WO2021141733A1

WO2021141733A1 - Method of modulating a fibrotic condition

Info

Publication number: WO2021141733A1
Application number: PCT/US2020/065002
Authority: WO
Inventors: Robert Joseph Isfort; Frederic Bard; Hui Hui WONG; Sze Hwee SEET
Original assignee: The Procter & Gamble Company; Agency For Science, Technology And Research (A*Star)
Priority date: 2020-01-07
Filing date: 2020-12-15
Publication date: 2021-07-15

Abstract

A method of modulating fibrosis in a tissue of a subject by increasing or decreasing the expression of C2orf69. C2orf69 expression has been found to inhibit the expression of genes involved in building, repairing, and maintaining the extra-cellular matrix. In particular, C2orf69 inhibits the collagen synthesizing of COL1A1. By modulating the expression of the C2orf69, collagen synthesis can be increased or decreased, which can provide a variety of cosmetic and medical benefits.

Description

METHOD OF MODULATING A FIBROTIC CONDITION

FIELD

The present disclosure is directed, generally, to a method of modulating a fibrotic process or condition by modulating the expression of Chromosome 2 Open Reading Frame 69 [SEQ ID NO: 1] and/or the activity of its gene expression products.

BACKGROUND

Fibrotic processes and conditions are generally related to the formation or development of fibrous connective tissue such as collagen by cells in an organ or tissue. Although fibrotic processes and conditions occur as part of normal tissue formation or repair (e.g., the formation of scar tissue when a wound heals), dysregulation of these processes can lead to altered cellular composition and excess connective tissue deposition that progressively impairs tissue or organ function resulting in an undesirable fibrotic condition sometimes referred to as fibrosis. Some undesirable fibrotic conditions such as pulmonary fibrosis are characterized as diseases. However, not all fibrotic processes or conditions are undesirable. For example, the formation of collagen in skin to help provide skin with strength and elasticity may be desirable. Thus, identifying compounds and methods useful for modulating fibrotic processes and conditions has long been a goal for pharmaceutical and cosmetic manufacturers.

Research into the genetic underpinning of fibrotic processes and conditions has led to the discovery of new genetic and biomolecular targets. For example, U.S. Patent No. 10,048,250 describes targets that play a role in the differentiation of macrophages into M2 macrophages, and in particular a suppression of the release or expression of CCL18 and/or CD206. U.S. Publication No. 2013/0209490 describes a method for inhibiting the fibrotic activity of a cell by using a BMP9 or BMP10 antagonist. U.S. Publication No. 2009/0220488 describes a process for treating or preventing scleroderma or other fibrotic disorders by administering an effective amount of a Wnt signaling antagonist. US 2009/0220488 also discloses treating or preventing scleroderma by administering an effective amount of an agent that decreases expression of a gene identified by the researchers as being altered in relation to the expression of scleroderma. U.S. Patent No. 9,481,915 describes a method of screening drugs that promote LOXL1 expression, which is believed to play a role in extracellular matrix protein crosslinking. However, there is still a need to identify new targets that are involved in fibrotic processes and conditions as well as compounds that modulate these targets.

One particular fibrotic process of interest to researchers in the medical and cosmetic skin care fields is the formation of collagen in skin. Collagen is the primary component in the extracellular matrix (ECM) and makes up approximately one-third of the protein in the human body. In human skin, collagen is generated by dermal fibroblasts and then secreted into the ECM, where it aggregates with the existing matrix to form an interlocking mesh of fibrous proteins. Skin quality and appearance depend to a great extent on the properties of the dermis and its extracellular matrix. Failure to maintain suitable collagen levels in skin is thought to underlie physical manifestations of skin aging such as wrinkles, sagginess, and laxity.

While significant research has been done on the collagen synthesis pathway, it is a complex biochemical process and there is still much that is not known. In particular, relatively large parts of collagen regulation are not well elucidated, especially regarding signaling pathways that inhibit steps in the collagen synthesis pathway. Thus, it may be desirable to modulate collagen synthesis by identifying and targeting specific genes that inhibit collagen synthesis and/or the expression products of such genes. Thus, there is still a need to identify targets and compounds that modulate collagen synthesis.

Accordingly, it would be desirable to understand molecular and cellular processes related to certain fibrotic conditions and to modulate the fibrotic process and/or treat the fibrotic conditions by targeting specific genes and gene expression products with compounds that are able to modulate the expression of the gene and/or activity of the gene expression product. It would also be desirable to provide a new method of identifying such compounds.

SUMMARY

The present disclosure describes novel methods of modulating a fibrotic process and/or treating a fibrotic condition in a tissue of a subject, comprising identifying a subject in need of treatment, administering to the subject an effective amount of a treatment compound over the course of a treatment period, wherein the effective amount of the treatment compound increases or decreases the expression of Chromosome 2 Open Reading Frame 69 (C2orf69) [SEQ ID NO: 1], thereby modulating fibrosis in the tissue of the subject.

A method of modulating a fibrotic process is provided that comprises administering to one or more cells a treatment compound over the course of a treatment period, wherein the effective amount of the treatment compound increases or decreases the expression of Chromosome 2 Open Reading Frame 69 (C2orf69) [SEQ ID NO: 1], relative to the expression of C2orf69 without the compound, thereby modulating the fibrosis process in the cells.

Wherein “cells” includes cells such as keratinocytes, fibroblasts and melanocytes, as well as other types of cells commonly associated with skin, such as, for example, myocytes, Merkel cells, Langerhans cells, macrophages, stem cells, sebocytes, nerve cells and adipocytes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. l is a table of collagen synthesizing genes.

FIG. 2 is a chart showing that depleting C2orf69 induces collagen synthesis in fibroblasts.

FIG. 3 is a chart showing that cells overexpressing C2orf69 are resistant to siC2orf69 down- regulation and fail to induce COL1A1 expression.

