immediately or in short time after using.In the modified-release dosage form, described miR-335, histone deacetylase (HDAC) inhibitor or belinostat are to delay sending after using described miR-335, histone deacetylase (HDAC) inhibitor or belinostat, or send in the long time.

[0288] Modified-release dosage forms include slow-release (ER, XR, or XL) formulations. They also include sustained-release (SR) formulations that release the miR-335, histone deacetylase (HDAC) inhibitor, or belinostat at a predetermined rate, thereby maintaining drug concentrations over a period of time.

[0289] Other formulations may include controlled delivery (CD), controlled release (CR), delayed release (DR), extended release (ER), immediate release (IR), long-acting (LA), prolonged release (LAR), modified release (MR), extended release (PR), sustained action (SA), sustained release (SR), timed release (TR), slow release (XL), slow release (XR), and extended release (XT) formulations.

[0290] Such formulations are known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Edn, 1995, Mack Publishing Co, Pennsylvania, USA.

[0291] Application

[0292] The miR-335, histone deacetylase (HDAC) inhibitor, or belinostat composition can be applied to the skin using any suitable treatment regimen.

[0293] The composition can be administered in a single dose or multiple doses. A single dose can be administered once a day or multiple times a day, or multiple times a week, or once a month or multiple times a month. The composition can be administered in a series of doses. The series of doses can be administered once a day or multiple times a day, once a week or multiple times a week, or once a month or multiple times a month. Thus, those skilled in the art will recognize that, depending on the subject's skin type, location, health status, etc., the compositions described herein can be administered over any given period of time until treatment, prevention, or alleviation of the dermatological condition is at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any range therebetween.

[0294] The miR-335, histone deacetylase (HDAC) inhibitor, or belinostat composition can be administered at least once a week, for example, at least once every two days, or at least once a day. For example, it can be administered twice a day.

[0295] Typically, the treatment using miR-335 as herein described, histone deacetylase (HDAC) inhibitors or belinostat compositions can continue indefinitely. Alternatively, repeated treatment can be performed only within a limited time period, such as several weeks or months. Repeated treatment can then be performed within a similar time period later.

[0296] After application to the skin, the composition can be rinsed off or left on the skin. If the composition is to be rinsed off after application, the composition can be left on for at least a period of time before rinsing. An exemplary period of time is greater than 30 seconds, such as greater than 1 minute, for example greater than 3 minutes.

[0297] During application, the product may be massaged into the skin, most commonly into the scalp, for example for at least 5 seconds, such as for at least 20 seconds.

[0298] Reagent test kit

[0299] We also describe a kit comprising (a) miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat (or a salt, hydrate, or solvate thereof), or a composition comprising miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat (or a salt, hydrate, or solvate thereof), e.g., preferably in a suitable container and / or provided in suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition according to the methods described herein for the purposes described.

[0300] The written instructions may also include a list of indications that the active ingredient is suitable for treating.

[0301] Detection and diagnostic methods

[0302] Detection of miR-335 expression

[0303] In the Examples, we show that miR-335 expression is downregulated in atopic dermatitis compared to normal skin.

[0304] We show that in healthy skin, proliferating cells in the epidermis express low levels of miR-335 in the basal layer, while high levels of miR-335 are expressed in the suprabasal layer. We also show that in AD lesional skin, miR-335 expression is lost in all layers, including the basal and suprabasal layers.

[0305] We therefore provide a method for diagnosing a dermatological condition such as atopic dermatitis in cells or tissues of an individual.

[0306] Detection of miR-335 expression, activity, or amount can be used to provide a method for determining a pathological state of a cell. The cell may comprise a suprabasal cell of the epidermis. Thus, a pathological cell is a suprabasal cell having a low level of miR-335 expression, activity, or amount compared to a normal suprabasal cell of the epidermis.

[0307] Such detection of miR-335 expression in, for example, suprabasal cells can also be used to determine whether a cell is likely to be diseased. Thus, detecting low levels of miR-335 expression, amount, or activity in a cell can indicate that the cell is likely to be diseased or to be diseased. Similarly, if a cell has normal or high levels of miR-335 expression, amount, or activity, the cell is not diseased or is unlikely to be diseased.

[0308] Detecting miR-335 expression, amount, or level, for example, in suprabasal cells, can be used to determine the likelihood that a particular therapy will be successful in an individual suffering from a dermatological condition such as atopic dermatitis.

[0309] The diagnostic methods described herein can be combined with the therapeutic methods described herein. Thus, we provide a method for treating, preventing, or ameliorating a skin condition such as atopic dermatitis in an individual, the method comprising detecting modulation of miR-335 expression, amount, or activity in cells of the individual, and administering appropriate treatment to the individual based on the expression, amount, or activity level.

[0310] As described in further detail below, the presence and amount of miR-335 nucleic acid can be detected in a sample. Thus, miR-335-associated diseases, including skin conditions such as atopic dermatitis, can be diagnosed by a method comprising determining abnormally decreased or increased expression, amount, or activity of a miR-335 nucleic acid, such as decreased expression, amount, or activity, in a sample from a subject.

[0311] The sample may include a cell or tissue sample from an organism or individual having or suspected of having a disease associated with reduced, decreased, or abnormal expression, amount, or activity of miR-335 (including spatial or temporal variations in the level or pattern of expression, amount, or activity). The sample may include epidermal basal cells or suprabasal cells. The level or pattern of expression, amount, or activity of miR-335 in an organism having or suspected of having such a disease can be effectively compared to the expression level or pattern, amount, or activity in a normal organism as a diagnostic method for the disease.

[0312] The sample may include a cell or tissue sample, such as a skin tissue sample or a cell sample, from an individual having or suspected of having a skin condition such as atopic dermatitis. The cell or tissue may include epidermal basal cells or suprabasal cells.

[0313] In some embodiments, a reduced level of expression, amount, or activity of miR-335 is detected in a sample. The level of miR-335 may be significantly reduced compared to normal cells or cells from an individual known not to have a skin condition such as atopic dermatitis. Such cells can be obtained from the individual being tested or from another individual, such as an individual matched to the individual being tested in terms of age, weight, lifestyle, etc.

[0314] In some embodiments, the level of expression, amount or activity of miR-335 is reduced by 10%, 20%, 30% or 40% or more. In some embodiments, the level of expression, amount or activity of miR-335 is reduced by 45% or more, such as 50% or more.

[0315] The expression, amount or activity of miR-335 can be detected by a variety of means as known in the art and as described in further detail below. Typically, the amount of miR-335 in a tissue sample from an individual is measured and compared to a sample from an unaffected individual.

[0316] Detection of the amount, activity, or expression of miR-335 can be used to grade skin conditions such as atopic dermatitis. For example, low levels of miR-335 amount, activity, or expression can indicate a more severe skin condition such as atopic dermatitis. Similarly, high levels of miR-335 amount, activity, or expression can indicate a milder or less severe skin condition such as atopic dermatitis. Such a grading system can be used in conjunction with established grading systems for skin conditions such as atopic dermatitis.

[0317] The expression level of miR-335 can be determined using a variety of different techniques.

[0318] Measurement of miR-335 expression at the RNA level

[0319] miR-335 gene expression can be detected at the RNA level.