FIG. 4 is a chart showing that when C2orfORF69 is overexpressed in unstimulated BJ-Tert fibroblasts, basal COL1A1 mRNA levels are reduced.

FIG. 5 is a chart showing that basal collagen levels detected in C2orf69 expressing cells were about 40% lower than those in cells overexpressing GFP.

FIG. 6 illustrates a summary of a protein purification process.

FIG. 7 is a chart showing that collagen induction is reduced when treated with recombinant UPF0565 protein C2orf69.

FIG. 8 is a chart showing the negative correlation of collagen expression with increasing concentration of UFP0565 protein C2orf69.

FIG. 9 demonstrates that collagen protein levels can be effected by depletion of extracellular UPF0565 protein C2orf69.

FIGS. 10A and 10B show that C2orf69 protein [SEQ ID NO: 2] blocks TGFp-mediated collagen synthesis.

DETAILED DESCRIPTION

Previous research into fibrotic processes and conditions has not adequately appreciated that it may be possible to regulate a fibrotic process or condition by modulating specific genes, or their gene expression products, which inhibit the activity of collagen inhibiting genes. Surprisingly, it has now been discovered that modulating the expression of Chromosome 2 Open Reading Frame 69 (“C2orf69”) [SEQ ID NO: 1] and/or the activity of its gene expression products may be useful for modulating collagen synthesis.

Reference within the specification to “embodiment s)” or the like means that a particular material, feature, structure and/or characteristic described in connection with the embodiment is included in at least one embodiment, optionally a number of embodiments, but it does not mean that all embodiments incorporate the material, feature, structure, and/or characteristic described. Furthermore, materials, features, structures and/or characteristics may be combined in any suitable manner across different embodiments, and materials, features, structures and/or characteristics may be omitted or substituted from what is described. Thus, embodiments and aspects described herein may comprise or be combinable with elements or components of other embodiments and/or aspects despite not being expressly exemplified in combination, unless otherwise stated or an incompatibility is stated.

All ingredient percentages are by weight of the corresponding composition, unless specifically stated otherwise. All ratios are weight ratios, unless specifically stated otherwise. All ranges are inclusive and combinable. The number of significant digits conveys neither a limitation on the indicated amounts nor on the accuracy of the measurements. All numerical amounts are understood to be modified by the word “about” unless otherwise specifically indicated. Unless otherwise indicated, all measurements are understood to be made at approximately 25 °C and at ambient conditions, where “ambient conditions” means conditions under about 1 atmosphere of pressure and at about 50% relative humidity. All numeric ranges are inclusive of narrower ranges; delineated upper and lower range limits are interchangeable to create further ranges not explicitly delineated.

The transcriptional profiles herein can comprise, consist essentially of, or consist of, data related to the genes in a subject gene signature (e.g., in the form of gene identifiers and direction of regulation) as well as other optional components described herein (e.g., metadata). As used herein, “consisting essentially of’ means that the relevant subject matter (e.g., composition, component, or other element) may include additional ingredients, components, or elements, but only if the they do not materially alter the basic and novel characteristics of the claimed composition or method. As used in the description and the appended claims, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The gene and protein designations disclosed herein correspond to their respective known sequences in the National Institute of Health’s genetic sequence database, GenBank®, as of lanuary 7, 2020 and are incorporated herein by reference. DEFINITIONS

“About” modifies a particular value by referring to a range equal to plus or minus twenty percent (+/- 20%) or less (e g., less than 15%, 10%, or even less than 5%) of the stated value.

“C2orf69 protein” herein means UPF0565 protein C2orf69 [SEQ ID NO: 2] coded for by C2orf69 [SEQ ID NO: 1]

“COL1A1 protein” herein means collagen alpha-l(I) chain [SEQ ID NO: 4] coded for by COL1A1 [SEQ ID NO: 3]

“Cosmetic agent” means any substance, as well any component thereof, intended to be rubbed, poured, sprinkled, sprayed, introduced into, or otherwise applied to a mammalian body or any part thereof. Cosmetic agents may include substances that are Generally Recognized as Safe (GRAS) by the US Food and Drug Administration, food additives, and materials used in non-cosmetic consumer products including over-the-counter medications. In some embodiments, cosmetic agents may be incorporated in a cosmetic composition comprising a dermatologically acceptable carrier suitable for topical application to skin. Some non-limiting examples of cosmetic agents or cosmetically actionable materials can be found in: the PubChem database associated with the National Institutes of Health, USA; the Ingredient Database of the Personal Care Products Council; and the 2010 International Cosmetic Ingredient Dictionary and Handbook, 13th Edition, published by The Personal Care Products Council; the EU Cosmetic Ingredients and Substances list; the Japan Cosmetic Ingredients List; the Personal Care Products Council, the SkinDeep database; the FDA Approved Excipients List; the FDA OTC List; the Global New Products Database (GNPD); and from suppliers of cosmetic ingredients and botanicals.

“Fibroblast” means a connective-tissue cell of mesenchymal origin that secretes proteins, especially molecular collagen, to form the extracellular fibrillar matrix of connective tissue.

“Fibrotic condition” refers to a biological condition resulting from an excessive or insufficient amount of collagen in a tissue. One example of a fibrotic condition is thinner, less elastic skin resulting from an age-induced reduction in collagen production. Another example of a fibrotic condition is pulmonary fibrosis.

“Fibrotic process” refers to a biological process related to the synthesis of collagen and/or incorporation of collagen into the extracellular matrix. An example of a fibrotic process is the expression of one or more collagen synthesizing genes.

“Gene expression profiling” and “gene expression profiling experiment” mean a measurement of the expression of multiple genes in a biological sample using any suitable profiling technology. For example, the mRNA synthesis of thousands of genes may be determined using microarray techniques. Other emerging technologies that may be used include RNA-Seq or whole transcriptome sequencing using NextGen sequencing techniques.

“Gene expression product” means an RNA (e.g., mRNA) or protein resulting from the expression of a gene.

“Immortalized fibroblasts” are fibroblasts that, due to mutation, evade normal cellular senescence and instead can keep undergoing division indefinitely, thereby allowing them to be grown for prolonged periods in vitro.

“Microarray” means any ordered array of nucleic acids, oligonucleotides, proteins, small molecules, large molecules, and/or combinations thereof on a substrate that enables gene expression profiling of a biological sample. Some non-limiting examples of microarrays are available from Affymetrix, Inc.; Agilent Technologies, Inc.; Ilumina, Inc.; GE Healthcare, Inc.; Applied Biosystems, Inc.; and Beckman Coulter, Inc.

“Parental cell,” when referring to immortalized or transformed fibroblasts herein, means the cell line that was modified (i.e., immortalized or transformed) to produce immortalized or transformed fibroblasts.

“Positive control” refers to a compound (or mixture of compounds) that has a known effect on a particular fibrotic process or condition and/or modulates the expression of one or more genes associated with the fibrotic process or condition in a known direction.

“Safe and effective amount” means an amount of a compound or composition sufficient to significantly induce a positive benefit (e.g., an increase in collagen synthesis by dermal fibroblasts), including independently or in combinations the benefits disclosed herein, but low enough to avoid serious side effects, i.e., to provide a reasonable benefit to risk ratio, within the scope of sound judgment of the skilled artisan.

“Skin” means the outermost protective covering of mammals that is composed of cells such as keratinocytes, fibroblasts and melanocytes. Skin includes an outer epidermal layer and an underlying dermal layer. Skin may also include hair and nails as well as other types of cells commonly associated with skin, such as, for example, myocytes, Merkel cells, Langerhans cells, macrophages, stem cells, sebocytes, nerve cells and adipocytes.

“Skin care active” means a compound or combination of compounds that, when applied to skin, provide an acute and/or chronic benefit to skin or a type of cell commonly found therein. Skin care actives may regulate and/or improve skin or its associated cells (e.g., improve skin elasticity; improve skin hydration; improve skin condition; and improve cell metabolism).

Upregulating the expression of collagen synthesizing genes typically leads to an increase in collagen production. Conversely, inhibiting expression of these collagen synthesizing genes is believed to lead to a decrease in collagen production. Enhancing or inhibiting the activity of the gene expression products of collagen synthesizing genes may also result in higher or lower amounts of collagen, respectively. Genes that inhibit collagen synthesis are known. For example, some genes inhibit collagen synthesis by suppressing activation of a collagen synthesizing gene and/or interfering with a gene expression product of a collagen synthesizing gene. Trying to modulate the various collagen synthesis genes directly to treat a fibrotic condition can be complex and unpredictable. Thus, it may be advantageous to modulate the expression a collagen inhibiting gene rather than the collagen synthesizing genes. Genes of interest that directly contribute to the synthesis of collagen are referred to herein as “collagen synthesizing genes” and are shown in the table in FIG 1.

It is believed without being limited by theory that C2orf69 [SEQ ID NO: 1] is known to downregulate certain collagen synthesizing genes. Accordingly, in some instances, the methods disclosed herein involve modulating the expression of C2orf69 [SEQ ID NO: 1] by administering a safe and effective amount of a treatment compound to a subject, thereby increasing or decreasing the collagen level in the tissue of the subject. As used herein, “gene modulation” generally refers to increasing or decreasing the expression of a gene, and thus increasing or decreasing the amount of a gene expression product produced by that gene. Gene modulation is sometimes referred to as gene regulation, where “upregulating” the gene means promoting and/or increasing gene expression and “downregulating” the gene means inhibiting and/or decreasing gene expression. Genes can be modulated at any step in the gene expression pathway (e.g., from DNA-RNA transcription to post- translational modification of a protein) by a variety of different mechanisms used by cells to increase or decrease the gene expression products of specific genes.

In some instances, it may be desirable to modulate expression of C2orf69 [SEQ ID NO: 1] by administering a treatment compound that increases or decreases the expression of C2orf69 [SEQ ID NO: 1] The compound may be a protein, RNA, small molecule, or DNA that interacts directly with the C2orf69 [SEQ ID NO: 1] promoter or interacts indirectly with the C2orf69 [SEQ ID NO: 1] promoter via a signaling pathway. For example, activation of the C2orf69 [SEQ ID NO: 1] promoter can be achieved by increasing the activity of a transcription factor associated with C2orf69 [SEQ ID NO: 1], increasing the activity of an enhancer, or decreasing the activity of a transcription repressor. Additionally or alternatively, the expression of C2orf69 [SEQ ID NO: 1] can be increased by decreasing the degradation of C2orf69 mRNA or C2orf69 protein [SEQ ID NO: 2] or increasing the stability of C2orf69 mRNA or C2orf69 protein [SEQ ID NO: 2] In addition, the level of C2orf69 protein [SEQ ID NO: 2] can be modulated by interfering with a C2orf69 [SEQ ID NO: 1] binding protein. It is believed, without being limited by theory, that increasing the expression of C2orf69 [SEQ ID NO: 1] can help lower collagen levels in the tissue of the subject, which may be useful for treating fibrosis. Alternatively, decreasing the expression of C2orf69 [SEQ ID NO: 1] may increase collagen levels in the tissue of a subject, which may be desirable for treating age-related skin conditions such as fine lines and wrinkles and promoting wound healing.