[0320] Thus, in one embodiment, we disclose a method for detecting the presence of miR-335 nucleic acid in a sample by contacting the sample with at least one nucleic acid probe specific for miR-335 and monitoring the presence of miR-335 in the sample. For example, the nucleic acid probe can specifically bind to miR-335 or a portion thereof, and the binding between the two can be detected; the presence of the complex itself can also be detected.

[0321] Thus, in one embodiment, the amount of miR-335 in a sample can be measured. miR-335 can be assayed by in situ hybridization, Northern blotting, and reverse transcriptase-polymerase chain reaction. Nucleic acid sequences can be identified by in situ hybridization, Southern blotting, single-stranded conformational polymorphism, PCR amplification using specific primers, and DNA chip analysis (Kawasaki, 1990; Sambrook, 1992; Lichter et al, 1990; Orita et al, 1989; Fodor et al, 1993; Pease et al, 1994).

[0322] MiR-335 RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis) or the RNeasy RNA preparation kit (Qiagen). Typical assay formats utilizing RNA hybridization include nuclear run-on assays, in situ hybridization, RT-PCR, and RNase protection assays (Melton et al. Nuc. Acids Res. 12:7035). Detection methods that can be used include radiolabeling, enzyme labeling, chemiluminescent labeling, fluorescent labeling, and other suitable labels.

[0323] Each of these methods allows for quantitative determination and is known in the art. An example of a detection method suitable for compositions and methods as described herein is in situ hybridization. The method and detailed protocol for detecting the in situ hybridization of microRNA are known in the art and are described in, for example, Nielsen (2012) MicroRNA In Situ Hybridization, pages 67-84 in Jian-Bing Fan (ed.), Next-Generation MicroRNA Expression Profiling Technology: Methods and Protocols, Methods in Molecular Biology, vol. 822 and Urbanek et al (2015) Small RNA Detection by in Situ Hybridization Methods, Int. J. Mol. Sci. 16, 13259-13286.

[0324] Thus, any method known in the art for polynucleotide quantification can be used to measure the reduction or increase in expression, amount or activity of miR-335 at the RNA level. Any suitable probe from the miR-335 sequence, such as any portion of a suitable human miR-335 sequence, can be used as a probe.

[0325] Typically, RT-PCR is used to amplify RNA targets. In this process, an enzyme called reverse transcriptase is used to convert RNA into complementary DNA (cDNA), which can then be amplified for detection.

[0326] Many methods of DNA amplification are known, most of which rely on enzymatic chain reactions (such as the polymerase chain reaction, the ligase chain reaction, or self-sustained sequence replication systems), or on replication of all or part of a cloned vector.

[0327] Many target and signal amplification methods have been described in the literature, for example, as reviewed in Landegren, U. et al. Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990).

[0328] For example, polymerase chain reaction can be used to detect miR-335.

[0329] "Polymerase chain reaction" or "PCR" is a nucleic acid amplification method, described in particular in U.S. Patents 4,683,195 and 4,683,202. PCR can be used to amplify any known nucleic acid in diagnostic settings (Mok et al., 1994, Gynaecologic Oncology 52:247-252). The self-sustaining sequence replication system (3SR) is a variant of TAS that involves isothermal amplification of a nucleic acid template by successive cycles of reverse transcriptase (RT), polymerase, and nuclease activity mediated by an enzyme mixture and appropriate oligonucleotide primers (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874). The ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two for each target strand. This technique is described in Wu, DY and Wallace, RB, 1989, Genomics 4:560. In the Qβ replicase technique, RNA replicase from bacteriophage Qβ, which replicates single-stranded RNA, is used to amplify target DNA as described in Lizardi et al., 1988, Bio / Technology 6:1197.

[0330] The PCR procedure mainly includes: (1) treating the extracted DNA to form a single-stranded complementary chain; (2) adding a pair of oligonucleotide primers, wherein one primer in the primer pair is substantially complementary to a portion of the sequence in the sense strand, and the other primer in each pair is substantially complementary to a different portion of the same sequence in the complementary antisense strand; (3) annealing the paired primers to the complementary sequence; (4) simultaneously extending the annealed primers from the 3' end of each primer to synthesize an extension product complementary to the strand annealed to each primer, wherein the extension product after separation from the complementary sequence serves as a template for synthesizing the extension product of the other primer of each pair; (5) separating the extension product from the template to produce a single-stranded molecule; and (6) amplifying the single-stranded molecule by repeating the annealing, extension and separation steps at least once.

[0331] Reverse transcription polymerase chain reaction (RT-PCR) can be used. Quantitative RT-PCR can also be used. Such PCR techniques are known in the art and any suitable primers from the miR-335 sequence can be used.

[0332] Alternative amplification techniques can also be utilized. For example, rolling circle amplification (Lizardi et al. 1998, Nat Genet 19:225) is a commercially available amplification technology (RCAT™) that is driven by DNA polymerase and can replicate circular oligonucleotide probes under isothermal conditions using linear or geometric kinetics. Another technique, strand displacement amplification (SDA; Walker et al., 1992, Proc. Natl. Acad. Sci. USA 80:392), initiates with a specific sequence unique to a specific target.

[0333] Detection of miR-335-induced polypeptide expression

[0334] As shown in the Examples, miR-335 inhibits the expression of SOX6.

[0335] Therefore, the expression of SOX6 can be used as a surrogate to detect the expression of miR-335 at the nucleic acid level or the polypeptide level.

[0336] diagnostic kits

[0337] We also provide diagnostic kits for detecting a dermatological condition such as atopic dermatitis in an individual, or detecting a susceptibility of an individual to a dermatological condition such as atopic dermatitis.

[0338] The diagnostic kit may include means for detecting the expression, amount, or activity of miR-335 in an individual by any of the methods described herein. Thus, the diagnostic kit may include any one or more of the following: miR-335 or a fragment thereof; or a complementary nucleotide sequence of a miR-335 nucleic acid or a fragment thereof.

[0339] The diagnostic kit may include instructions for use or other markings. The diagnostic kit may also include a tool for treating or preventing a dermatological condition such as atopic dermatitis, such as any composition described herein, or any tool known in the art for treating a dermatological condition such as atopic dermatitis. The diagnostic kit may include a therapeutic composition comprising miR-335, a histone deacetylase (HDAC) inhibitor, or belinostat.

[0340] Example

[0341] Example 0. Overview

[0342] Here, we investigated microRNAs that are critical for maintaining skin barrier function and identified miR-335 as a key driver of keratinocyte differentiation and cornification.

[0343] In silico prediction, followed by experimental validation, identified the transcription factor SOX6 as a direct target of miR-335 repression. This regulatory relationship, which promotes epidermal differentiation, is disrupted in AD.

[0344] In healthy epidermis, miR-335 is abundantly expressed, whereas SOX6 is absent; in contrast, in patient skin biopsies with lesions, miR-335 loss is observed to coincide with aberrant SOX6 upregulation. SOX6 inhibits epidermal differentiation by recruiting components of the SMARCA complex, which epigenetically silence key genes involved in keratinocyte differentiation. The resulting skin barrier defect can be therapeutically reversed by restoring miR-335 expression. miR-335 is epigenetically regulated by histone deacetylases (HDACs), and screening for suitable HDAC inhibitors identified belinostat as a drug candidate that can restore epidermal miR-335 expression. This has clinical significance not only as a treatment for AD but also as a potential means of preventing the progression and further development of atopy in this systemic allergic disease.