In some instances, it may be desirable to administer a treatment compound that interferes with the activity of a gene expression product of C2orf69 [SEQ ID NO: 1] to modulate collagen synthesis. In particular, it may be desirable to select a treatment compound that interferes with the activity of C2orf69 [SEQ ID NO: 1] messenger RNA (“mRNA”) or C2orf69 protein [SEQ ID NO: 2] Interfering with the activity of a gene expression product generally involves inhibiting or facilitating the ability of the gene expression product to chemically interact with its intended biochemical target. For example, the treatment compound may block the interaction of C2orf69 protein [SEQ ID NO: 2] with its receptor. The treatment compound can be a small molecule, protein, DNA, etc. Some non-limiting examples of small molecules that may be suitable for use as a treatment compound include antagonists and inverse agonists. Some non-limiting examples of proteins that may be suitable for use as a treatment compound include antibodies (or fragments thereof), binding proteins (or fragments thereof), soluble receptors (or fragments thereof), combinations of these and the like. A non-limiting example of DNA that may be suitable for use as a treatment compound is an aptamer.

Treatment compounds for use in the present method are not particularly limited as long as they are able to modulate collagen inhibiting genes such as C2orf69 [SEQ ID NO: 1] Compounds for cosmetic use should generally recognized as safe (GRAS) for administering to humans. Compounds for medical use should have a suitable therapeutic index. In other words, the amount of the treatment compound that causes the therapeutic effect should be suitable relative to the amount that causes toxicity. Treatment compounds may be in the form of small molecules, nucleic acids (e.g., small interfering RNA, micro RNA, and small activating RNA), antibodies, plant extracts, vitamins, minerals, and cosmetic agents that are capable of modulating C2orf69 [SEQ ID NO: 1] expression and/or interfering with the activity of its gene expression products. Some non-limiting examples of methods of identifying compounds that modulate collagen inhibiting genes such as C2orf69 [SEQ ID NO: 1] are described in co-pending U.S. Provisional App. Serial No. 62/890,641, titled “Method of Modulating a Fibrotic Condition,” filed on August 23, 2019. Changes in gene expression and/or protein level may be determined by any suitable method known in the art such as, for example, protein quantitation, reporter gene activity, and/or gene transcriptomic analysis.

In some instances, it may be desirable to determine the collagen synthesis activity of an extracellular matrix gene such as COL1A1 [SEQ ID NO: 3] and/or another member of the collagen family of genes by measuring the activity of the gene(s) via a reporter gene inserted downstream of an endogenous promoter for the gene of interest. The reporter gene may encode for a protein that emits at a defined fluorescent wavelength when excited by a specific wavelength range. In some instances, it may be desirable to use an exogenous collagen synthesizing gene and/or collagen inhibiting gene promoter linked to a reporter gene. For example, a collagen synthesizing gene and/or collagen inhibiting gene promoter may be cloned into a plasmid, linked to a reporter construct and transfected into an immortalized/transformed fibroblast cell line. The amount of fluorescent protein present in the fibroblast sample can be quantitated and directly correlated to the activity of the gene of interest. The fluorescent protein may be quantitated using a suitable fluorescence spectroscopy technique (e.g., using a fluorometer according to the manufacturer’ s instructions). The fluorescent protein used herein, or the gene that codes for it, is not particularly limited and includes green fluorescent proteins (“GFP”) and red fluorescent proteins (“RFP”). Some non-limiting examples of RFPs are mCherry, mStrawberry, mOrange, and dTomato. In a more specific example, it may be desirable to use an immortalized or transformed fibroblast cell line modified via a gene editing technique (e.g., zinc finger nuclease, transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) to include a nuclear-localizing signal mCherry gene (“NLS- mCherry”) that codes for the mCherry RFP when a gene of interest is activated.

In some instances, it may be desirable to use transcriptomic analysis to measure gene activity of cells contacted with a test compound relative to a control. For example, the ability of a test compound to modulate C2orf69 [SEQ ID NO: 1] may be determined by comparing the transcriptional profiles of the test sample and control sample to one another and measuring the change in expression of C2orf69 [SEQ ID NO: 1] It may also be desirable to use transcriptomic analysis to determine the change in collagen production caused by modulating C2orf69 [SEQ ID NO:l], for example, by comparing the expression of one or more collagen synthesizing genes to a control. In some instances, messenger ribonucleic acid (“mRNA”) encoded by one or more genes of interest in a gene signature may be measured and compared to a control. Any suitable quantitative nucleic acid assay may be used to conduct that transcriptomic analysis. For example, conventional quantitative hybridization, Northern blot, and polymerase chain reaction procedures may be used for quantitatively measuring the amount of an mRNA transcript or cDNA in a biological sample. Optionally, the mRNA or cDNA may be amplified by polymerase chain reaction (PCR) prior to hybridization. The mRNA or cDNA sample is then examined by, e.g., hybridization with oligonucleotides specific for mRNAs or cDNAs encoded by the one or more of the genes of interest (e g., collagen inhibiting genes), optionally immobilized on a substrate (e.g., an array or microarray). Binding of the nucleic acid to the oligonucleotide probes specific for the gene of interest allows identification and quantification of the expression level of that gene. Suitable examples of transcriptomic methods of quantifying gene expression are disclosed in U.S. Patent Nos. 9,434,993; 10,036,741; and 10,282,514.

In some instances, it may be desirable to determine collagen synthesis activity by protein quantitation, for example, using a conventional assay such as an enzyme-linked immunosorbent assay (ELISA), a Western blot, mass spectrometry, a UY absorption, a bicinchoninic acid, a Bradford assay, a Kjeldahl assay, or a Folin-Lowry assay. Other non-limiting examples of methods for quantitating collagen are described in L. C. U. Junqueira, et al., (1979) “A Simple and Sensitive Method for the Quantitative Estimation of Collagen;” Analytical Biochemistry 94; 96 - 99; and R.F. Diegelmann, et al., (1990) “A Microassay to Quantitate Collagen Synthesis by Cells in Culture;” Analytical Biochemistry 186(2); 296 -300.

COMPOSITIONS

The compositions herein include a safe and effective amount of a treatment compound. The amount of treatment compound should be sufficient to modulate the expression of C2orf69 [SEQ ID NO: 1] Additionally, the amount of treatment compound should be sufficient to improve a fibrotic condition after a suitable course of treatment (e.g., 2, 4, 8 weeks or more up to year). Optional ingredients included in the present compositions are not particularly limited as long as they do not unacceptably alter the ability of the treatment compound to modulate the expression of C2orf69 [SEQ ID NO: 1] and/or improve the fibrotic condition. The optional components, when present, should be suitable for use with human tissue without undue toxicity, incompatibility, instability, allergic response, and the like. The optional ingredients may be present at 0.0001% to 50% (e.g., 0.001% to 20% or even 0.01% to 10%). The amounts listed herein are only to be used as a guide, as the optimum amount of the optional ingredients used in a composition will depend on the specific ingredient selected since their potency and/or function can vary considerably. The form of the composition should be tailored for the desired administration route of the treatment compound (e.g., topical application, oral ingestion, infusion or injection). For example, the composition may be in the form of a solution, suspension, dispersion, emulsion, powder, tablet, capsule, biodegradable polymer, nanoparticles, lotion, cream, gel, toner, spray, aerosol, ointment, cleansing liquid wash, solid bar, shampoo, hair conditioner, paste, foam, powder, mousse, shaving cream, wipe, strip, patch, wound dressing, adhesive bandage, hydrogel, film-forming product, facial and skin mask (with and without insoluble sheet). The composition may be provided in a package sized to store a sufficient amount of the composition for a treatment period.

The present compositions may be prepared by conventional methods for making compositions of the type desired. These methods typically involve mixing the ingredients in one or more steps to a relatively uniform state, with or without heating, cooling, application of vacuum, and the like. The compositions of the present invention are generally prepared by conventional methods such as are known in the art of making topical compositions. Such methods typically involve mixing of the ingredients in one or more steps to a relatively uniform state, with or without heating, cooling, application of vacuum, and the like. The compositions herein are prepared to provide desirable stability (physical stability, chemical stability, photostability) and/or delivery of the active materials. Providing good stability to the composition may include adjusting the pH (e.g., less than 7), exclusion of materials that can complex with the active agent and thus negatively impact stability or delivery (e.g., exclusion of contaminating iron), use of approaches to prevent complex formation (e.g., appropriate dispersing agents or dual compartment packaging), use of appropriate photostability approaches (e.g., incorporation of sunscreen/sunblock, use of opaque packaging), etc.

In some instances, the treatment compound is mixed with a suitable acceptable carrier. For example, the carrier emulsion carriers, including, but not limited to, oil -in-water, water-in-oil, silicone- in-water, water-in-silicone, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, may suitable forms for the carriers. While not particularly limited, the carrier should have good aesthetic properties, be compatible with the ingredients in the composition, and not cause any unreasonable safety or toxicity concerns to the user. The carrier may contain one or more hydrophobic or hydrophilic diluents in which the treatment compound can be dispersed, dissolved, or otherwise incorporated. Some non limiting examples of hydrophilic diluents include water, organic hydrophilic diluents such as lower monovalent alcohols (e.g., Ci - C4) and low molecular weight glycols and polyols, including propylene glycol, polyethylene glycol (e.g., molecular weight of 200 to 600 g/mole), polypropylene glycol (e.g., molecular weight of 425 to 2025 g/mole), glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, sorbitol esters, butanediol, ether propanol, ethoxylated ethers, propoxylated ethers and combinations thereof. Some non-limiting examples of hydrophobic diluents include volatile and non-volatile hydrocarbon oils and waxes, silicone oils and waxes, and botanical oils.

The carrier may be present at 1% to 95% (e.g., 10% to 90%, 30% to 70%, 50% to 60%) by weight of the composition. The carrier may be aqueous or anhydrous. For example, suitable carriers may include water, water miscible solvents, and oils. Suitable water miscible solvents include monohydric alcohols, dihydric alcohols, polyhydric alcohols, glycerol, glycols, polyalkylene glycols such as polyethylene glycol, and mixtures thereof. Suitable oils include silicones, hydrocarbons, esters, amides, ethers, and mixtures thereof. The oils may be volatile or nonvolatile.

Optional ingredients that may be added to the present composition include known compounds that inhibit collagen production (“anti-fibrotic compounds”). Some non-limiting examples of anti- fibrotic compounds are disclosed in U S. Publication No. 2013/0209490; U.S. Patent No. 7,026,283; and Wynn, et af, Journal Clin. Invest., Vol 117 Number 3, March 2007, p 524. In some instances, the composition may optionally include compounds that promote collagen production (“pro-fibrotic compounds”), for example, by acting on the same or similar biochemical pathways as the anti-fibrotic actives, but with opposite effect.

Method of Use

The compositions herein are useful for modulating the expression of C2orf69 [SEQ ID NO: 1] and/or improving a fibrotic condition. Accordingly, compositions comprising an effective amount of a treatment compound may be administered to a person in need of treatment or who desired treatment. A person in need of treatment is one who exhibits symptoms of a fibrotic condition or who is diagnosed with a fibrotic condition. Of course, it is to be appreciated that a person who does not exhibit symptoms of a fibrotic condition or has not been diagnosed with a fibrotic condition may still desire treatment, for example, as a preventative measure. Generally, the compositions herein may be administered in accordance with conventional methods of administering compositions of the type. For example, the compositions may be administered intravenously, intradermally, subcutaneously, orally (e.g., via inhalation or ingestion), topically, and/or transmucosally. In one specific example, the present compositions may be administered topically by applying an effective amount of the composition (e.g., 0.1 mg/cm² to about 20 mg/cm²) to a target area of skin where treatment is needed or desired. Some non-limiting examples of routes for administering the compositions herein are disclosed in U.S. Pat. Nos. 7,175,844 and 5,328,470. Suitable release rates and dosages of the treatment compound can be determined by those skilled in the art.