[0345] We identified a microRNA (miR-335) as a key driver of keratinocyte differentiation and cornification, which are required for establishing a healthy skin barrier.

[0346] However, miR-335 expression was significantly downregulated in AD lesional skin. In the absence of miR-335, we observed sustained expression of its downstream targets SOX6 and CASP7.

[0347] To elucidate this mechanism, we demonstrate how SOX6-mediated recruitment of the SMARCA complex leads to epigenetic silencing of genes critical for epithelial differentiation, thereby causing barrier defects.

[0348] Furthermore, in the absence of miR-335, sustained expression of pro-inflammatory CASP7 promoted increased expression of inflammatory cytokines, thereby forming an inflammatory microenvironment.

[0349] Thus, miR-335 drives keratinocyte differentiation and concurrently plays a role in the resolution of inflammation.

[0350] Understanding how these are dysregulated in atopic dermatitis, we aim to restore miR-335, repair barrier defects and suppress inflammation.

[0351] Our data also indicate that miR-335 biogenesis is epigenetically regulated through histone deacetylases (HDACs).

[0352] Example 1. Materials and Methods: Cell Culture and Transfection

[0353] N / TERT-1 keratinocytes (a gift from James Rheinwald) were grown in serum-free keratinocyte medium (KSFM) (Life Technologies) supplemented with 0.2 ng / mL epidermal growth factor (EGF), 25 μg / mL bovine pituitary extract (BPE), 0.4 mM calcium chloride (CaCl2), and 1% penicillin / streptomycin. When experiments required high-density culture, N / TERT-1 cells were grown in DF-K medium (Dulcerative Colitis Modified Eagle Medium; Gibco) without glutamine and in KSFM supplemented with 0.2 ng / mL EGF, 25 μg / mL BPE, 2 mM L-glutamine, 0.15 mM CaCl2, and 1% penicillin / streptomycin. HEK293T cells (Clontech) were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco), 4 mM L-glutamine, and 1 mM sodium pyruvate. Cultures were maintained at 37°C in a humidified incubator containing 5% CO2.

[0354] Before transfection, N / TERT-1 keratinocytes were cultured in 6-well plates to 30% to 40% confluency. For miR-335 overexpression studies, the cells were cultured using the manufacturer's protocol. RNAiMAX reagent (Life Technologies) was used to transfect N / TERT-1 cells with miR-335 mimics or negative control mimics (Dharmacon) at a final concentration of 30 nM. For knockdown studies, short interfering RNA (siRNA) (Dharmacon) targeting SOX6, CASP7, MEST, HELLS, SMARCA4, PNN, or SMARCC1 was transfected into N / TERT-1 cells in the same manner as the miRNA transfection. Non-targeting siRNA was transfected as a negative control. Total RNA and / or protein were collected 48 hours after transfection and subjected to quantitative real-time PCR and / or Western blotting.

[0355] Example 2. Materials and Methods: Preparation of Lentiviral Stock and Transduction

[0356] Third generation lentiviral particles were produced in HEK293T cells (Clontech) transfected with calcium phosphate using a lentiviral vector and a packaging mixture consisting of three constructs, namely pMDLg / pRRE (#12251, Addgene), pRSV-Rev (#12253, Addgene), and pMD2.G (#12259, Addgene). Control viruses were prepared using an empty vector construct. For the pTRIPZ plasmid, a trans-lentiviral packaging mixture (Dharmacon) was co-transfected into HEK293T cells using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer's instructions. Supernatants containing viral particles were collected 48 and 72 hours after transfection. The viral supernatant was filtered through a 0.22 μM membrane to remove non-adherent cells and debris, and then concentrated by ultracentrifugation at 19,600 rpm for 4 hours at 4°C. The viral particles were then resuspended in Hank's balanced salt solution (Sigma-Aldrich). To determine the titer of each virus, viral titration was performed in HEK293T cells after transducing HEK293T cells with serial dilutions of the lentiviral stock solution according to the protocol described by Tiscornia et al. (2006). Viral titer was calculated based on the percentage of GFP-positive cells shown by flow cytometry analysis. Lentiviral transduction was performed by incubating 10 μg / mL of polybrene and purified lentiviral particles with N / TERT-1 cells at an infection rate of 4 transduction units (TU) per cell. Stably transduced cells were selected using GFP sorting (for pCDH constructs) or puromycin treatment for 5 days (for control constructs).

[0357] Example 3. Materials and Methods: miRNA Profiling

[0358] use Total RNA was extracted from skin biopsy tissue using reagents and then purified using an Exiqon miRCURY RNA column. Total RNA was labeled using the miRCURY LNATM microRNA Hi-Power Labeling Kit (Exiqon) according to the manufacturer's protocol. Labeled samples were hybridized on the miRCURY LNATM microRNA array (6th generation) with probes targeting 1223 known human mature miRNAs. Background was subtracted from the raw intensity of all samples, standardized using a global local weighted scatter plot smoothing (lowess) regression method, and log2 transformed. Differential miRNA expression analysis was performed using Partek Genomics Suite software.

[0359] Example 4. Materials and Methods: Microarray Analysis

[0360] Total RNA was converted into biotinylated cRNA using the TargetAmp Nano-g Biotin-aRNA Labeling Kit (Epicenter). cRNA was purified using the RNeasy Mini Kit (Qiagen). cRNA was hybridized on the HumanHT-12V4 Expression BeadChip Kit (Illumina) according to the manufacturer's instructions. Raw data were extracted using Illumina BeadStudio, background subtracted, and standardized. Differential gene expression analysis was performed using Partek Genomics Suite software.

[0361] Example 5. Materials and Methods: miRNA in situ hybridization

[0362] Paraffin-embedded skin tissue sections (5 μm) were dewaxed, rehydrated, and boiled in pretreatment solution (Panomics) for 5 minutes, followed by protease treatment (Panomics) at 37°C for 30 minutes. Locked nucleic acid (LNA) probes were then added to the sections and incubated at 51°C for 4 hours. The LNA probes used were specific for miR-335 and a scrambled (non-targeting) sequence and were labeled with 5' and 3' digoxigenin (DIG) markers. After sequential washing with 5x sodium citrate (SSC), 1xSSC, and 0.3xSSC buffer, the sections were blocked with 10% goat serum and incubated with anti-DIG alkaline phosphatase (Roche) overnight at 4°C. LNA probes bound to miRNAs were detected using Fast Red Substrate (Panomics). Tissue sections were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich) and mounted with FluorSave™ reagent (EMD Millipore). Slides were examined using an FV1000 inverted confocal microscope (Olympus), and images were acquired using an Olympus FluoView with TRITC and DAPI filters.