Compositions containing an effective amount of a treatment compound may be administered once a day, twice a day, or on a more frequent daily basis, over the course of a treatment period. The treatment period is ideally of sufficient time for the treatment compound to provide the desired benefit. For example, the treatment period may be of sufficient time for the treatment compound to provide a noticeable and/or measurable improvement in a fibrotic condition or change in a fibrotic process. The treatment period may last for at least 1 week (e.g., about 2 weeks, 4 weeks, 8 weeks, or even 12 weeks). In some instances, the treatment period will extend over multiple months (i.e., 3-12 months) or multiple years. In some instances, a composition containing an effective amount of a treatment compound may be administered most days of the week (e g., at least 4, 5 or 6 days a week), at least once a day or even twice a day during a treatment period of at least 2 weeks, 4 weeks, 8 weeks, or 12 weeks, up to one year or more.

EXAMPLES

Non-limiting example of various aspects of the methods described herein are provided below. The examples are given solely for the purpose of illustration and are not intended to be construed as limiting the invention, as many variations thereof are possible.

Example 1 - Identification of C2orf69 [SEQ ID NO: 1] as a novel collagen inhibitor.

This example demonstrates that C2orf69 [SEQ ID NO: 1] modulates COL1A1 protein [SEQ ID NO: 4], which is a major component of the extracellular matrix. Expression of COL1A1 [SEQ ID NO: 3] is maintained at a relatively low, steady state in quiescent, unstimulated fibroblasts. However, in a genetic screen for collagen modulators, it was observed that siRNA-mediated depletion of an uncharacterized gene mRNA, C2orf69 [SEQ ID NO: 1], upregulates collagen transcription in Red- COL1A1, a collagen I reporter cell line derived from the immortalized human dermal fibroblast line BJ-Tert. It was found that silencing C2orf69 [SEQ ID NO: 1] consistently enhanced COL1A1 [SEQ ID NO: 3] mRNA expression in the parental BJ-Tert cell line, as well as in 4 primary normal human fibroblast cell lines - NF131, NF175, NF186, NF190. The results are summarized in Table 1.

Table 1 - COL1A1 mRNA Expression

Example 2 - Depletion of C2orf69 [SEQ ED NO: 1] mRNA induces collagen synthesis in immortalized human dermal fibroblasts and represses basal collagen expression.

This example demonstrates the ability of C2orf69 [SEQ ED NO: 1] to repress COL1A1 [SEQ ID NO: 3] expression by depleting C2orf69 [SEQ ID NO: 1] mRNA using siRNA and then probing for COL1A1 [SEQ ID NO: 3] transcript levels over a time course of 144 hours (5 days). BJ-Tert fibroblasts were transfected with non-targeting control siRNA (siNT) or siRNA targeting C2orf69 (siC2orf69). Cells were harvested at 24, 48, 72, 96 and 144 hours post transfection for analysis of C2orf69 [SEQ ED NO: 1] and COL1A1 [SEQ ID NO: 3] mRNA levels. FIG. 2 illustrates the results of rtPCR analysis of C2orf69 [SEQ ID NO: 1] and COL1 A1 [SEQ ED NO: 3] mRNA levels at the indicated time points. Fold change were calculated with reference to siNT samples b-actin was used as an internal control.

Detection of C2orf69 [SEQ D NO: 1] mRNA confirmed that C2orf69 [SEQ ED NO: 1] mRNA was efficiently depleted (>90%) during the 144-hour time period, as illustrated in FIG. 2. COL1A1 [SEQ ED NO: 3] mRNA levels in treated BJ-Tert fibroblasts increased approximately 2-fold from 24 to 96 hours post siRNA treatment. The knockdown efficiency of C2orf69 [SEQ ED NO: 1] was reduced to 70% at 144 hours and was accompanied by a corresponding reduction in COL1A1 protein [SEQ ED NO: 4] levels, demonstrating the link between C2orf69 protein [SEQ ED NO: 2] levels and COL1A1 [SEQ ED NO: 3] mRNA levels. The western blot analysis of total cell lysate at 96 hours post transfection confirmed that the reduction in C2orf69 protein [SEQ ID NO: 2] correlates with increased COL1 A1 protein [SEQ ID NO: 4] levels, consistent with the mRNA data.

Example 3 - Confirming that C2orf69 protein [SEQ ED NO: 2] level correlates with COL1A1 protein [SEQ ED NO: 4] level.

This example demonstrates the correlation between C2orf69 protein [SEQ ED NO: 2] level and COL1 A1 protein [SEQ ED NO: 4] level. In this example, designed rescue experiments were conducted by repeating the RNAi experiments in cells exogenously expressing control GFP or C2orf69 protein [SEQ ED NO: 2] BJ-Tert fibroblasts overexpressing green fluorescent protein or C2orf69 protein [SEQ ED NO: 2] were transfected with non-targeting siRNAs (NT, siNT) and C2orf69-targeting siRNAs (C2, siC2orf69). Five days after transfections, the cells were analyzed for COL1A1 protein [SEQ ID NO: 4], C2orf69 protein [SEQ ID NO: 2], and GFP expression by western blot analysis and protein levels were quantified using ImageJ analysis software (available on the National Institute of Health website) b-tubulin protein was used as a control. The data were normalized to GFP expression cells transfected with the respective siNT controls. The results of this test are illustrated in FIGS. 3, 4, and 5.