[0363] Example 6. Materials and Methods: Immunohistochemistry

[0364] Paraffin-embedded skin tissue sections (5 μm) were dewaxed and rehydrated by reducing the ethanol concentration. Endogenous peroxidase in the tissue sections was quenched with a 3% H2O2 solution in anhydrous methanol for 30 minutes. The sections were then boiled in antigen retrieval solution (pH 6, DAKO). The sections were then blocked in 10% goat serum for 30 minutes and incubated with the primary antibody at 4°C overnight. After incubation with the primary antibody, the tissue was incubated with anti-rabbit- or anti-mouse-Envision labeled polymer reagent (DAKO) at room temperature for 1 hour. The staining was visualized by a 3,3'-diaminobenzidine (DAB) substrate development kit (DAKO). The slides were counterstained with hematoxylin and examined using a Zeiss AxioImager Z1 upright light microscope after mounting with CytosealTM60 mounting medium (Richard Allan Scientific). Images were acquired using Zeiss Zen software.

[0365] Example 7. Materials and Methods: Immunocytochemistry

[0366] The cells were fixed and permeabilized with ice-cold acetone / methanol for 10 minutes. After washing in phosphate buffered saline (PBS), the cells were incubated with 10% goat serum for 30 minutes. Subsequently, the cells were incubated with a primary antibody at room temperature for 2 hours or at 4 ° C overnight. After washing 3 times with PBS containing 0.1% Tween 20, the cells were incubated with Alexa Fluor secondary antibodies (Invitrogen) in the dark for 1 hour. DAPI was used to stain the nucleus. After washing with PBS, the cells were fixed on a slide with FluorSave reagent (EMD Millipore). Images were taken using an Olympus FluoView inverted confocal microscope (Olympus) using an FV1000.

[0367] Example 8. Materials and Methods: Quantitative Real-Time PCR

[0368] Total RNA was isolated using the miRCURY™ RNA Isolation Kit (Exiqon). For miRNA quantification, the cDNA was synthesized using a miRNA reverse transcription kit (Life Technologies), and miRNA expression levels were quantified using TaqMan gene expression assays (Applied Biosystems) in a 7900 Fast RT-PCR system (Applied Biosystems) using TaqMan miRNA-specific primers. For mRNA expression, the mRNA expression was determined using the TaqMan gene expression assays (Applied Biosystems) according to the manufacturer's protocol. cDNA was synthesized using Sigma-Aldrich III reverse transcriptase (Life Technologies). SYBR green PCR Master Mix (Applied Biosystems) was used to measure mRNA expression levels. U6 and RPLP0 were used as endogenous normalization controls for miRNA and protein-coding genes, respectively. Gene / miRNA expression was defined from the threshold cycle (Ct), and relative transcript abundance was calculated using the ddCT method. All reactions were repeated three times. Primers were designed using Primer3 software and the NCBI primer design tool. Primers were checked for GC content and any secondary structure formation using OligoCalc.

[0369] Example 9. Materials and Methods: Western Blot

[0370] Whole-cell lysates were extracted using RIPA buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1% Nonidet P-40 (NP40), 1% sodium deoxycholate, 0.05% sodium dodecyl sulfate (SDS), and 10% glycerol) supplemented with protease inhibitors (Calbiochem). Protein sample concentrations were measured using the Bradford protein assay (Bio-Rad). 20 to 30 μg of protein sample was resuspended in 2× Laemmli buffer, separated by SDS-PAGE on 4-15% TGX™ precast protein gels (Biorad), and then electrophoretically transferred to a PVDF membrane (Millipore). After incubation with 5% nonfat milk in Tris-buffered saline containing 1% Tween (TBST) for 30 minutes, the membrane was incubated with the primary antibody overnight at 4°C. The membrane was washed three times for 15 minutes each and incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies. The blots were washed three times with TBST and developed by autoradiography using ECL western detection reagent (Millipore Crescendo). β-actin was used as a protein loading control.

[0371] Example 10. Materials and Methods: Dual-Luciferase Reporter Gene Assay

[0372] The wild-type 3'UTR reporter constructs of SOX6 and CASP7 were co-transfected with pCDH plasmid or pCDH-335 plasmid into HEK293T cells using Lipofectamine 2000 (Thermo Scientific). 24 hours after transfection, firefly and Renilla luminescence were detected using a dual-luciferase reporter assay (Promega).

[0373] Example 11. Materials and Methods: Chromatin Immunoprecipitation

[0374] The cells were cross-linked at 37°C for 10 minutes with 1.5% formaldehyde. The reaction was terminated by adding glycine to a final concentration of 125mM at 37°C for 5 minutes. The fixed cells were washed twice with ice-cold PBS, scraped and centrifuged. The precipitate was then incubated on ice for 30 minutes with buffer A (10mM HEPES Ph7.9, 10mM KCl, 0.1mM EGTA, 1mM DTT and 0.5mM PMSF) supplemented with 1x protease inhibitor cocktail (Roche) and 10% glycerol, and then incubated with 0.5% NP-40 for 5 minutes to lyse the cytoplasmic membrane. The cells were then centrifuged at 1500x g for 5 minutes to precipitate the nuclei. The nuclear precipitate was then lysed in ChIP buffer (50mMTris pH 7.4, 150mM NaCl, 5mM EDTA, 0.5% NP-40, 1% Triton X-100, 0.05% SDS, 1x protease inhibitor cocktail). By Nuclear chromatin was sheared by sonication in a waterbath sonicator for 15 minutes (30 seconds on and 30 seconds off). The sonicated samples were centrifuged and the supernatant containing the sheared chromatin was collected. For each ChIP reaction, 250 μl of chromatin extract was incubated with the appropriate antibody overnight at 4°C on a rotating wheel. 50 μl of protein A agarose resin (pre-coated with 0.5% BSA and 0.2 mg / mL tRNA) was then added to the immunoprecipitated sample and incubated for 2 hours. The sample was then washed five times with ChIP buffer and finally with PBS. 100 μl of 10% Chelex slurry was added to each reaction and boiled for 5 minutes. The supernatant was collected and eluted a second time with 100 μl of nuclease-free water. The sample was then treated with RNase A (Roche) at 37°C for 2 hours and then with proteinase K (Roche) at 55°C overnight. The eluted sample was purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's protocol. For total chromatin input, 25 μl of chromatin extract was washed with ice-cold 75% ethanol and spun at 16,000 x g at 4°C for 10 minutes. 100 μl of 10% Chelex slurry was added to each sample and boiled for 5 minutes. The eluted samples were then treated with RNase A and Proteinase K as described above. The resulting DNA was diluted 5-fold, and 4 μl of each sample was used for real-time PCR. Fold enrichment of genes was calculated using the ddCt method, with the input Ct value used as a normalization control.

[0375] Example 12. Materials and Methods: Co-immunoprecipitation

[0376] Dynabeads Protein A (Invitrogen) were prepared according to the manufacturer's protocol. Nuclear lysates were incubated with Dynabeads Protein A coated with appropriate antibodies at 4°C for 4 hours. After washing the beads five times in wash buffer (PBST) to remove nonspecific proteins, proteins bound to the beads were analyzed by mass spectrometry. To verify the co-immunoprecipitated proteins by Western blotting, the proteins were eluted in 2x Laemli buffer at 95°C for 5 minutes. The eluted proteins were subjected to Western blotting according to standard protocols.

[0377] Example 13. Materials and Methods: Mass Spectrometry

[0378] DynaBeads-bound proteins were digested on the beads according to the method of Duan et al. (2009). Digested peptides were analyzed on an Orbitrap Fusion Tribrid mass spectrometer coupled to a Proxeon EASY-nLC 1000 liquid chromatograph. Raw data were analyzed using Proteome Discoverer. Candidates exhibiting >2 unique peptides and >15 peptide spectrum matches (PSMs) were selected and validated for co-immunoprecipitation.

[0379] Example 14. Materials and Methods: Corneal Envelope Analysis

[0380] N / TERT-1 keratinocytes were grown in DF-K medium until confluent and then transferred to a high Ca 2+ Conditions (1.5 mM) were then cultured in fresh DF-K medium for 7 days to induce terminal differentiation and cornified envelope formation, and the number of cornified envelopes (CEs) was assessed according to the method of Rice et al. Briefly, cells were trypsinized and plated at 2.0 × 10 6 Cells were resuspended at 100 cells / ml in dissociation buffer (containing 0.1 M Tris-HCl buffer, pH 8.0, 2% SDS, and 20 mM dithiothreitol). CE was collected by boiling the sample at 100°C for 15 minutes. Detergent- and reducing agent-resistant CE was collected as an insoluble precipitate by centrifugation at 4000 x g for 10 minutes. CE was counted using a hemocytometer and expressed as a percentage of the input cell number.

[0381] Example 15. Materials and Methods: HDAC Inhibitor Screening

[0382] A panel of 42 HDAC inhibitors (HDACi), encompassing most type I, type II, and pan-HDACi, was purchased from Selleckchem. N / TERT-1 cells grown to confluency were treated with HDACi at a final concentration of 1 μM from a 100 μM stock solution. Control wells were treated with DMSO at a final concentration of 1%. After 48 hours of treatment, cells were either processed for total RNA isolation or fixed and stained for human involucrin by immunocytochemistry.

[0383] Example 16. Materials and Methods: Ex-vivo Human Skin Organ Culture Model

[0384] Human skin organ cultures were performed according to the method of Moll et al.

[27] . Briefly, abdominal skin discarded from the clinic was collected within 3 h after surgery. Excess fat was trimmed with scissors, and 8 mm circular explants were cut using a commercial biopsy punch. The explants were placed on 6-well membrane inserts (4 μM PET membrane) and cultured at the air-liquid interface. Belinostat and mocetinostat were dissolved in acetone (final concentration of 1 mM), and 10 μl of this mixture was applied directly to the epidermal side of the biopsy tissue and allowed to dry. Biopsies treated with 10 μl of acetone alone were used as mock treatments. Organ cultures were treated daily for six days. Thereafter, skin biopsies were either processed for total RNA isolation as described previously

[52] or fixed in 10% neutral buffered formalin and processed into FFPE sections.

[0385] Example 17. Materials and Methods: Bioinformatics Analysis Software

[0386] DAVID (https: / / david.ncifcrf.gov / ) was used to perform gene ontology analysis. TargetScan (http: / / www.targetscan.org / ), miRANDA (http: / / www.microrna.org), and PITA (https: / / genie.weizmann.ac.il / pubs / mir07 / mir07_data.html) were used for miRNA target gene prediction. ProteINSIDE (www.proteinside.org) was used to assess nuclear protein enrichment in mass spectrometry datasets.

[0387] Example 18. Materials and Methods: Statistical Analysis

[0388] All quantitative data are expressed as mean ± SD. Statistical analysis was performed using a two-sided Student's t-test when comparing two samples. A p-value < 0.05 was considered statistically significant.

[0389] Example 19. Results: Screening of miRNAs in AD identifies miR-335 as an epithelial differentiation factor

[0390] MicroRNA microarray analysis comparing AD lesions with normal healthy skin revealed multiple differentially expressed miRNAs ( Figure 7 A).

[0391] The microarray data were cross-checked using quantitative real-time PCR (qRT-PCR) of RNA isolated from 10 lesional skin samples and 7 healthy controls. We found that miR-335 was the most consistently differentially expressed miRNA across these samples—miR-335 was significantly downregulated in AD lesions relative to healthy skin (Student's t-test, P < 0.01; Figure 1 A).

[0392] To characterize the expression pattern of miR-335, we performed in situ hybridization on sections from AD lesional and healthy skin using a locked nucleic acid (LNA) probe highly specific for mature miR-335. Significant expression of miR-335 was evident in the epidermis of unaffected individuals, whereas, in contrast, little or no miR-335 was detected in sections from AD lesions. Figure 1 B).

[0393] Interestingly, high expression of miR-335 was restricted to the suprabasal (differentiated) layer of the epidermis, whereas in the basal (undifferentiated) layer of the epidermis of normal healthy skin, the miR-335 signal was much lower ( Figure 1 B) miR-335 expression only in committed differentiating epidermal cells suggests a role for miR-335 in differentiation and maintenance of epidermal homeostasis.

[0394] We evaluated the role of miR-335 in epidermal differentiation by performing microarray analysis on N / TERT-1 cells transfected with miR-335 mimics. N / TERT-1 is an immortalized human keratinocyte cell line

[17] that does not express miR-335 in its undifferentiated state ( Figure 7 B).

[0395] Total RNA from N / TERT-1 cells transfected with control or miR-335 mimics ( Figure 7C) Microarray analysis and gene expression profile comparison were performed. Gene set enrichment analysis revealed signatures of human keratinocyte differentiation that were enriched in N / TERT-1 cells transfected with miR-335 mimics. This included a subset of genes significantly enriched in GO terms related to cornification and peptide cross-linking ( Figure 1 C).

[0396] A set of genes essential for cornified envelope formation, including IVL, SPRR1A, SPRR1B, SPRR2E, SPRR2F, and TGM1, all genes in the epidermal differentiation complex on chromosome 1q, were significantly upregulated in N / TERT-1 cells expressing miR-335 compared with control cells. Figure 1 D).

[0397] The microarray results were further validated by qRT-PCR using biological replicates of this gene subset ( Figure 1 E) We performed cornified envelope analysis on N / TERT-1 cells transfected with control or miR-335 mimics to investigate the role of miR-335 in keratinocyte differentiation and cornification.

[0398] Corneal envelopes are a well-established feature of terminal keratinocyte differentiation.

[18] They are formed by transglutaminase-catalyzed cross-linking of keratinocyte differentiation proteins, including involucrin (IVL), small proline-rich protein (SPRR), and other proteins.

[0399] Phase contrast microscopy revealed an increase in the number of terminally differentiated cells, visible as cornified envelopes, in cells transfected with miR-335 mimics compared to control cells ( Figure 1 F and Figure 1 G), which supports our hypothesis that miR-335 is essential for keratinocyte terminal differentiation and barrier formation.

[0400] Example 20. Results: SOX6 is a direct target of miR-335

[0401] To further evaluate the molecular pathways used by miR-335 to induce keratinocyte differentiation and cornification, we used integrative genomic, bioinformatic, and experimental approaches to identify its targets.

[0402] We collated microarray expression data of genes significantly downregulated by miR-335 and overlapped these hits with miR-335 predicted target genes from available algorithms and screened out 30 genes, including the transcription factor SOX6.

[0403] As shown in the heat map, we identified SOX6 as a bona fide target of miR-335.

[0404] SOX6 was significantly downregulated in N / TERT expressing miR-335 compared to those transfected with scrambled control ( Figure 2 A), and has a miR-335 binding site in its 3'-UTR ( Figure 2 B).