As illustrated in FIG. 3, cells overexpressing the C2orf69 protein [SEQ ID NO: 2] are resistant to siC20RF69 down-regulation and fail to induce COL1A1 [SEQ ID NO: 3] expression, in contrast to the induction of COL1A1 protein [SEQ ID NO: 4] by siC2orf69 in control GFP cells. Conversely, when C2orf69 [SEQ ID NO: 1] is overexpressed in unstimulated BJ-Tert fibroblasts, basal COL1A1 mRNA levels are nearly halved (52%) compared to control cells overexpressing GFP, as illustrated in FIG. 4. This result is consistent with the decrease in COL1 A1 protein expression, where basal collagen levels detected in C2orf69 [SEQ ID NO: 1] expressing cells were 40% lower than those in cells overexpressing GFP, as illustrated in FIG. 5. These results demonstrate that the activation of COL1 A1 expression is correlated to the level of C2orf69 [SEQ ID NO: 1] expression.

Example 4 - The role of secreted C2orf69 protein [SEQ ID NO: 2] in the control of collagen synthesis.

Sequence analysis of C2orf69 [SEQ ID NO: 1] revealed the presence of a signal peptide, suggesting that it may function as a secreted protein. In order to evaluate the role of a secreted C2orf69 protein [SEQ ID NO: 2] in the regulation of collagen synthesis, C2orf69 [SEQ ID NO: 1] was expressed in bacteria and the resulting recombinant protein purified by Nickel-affinity chromatography. As a control, lysate from bacteria transformed with empty control plasmid was purified in parallel. A summary of the purification process is illustrated in FIG. 6. BJ fibroblasts were first transfected with siRNAs 24 hours prior to treatment of the BJ fibroblast with 2nM of recombinant C2orf69 protein [SEQ ID NO: 2] or purified control lysate. Consistent with previous results, in cells treated with purified control lysates, depletion of C2orf69 [SEQ ID NO: 1] using siC2orf69 induced collagen expression by about 2.5-fold. As illustrated in FIG. 7, this induction was abolished when cells were treated with 2 nM of recombinant C2orf69 protein [SEQ ID NO: 2] in the culture media. An independent immunofluorescence-based assay also showed that supplementing C2orf69-depleted cells with recombinant C2orf69 protein [SEQ ID NO: 2] blocks collagen induction in a dose responsive manner. However, these effects were not observed when siC2orf69 treated cells were similarly treated with a control recombinant protein.

Example 5 - Dose-response of C20RF69 protein [SEQ ID NO: 2] on basal collagen synthesis. BJ-Tert fibroblasts were treated with rh-C2orf69 protein at serially diluted concentrations ranging from 0.25 to 8 nM and COL1A1 protein [SEQ ID NO: 4] levels were analyzed by western blot and then quantified using ImageJ software b-tubulin was included as a loading control. As illustrated in FIG. 8, collagen expression negatively correlates with increasing concentration of C2orf69 protein [SEQ ID NO: 2] as compared to untreated cells.

Example 6 - Intrinsically expressed extracellular C2orf69 protein [SEQ ID NO: 2] modulates collagen expression.

In this example, the effect of C2orf69 [SEQ ID NO: 1] neutralizing antibodies on collagen expression was examined. BJ-Tert fibroblasts were treated with 10pg/mL of either rabbit IgG control antibodies (Control IgG) or rabbit polyclonal antibodies against C2orf69 [SEQ ID NO: 1] (Anti- C2orf69) for 24 and 48 hours. Cells were analyzed for COL1A1 [SEQ ID NO: 3] expression in the total cell lysate by western blot analysis. B-Actin (a -b-Actin) was included as a loading control.

As can be seen in FIG. 9, at 24 and 48 hours post-treatment, fibroblasts cultured in media supplemented with antibodies specific to C2orf69 [SEQ ID NO: 1] had elevated COL1A1 protein [SEQ ID NO: 4] levels when compared to treatment with control IgG antibodies. The data show that specifically depleting the extracellular pool of C2orf69 protein [SEQ ID NO: 2] can effectively increase collagen synthesis, which suggests that extracellular C2orf69 protein [SEQ ID NO: 2] acts in an autocrine manner to modulate collagen synthesis. This also demonstrates that two methods of decreasing C2orf69 protein [SEQ ID NO: 2] levels, antisense RNA and neutralizing antibodies, both result in increased collagen production.

Example 7 - C2orf69 protein [SEQ ID NO: 2] blocks TGFb-mediated collagen synthesis.

During wound healing, in fibrosis, and in some cancers, collagen synthesis in fibroblasts is activated in the presence of stimulatory cytokines such as transforming growth factor beta (TϋRb), which is one of the most potent inducers of collagen synthesis known. In order to understand if neutralizing C2orf69 protein [SEQ ID NO: 2] would block TGFb-mediated collagen synthesis, BJ- Tert fibroblasts were stimulated with and 1.3, 2.5, 5 and 10 ng/mL of TGFfl and then co-treated with either 2nM of control recombinant protein (CP, tPE) or 2 nM of rh-C2orf69 for 72 hours. Western blot analysis was used to quantitate COL1A1 protein [SEQ ID NO: 4] levels (b-tubulin was included as a loading control), and quantitative PCR (qPCR) was used to analyze COL1A1 [SEQ ID NO: 3] mRNA expression. The qPCR analysis (sometimes referred to as real time PCR) was conducted using standard laboratory techniques which are well-known to those skilled in the art. The results of the testing are illustrated in FIGS. 10A (western blot) and 10B (qPCR).

As can be seen in FIGS. lOAand 10B, in the presence of control protein (CP), COL1A1 protein [SEQ ID NO: 4] and mRNA levels were strongly induced by TGF . However, the induced COL1 A1 protein [SEQ ID NO: 4] and COL1 A1 [SEQ ID NO: 3] mRNA levels were completely abolished when rh-C2orf69 was introduced. The data suggest that C2orf69 protein [SEQ ID NO: 2] is an inhibitor of both basal and TGFp-induced collagen synthesis.

Example 8 - Reducing C2orf69 [SEQ ID NO: 1] expression increases multiple ECM components.