[0405] To confirm that SOX6 is a direct target of miR-335, we cloned its 3'-UTR into a luciferase reporter construct. Co-transfection of the miR-335 mimic with the SOX6 wild-type 3'-UTR significantly reduced the activity of the luciferase reporter. In contrast, when the miR-335 binding site was mutated, we did not see a change in reporter activity, confirming that miR-335 binds directly to the target site ( Figure 2 C) Up.

[0406] A clear negative correlation in the expression patterns of miR-335 and SOX6 was observed in normal skin sections not affected by AD, with strong nuclear immunohistochemical staining of SOX6 in the basal layer of the epidermis showing little or no overlap with that of miR-335 ( Figure 2 D, upper panel). Nuclear staining of SOX6 is absent in the suprabasal layers of normal epidermis.

[0407] In AD lesional skin with loss of miR-335 expression, nuclear SOX6 was expressed throughout the epidermis ( Figure 2 D, lower panel). We also observed that SOX6 transcript and protein abundance were decreased in N / TERT-1 cells in response to transfection of miR-335 mimics ( Figure 3 A and Figure 3 B).

[0408] Example 21. Results: Transcriptomic profile of miR-335 inducing epidermal differentiation by targeting SOX6

[0409] Having identified SOX6 as a direct target of miR-335, we set out to evaluate its functional role in the epidermis.

[0410] Endogenous SOX6 was knocked down in the N / TERT-1 cell line using specific short hairpin RNA (shRNA). Figure 8 A and Figure 8 B). ShSOX6 knockdown significantly reduced cell proliferation relative to that observed in scrambled controls ( Figure 3 C and Figure 3 D).

[0411] We used genome-wide expression profiling of control and shSOX6 cells to elucidate the molecular mechanisms by which SOX6 regulates differentiation. This analysis revealed differential expression of a subset of genes significantly enriched in GO terms related to terminal differentiation (epidermal development, differentiation, keratinization, and peptide cross-linking). Figure 3 E).

[0412] The microarray results were validated by qRT-PCR using biological replicates of an overlapping subset of genes, including IVL, SPRR1A, SPRR1B, SPRR2E, SPRR2F, and TGM1 ( Figure 3 F) From these data, we conclude that SOX6 represses molecular pathways that drive epidermal differentiation. SOX6 knockdown in N / TERT-1 cells mimics the phenotypes observed with miR-335 transfection.

[0413] The transcriptomic response to SOX6 knockdown was reflected in the morphology of confluent shSOX6 keratinocytes, which exhibited more stratified cells upon SOX6 silencing, implying a higher differentiation propensity of these cells.

[0414] To investigate whether SOX6 knockdown could promote keratinization, we measured corneal envelope formation in keratinocytes transduced with shSOX6 and scrambled control. ShSOX6 cells produced more corneal envelopes compared to the control group, indicating that SOX6 inhibits differentiation ( Figure 3 G). Therefore, downregulation of SOX6 in the epidermis may be critical for skin maturation and the formation of a healthy barrier.

[0415] Given these findings that SOX6, a known transcription factor, can inhibit epidermal differentiation, we sought evidence that SOX6 directly binds to the promoters of differentiation-related genes. The promoters of these genes were screened for consensus SOX6-binding sequences

[19] .

[0416] We found SOX6 binding sites upstream of putative transcription start sites in the promoters of IVL (involucrin), SPRR2F (small proline-rich protein 2F), and TGM1 (transglutaminase-1).

[0417] Chromatin immunoprecipitation (ChIP) was used to investigate the occupancy of SOX6 at these sites; ChIP was performed on N / TERT-1 nuclear extracts using an antibody against SOX6. Quantitative PCR analysis of these samples revealed SOX6 enrichment at the promoters of all three genes ( Figure 3 H).

[0418] We confirmed the specificity of this interaction by showing that SOX6 is not enriched upstream of the control genes RPLP0 and KRT14, which are abundantly expressed in the epidermis ( Figure 3 H).

[0419] Together, these data support a model in which SOX6 represses transcription of a subset of genes critical for epidermal differentiation ( Figure 3 I).

[0420] Example 22. Results: SOX6 interacts with the SMARCA chromatin remodeling complex and stalls epidermal cell differentiation

[0421] SOX6 inhibition of keratinocyte differentiation may require additional interacting proteins; SOX6 itself lacks regulatory domains and is known to interact with other proteins to carry out its function

[20] .

[0422] To gain insight into the interacting partners of SOX6, we performed co-immunoprecipitation followed by mass spectrometry (MS). To this end, we generated a HEK293T cell line with doxycycline-inducible SOX6 expression fused to an N-terminal MYC tag. Nuclear lysates from doxycycline-treated and untreated cells were immunoprecipitated using rabbit IgG, anti-SOX6 antibody, and anti-MYC antibody ( Figure 9 A) Immunoprecipitated proteins were subsequently subjected to MS analysis to identify potential SOX6 interacting partners.

[0423] After filtering out nonspecific interactors, a total of 26 putative SOX6-interacting proteins involved in transcriptional regulation and chromatin remodeling were identified ( Figure 9 B). Several subunits of the human switch / sucrose non-fermentable (SWI / SNF) type ATP-dependent chromatin remodeling complex were identified in the list of SOX6 interacting proteins. These include SMARCA4 (also known as BRG1), SMARCD2 (also known as BRG1-associated factor, BAF60b), SMARCC1 (BRG1-associated factor, BAF155), and ACTL6a (BRG1-associated factor, BAF53a)

[21] ( Figure 4 A). HELLS (SMARCA6) was also identified as a candidate SOX6 interacting partner.

[0424] The SWI / SNF complex controls the accessibility of DNA to sequence-specific transcription factors through nucleosome remodeling, leading to the activation or repression of their target genes.[22,23] SWI / SNF proteins lack sequence specificity and are known to interact with transcription factors to reach specific target sites.

[0425] Therefore, we hypothesized that the interaction of SOX6 with SMARCA proteins could help recruit chromatin remodeling complexes to specific genomic loci.

[0426] Validation by co-immunoprecipitation / co-immunolocalization confirmed the specific interaction of SOX6 with SMARCA4, SMARCA6, and SMARCC1 in keratinocytes ( Figure 4 B). No colocalization was observed between SOX6 and the negative control protein pinin 1 (PNN1) ( Figure 4 B) The degree of colocalization of all tested proteins was quantified by calculating the Pearson colocalization coefficient in Z-stack confocal images ( Figure 4 C). Loss-of-function studies support the hypothesis that the transcription factor SOX6 interacts with a complex containing SMARCC1 and SMARCA4 to exert its function during epidermal differentiation. Knockdown of SMARCC1 significantly increased the expression of epidermal differentiation factors IVL, SPRR2F, and TGM1 ( Figure 4 D).

[0427] Combined with the above findings, we can fully understand the relationship between miR-335, SOX6 and epidermal differentiation-related proteins during skin maturation.

[0428] In healthy skin, proliferating cells in the basal layer of the epidermis express low levels of miR-335. SOX6 is expressed and recruits components of the SMARCA complex to inhibit the production of epidermal differentiation factors in the basal epidermis. High levels of miR-335 in the suprabasal layer directly block SOX6 translation. This relieves transcriptional repression of IVL, SPRR2F, and TGM1, allowing terminal keratinocyte differentiation to occur.

[0429] In contrast, epidermal miR-335 expression is lost in all layers of AD-lesional skin. This loss of miR-335 leads to sustained expression of SOX6 and aberrant loss of differentiation-related proteins. The epidermis fails to fully mature, leading to barrier defects and their associated impact on allergen sensitization.

[0430] Example 23. Results: Epigenetic Regulation of miR-335 by Histone Deacetylase 2 (HDAC2)

[0431] So far, we have demonstrated how downregulation of miR-335 leads to barrier defects and an increased inflammatory microenvironment in AD. However, the molecular mechanisms controlling miR-335 expression remain poorly understood.

[0432] miR-335 is an intronic miRNA encoded by the second intron of the mesoderm-specific transcript (MEST) ( Figure 5 A) Intronic miRNAs can share the same promoter as their host gene or have their own independent promoter.

[0433] RNA seq data analysis showed that miR-335 is expressed in normal human epidermal keratinocytes. However, the CAGE tag was only present in the upstream region of MEST, but not in miR-335, indicating that miR-335 does not have an independent promoter ( Figure 10 ).

[0434] Given the role of miR-335 in directing keratinocyte differentiation and its specific expression in the suprabasal layer of the epidermis, we investigated the expression of miR-335 under specific conditions that promote keratinocyte differentiation. Histone deacetylase (HDAC) inhibitors have been shown to induce keratinocyte differentiation, and recent reports support their role in alleviating AD-like phenotypes in mouse models [24,25].

[0435] We observed that treatment of N / TERT-1 keratinocytes with the broad-spectrum HDAC inhibitor sodium butyrate (NaB) resulted in a significant upregulation of miR-335 ( Figure 5 B).

[0436] NaB treatment also significantly increased the expression of MEST, supporting our hypothesis that miR-335 is transcribed in tandem with its host gene ( Figure 5 C). After treatment with NaB, the significant increase in H3K4ac enrichment in the upstream regions of MEST and miR-335 further confirmed the role of HDAC as a transcriptional regulator of miR-335 and MEST ( Figure 5 D).

[0437] However, it is likely that only miR-335 mediates NaB-induced keratinocyte differentiation—knockdown of MEST did not significantly affect the production of KRT1 (a suprabasal expression marker of keratinocyte differentiation), IVL, or TGM1 ( Figure 5 E). On the other hand, NaB treatment resulted in a significant upregulation of differentiation markers such as IVL, SPRR2F, and TGM1 ( Figure 5 F).

[0438] These observations suggest that NaB-induced HDAC inhibition enables miR-335 expression and its pro-differentiation effects. Therefore, we infer that miR-335 inhibition may be caused by HDAC activity, and that using HDAC inhibitors to restore miR-335 expression in atopic dermatitis may represent a potential therapeutic strategy to restore skin barrier defects.

[0439] Example 34. Results: Belinostat restores barrier function and epidermal homeostasis via the miR-335 network

[0440] HDAC inhibitors (HDACi) can reprogram cellular machinery to induce cell cycle arrest, differentiation, and apoptosis by altering the acetylation status of a range of substrates, including histones, transcription factors, or chaperone proteins. Interestingly, HDAC inhibitors can alter the expression profile of miRNAs

[26] .

[0441] Since miR-335 expression is regulated by HDACs, we attempted to identify HDAC inhibitors that could induce miR-335 expression.

[0442] We treated N / TERT-1 cells with a panel of 42 HDAC inhibitors and identified five that stimulated miR-335 and IVL expression, leading to keratinocyte differentiation. Notably, HDAC inhibitors that induced miR-335 also showed growth arrest and spontaneous differentiation, although some of these inhibitors exhibited high toxicity.

[0443] Based on the significant, consistent, and low toxicity of belinostat, we identified belinostat as a potential candidate drug targeting miR-335 ( Figure 6 A).

[0444] Induction of miR-335 expression in N / TERT-1 cells treated with belinostat was confirmed by in situ hybridization ( Figure 6 B, upper panel). Immunocytochemistry of N / TERT-1 cells showed significant expression of IVL after belinostat treatment compared to control cells ( Figure 6 B, lower panel). In addition, cornified envelope analysis further showed that the number of CEs was increased in cells treated with belinostat compared with controls, indicating that belinostat could restore epidermal homeostasis by stimulating miR-335 expression ( Figure 6 C and Figure 6 D) These findings highlight the importance of HDAC inhibitors for post-transcriptional regulation by inducing the expression of specific miRNAs.

[0445] To test the therapeutic potential of belinostat, we employed an ex vivo human organ culture model, which is more suitable for evaluating the effects of single small molecule drugs through local penetration or transdermal absorption. We selected ex vivo organ cultures from human elective surgery and skin slices with normal skin barrier function and a mature stratum corneum for our experiments.

[0446] Punch biopsies from these skin samples were cultured at the air-liquid interface

[27] . The biopsies were topically treated with acetone alone or with HDAC inhibitors dissolved in acetone. Quantitative RT-PCR analysis showed significant expression of miR-335 after treatment with belinostat compared to other HDAC inhibitors ( Figure 6 E).

[0447] This was further confirmed by in situ hybridization, where belinostat-treated skin showed higher miR-335 expression in the epidermis compared to control mock-treated skin ( Figure 6 F).

[0448] Furthermore, immunohistochemistry revealed that belinostat treatment led to a significant increase in the levels of IVL, a bona fide downstream target gene of miR-335 ( Figure 6 G).

[0449] In addition, belinostat also induced significant levels of filaggrin (FLG) ( Figure 11 ). FLG is a natural moisturizing factor that is frequently mutated or downregulated in AD.

[0450] All of the above indicate that belinostat can effectively stimulate the expression of miR-335 and restore epidermal homeostasis in a dry skin model that mimics AD-like phenotypes.

[0451] In conclusion, the HDAC inhibitor belinostat alleviates inflammation and barrier defects in AD through the miR-335 network.

[0452] Example 25. Discussion

[0453] Atopic dermatitis is thought to be caused by genetic and environmental factors. However, the precise molecular mechanisms regulating the pathophysiology of AD remain unclear.

[0454] The two main pathophysiological hallmarks of AD are epidermal barrier dysfunction with altered keratinocyte proliferation / differentiation in the lesions

[28] and immune cell infiltration in the lesions[11,29,30]. AD has traditionally been considered an immune-driven disease, in which altered immune responses are considered the primary defect and a defective skin barrier is considered a secondary effect of local inflammation (the “inside-out hypothesis”)

[11] .

[0455] However, increasing evidence supports the opposite “outside-in” hypothesis, whereby genetic abnormalities in epidermal structure and enzyme proteins impair skin barrier function, leading to increased antigen penetration, allergen sensitization, and inflammation

[31] .

[0456] Epidermal differentiation is a multistep process characterized by the tightly controlled, sequential expression of a unique set of genes that are turned on and off in a manner that drives proliferation and specialized differentiation. These genes include those encoding structural cytoskeletal proteins (KRT5, KRT14, KRT1, KRT10) and proteins required for the formation of an effective skin barrier (IVL, TGM1, FLG, LOR, SPRR).

[0457] The skin barrier function is conferred by the stratum corneum, which is composed of corneocytes embedded in a multilayer of lipids. Each corneocyte is surrounded by a cornified envelope (CE) and connected to its neighbors by corneal desmosomes

[32] . The CE is formed by cross-linking insoluble structural proteins (including involucrin (IVL), loricrin (LOR), trichomexin, envoplakin, periplakin, and small proline-rich protein (SPRR)) through γ-glutamyl-ε-lysine bonds formed by transglutaminase (TGM1)

[33] .

[0458] The corneocyte layer in the stratum corneum provides a high elasticity that enables the stratum corneum to perform its function as a mechanical barrier against invading pathogens. This, combined with the presence of surrounding intercellular lipids, confers the epidermis with its impermeability.

[0459] MiRNAs are known to play important roles as post-transcriptional gene regulators in skin development and disease. A growing number of miRNAs, such as miR-203

[34] , miR-217

[35] , and miR-17

[36] , have been implicated in regulating keratinocyte differentiation. Our findings provide insights into the role of miR-335 in maintaining epidermal homeostasis.

[0460] In situ hybridization showed that miR-335 was abundantly expressed in the differentiated layers of the epidermis, which is consistent with a previous report that miR-335 showed higher expression in terminally differentiated keratinocytes compared with proliferating keratinocytes isolated from human skin

[37] .

[0461] Furthermore, we observed that miR-335 enhances cornified envelope formation by regulating the expression of a specific differentiation-related gene signature, indicating that miR-335 is a key regulator of keratinocyte differentiation. In AD lesional skin, HDAC-mediated downregulation of miR-335 leads to sustained expression of SOX6, thereby abolishing the expression of differentiation-related proteins such as IVL, SPRR, and TGM1. This impairs differentiation and keratinization, resulting in barrier defects.

[0462] SOX6 has been reported to be a multifaceted transcription factor that regulates the terminal differentiation of many cell lineages of mesodermal, ectodermal, and endodermal origin

[38] . SOX6 with a long 3'UTR has been shown to be post-transcriptionally regulated by miRNAs. For example, miR-499 and miR-219 have been shown to regulate SOX6 expression during the differentiation of skeletal muscle and oligodendrocytes, respectively [39,40].

[0463] We did not detect differential expression of miR-499 and miR-219 in the array data between healthy and AD skin samples, which is not surprising because miR-499 and miR-219 have been identified as muscle-specific miRNAs and brain-specific miRNAs, respectively.

[0464] We found that SOX6 is primarily expressed in the proliferative layer of the epidermis and that SOX6 knockdown enhanced keratinocyte cornification. Our transcriptome analysis revealed that upon SOX6 knockdown in keratinocytes, the expression of several keratinocyte differentiation-related genes, such as CNFN, IVL, SPRR, and TGM1, was upregulated, highlighting the role of SOX6 as a transcriptional repressor of keratinocyte differentiation and cornification.

[0465] In the absence of specific transactivation or transrepression domains, SOX6 interacts with a variety of cofactors, such as components of the transcriptional machinery and chromatin remodeling proteins, to carry out its functions. For example, SOX9 and SOX5 have been shown to cooperate with SOX6 in activating three extracellular matrix-associated proteins during chondrogenesis: CO12A1

[41] , AGC1

[42] , and COMP

[43] . The transcriptional co-repressor CtBP2 has also been shown to be a SOX6-interacting partner that inhibits fibroblast growth factor 3 (Fgf-3) transcription

[44] .

[0466] Our results show that transcriptional repression of specific genes associated with keratinocyte differentiation is mediated by the interaction of SOX6 with the chromatin remodeling proteins SMARCA4, SMARCA6, and SMARCC1. Notably, SMARCA4 and SMARCC1 are part of a highly conserved multi-subunit chromatin remodeling complex known as the SWI / SNF protein family. The SWI / SNF family of nucleosome remodeling complexes is known to play a key role in gene expression. Recent studies have identified transcriptional regulation mediated by biochemically distinct SWI / SNF complexes

[45] and possible mechanisms by which SWI / SNF targets specific promoters.

[0467] Surprisingly, multiple studies have shown that, in addition to activation, SWI / SNF is also required for transcriptional repression of specific genes, including Blimp-1

[46] and cyclin D1

[47] . Here, we demonstrate SOX6-mediated spatiotemporal transcriptional repression of specific differentiation-associated genes by distinct SWI / SNF complexes.

[0468] miR-335 belongs to a group of miRNAs regulated by HDACs. Previous studies have demonstrated that multiple HDAC isoforms can regulate miRNA expression [48,49]. Our ChIP data showed that HDAC1 and HDAC2 were highly enriched in the promoter region of miR-335, while the enrichment of HDAC2 was reduced when keratinocytes were treated with NaB, suggesting that HDAC2 can regulate miR-335 expression.

[0469] We have examined HDAC1 and HDAC2 in ChIP experiments, and whether any other HDACs are involved in the regulation of miR-335 requires further investigation. Treatment with HDAC inhibitors resulted in increased expression of keratinocyte differentiation markers such as IVL, TGM1, and SPRR2F.

[0470] Consistent with these observations, other studies have shown that treatment with HDAC inhibitors leads to premature expression of differentiation markers.[50,51] Although the role of HDACs in AD remains to be elucidated, two independent studies have demonstrated that TSA can inhibit the development of AD-like symptoms in mice.[24,25]

[0471] This prompted us to hypothesize that HDAC inhibitors could be effective in treating AD, in part by upregulating miR-335 expression to induce a differentiation-related transcriptional signature and restore barrier function.

[0472] We identified belinostat as a highly consistent and low-toxic HDACi that effectively restored miR-335 expression and induced the expression of proteins involved in epidermal differentiation.

[0473] We envision developing a topical formulation of belinostat that could provide a therapeutic approach to improve barrier defects and provide relief for refractory atopic dermatitis.

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[0527] In this document and in the claims, the verb "to comprise" and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Furthermore, reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element be present. Thus, the indefinite article "a" or "an" generally means "at least one."

[0528] Each application and patent cited herein, and each document cited or referenced in each of the aforementioned applications and patents, including the disclosures in each application and patent (“application-cited documents”) and any manufacturer’s instructions or catalogs for any products cited or referenced in each application and patent and in any application-cited document, are hereby incorporated by reference. Furthermore, all documents cited herein, and all documents cited or referenced in documents cited herein, and any manufacturer’s instructions or catalogs for any products cited or referenced herein, are hereby incorporated by reference.

[0529] Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and essence of the invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, it will be apparent to those skilled in the art of molecular biology or related fields that various modifications of the described modes for implementing the present invention are intended to fall within the scope of the claims.

Claims

1. Use of belinostat in the preparation of a medicament for treating, preventing or alleviating atopic dermatitis, wherein the PubChem CID of belinostat is 6918638.

2. The use according to claim 1, wherein Belinostat is formulated with a pharmaceutically acceptable excipient, carrier, or diluent.

3. The use according to claim 1 or 2, wherein Belinostat is formulated for topical administration.

4. The use according to claim 1 or 2, wherein The treatment, prevention or alleviation includes upregulating the activity or expression level of miR-335.

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