This example demonstrates the ability of C2orf69 [SEQ ID NO: 1] to modulate collagen synthesizing genes as well as other genes believed to be involved in fibrotic conditions, such as cellular communication network factor 2 (CCN2) [SEQ ID NO: 11], actin alpha 2, smooth muscle (ACTA2) [SEQ ID NO: 13] AND serpin family E member 1 (SERPINE1) [SEQ ID NO: 33] CCN2 [SEQ ID NO: 11] (a.k.a. CTGF) and ACTA2 [SEQ ID NO: 13] are believed to is believed to be mediators of ECM production. SERPINEl [SEQ ID NO: 22] is believed to play a role in cardiac fibrosis. In this example, C2orf69 [SEQ ID NO: 1] mRNA was depleted in BJ-Tert fibroblasts with siRNA as described in example 2 above. mRNA levels of the collagen synthesizing genes shown in Table 2 were quantitated in triplicate for each using qPCR. The results are illustrated in Table 2. Fold change in expression is relative to non-specific siRNA treatment of BJ fibroblast cells. Table 2

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.”

Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

CLAIMS What is claimed is:

1. A method of modulating a fibrotic process, comprising: a) administering to one or more cells a treatment compound over the course of a treatment period, wherein the effective amount of the treatment compound increases or decreases the expression of Chromosome 2 Open Reading Frame 69 (C2orf69) [SEQ ID NO: 1], relative to the expression of C2orf69 without the compound, thereby modulating the fibrosis process in the cells.

2. The method according to claim 1, wherein the compound is selected from the group consisting of small molecules, botanical extracts, microbiological extracts, nucleic acids, nucleic acid analogues, proteins, peptides, antibodies, nanobody aptamers, fragments of these and combinations thereof.

3. The method according to claim 2, wherein the compound is a nucleic acid selected from the group consisting of lariat-form RNA, antisense RNA, short temporary RNA, small interfering RNA, short hairpin RNA, micro RNA, aberrant RNA containing mismatches, double stranded RNA (dsRNA), and long deoxyribonucleotide containing RNA.

4. The method according to claims 1 to 3, wherein the compound increases or decreases transformative growth factor-b (TBG-b) induced collagen synthesis relative to a control.

5. The method according to any of the previous claims, wherein the effective amount of the compound increases C2orf69 [SEQ ID NO: 1] expression to treat a fibrotic condition selected from the group consisting of vascular fibrosis, pulmonary fibrosis, pancreatic fibrosis, liver fibrosis, renal fibrosis, musculoskeletal fibrosis, cardiac fibrosis, skin fibrosis, eye fibrosis, glaucoma, progressive systemic sclerosis, chronic graft versus-host disease, scleroderma, Peyronie's disease, post-cystoscopic urethral stenosis, idiopathic and pharmacologically induced retroperitoneal fibrosis, mediastinal fibrosis, progressive massive fibrosis, proliferative fibrosis, neoplastic fibrosis, radiation-induced fibrosis, autoimmune-related fibrosis, adult respiratory distress syndrome, inflammatory disorders, keloids, hypertrophic scars, and combinations of these.

6. The method according to any of the previous claims, wherein the effective amount of the compound decreases C2orf69 [SEQ ID NO: 1] expression to treat a fibrotic condition selected from the group consisting of dermal thinning, skin wrinkling, skin laxity, gum recession, vaginal atrophy, and combinations of these.

7. The method according to any of the previous claims, wherein he effective amount of the compound decreases expression of C2orf69 [SEQ ID NO: 1] by an amount sufficient to increase expression of one or more genes selected from the group consisting of asporin [SEQ ID NO: 5], biglycan [SEQ ID NO: 6], collagen type I alpha 1 chain [SEQ ID NO: 3], collagen type I alpha 2 chain [SEQ ID NO: 7], collagen type III alpha 1 chain [SEQ ID NO: 8], collagen type IV alpha 1 chain [SEQ ID NO: 9], collagen type V alpha 1 chain [SEQ ID NO: 10], cellular communication network factor 2 [SEQ ID NO: 11], decorin [SEQ ID NO: 12], actin alpha 2 [SEQ ID NO: 13], fibronectin 1 [SEQ ID NO: 14], laminin subunit alpha 3 [SEQ ID NO: 15], laminin subunit beta 1 [SEQ ID NO: 16], laminin subunit gamma 1 [SEQ ID NO: 17], matrix metallopeptidase 1 [SEQ ID NO: 18], matrix metallopeptidase 2 [SEQ ID NO: 19], matrix metallopeptidase 19 [SEQ ID NO: 20], nidogen 1 [SEQ ID NO: 21], serpin family E member 1 [SEQ ID NO: 22], TIMP metallopeptidase inhibitor 1 [SEQ ID NO: 23], tenascin C [SEQ ID NO: 24], versican [SEQ ID NO: 25], and vimentin [SEQ ID NO: 26] by a fold change of at least about 1.20.

8. The method according to any of the previous claims, wherein the effective amount of the compound decreases expression of C2orf69 [SEQ ID NO: 1] by an amount sufficient to decrease expression of one or more genes selected from the group consisting of integrin subunit alpha 1 [SEQ ID NO: 27], integrin subunit beta 1 [SEQ ID NO: 28], laminin subunit alpha 1 [SEQ ID NO: 29], matrix metallopeptidase 3 [SEQ ID NO: 30], thrombospondin 1 [SEQ ID NO: 31], thrombospondin 2 [SEQ ID NO: 32], and TIMP metallopeptidase inhibitor 2 [SEQ ID NO: 33] by a fold change of less than about 0 8

9. The method according to any of the previous claims, wherein the compound is administered over the course of a treatment period of greater than 1 week.

10. The method according to any of the previous claims, wherein the compound is administered intradermally, topically, orally, subcutaneously, or as an inhalant.

11. A method of identifying compounds that modulate the expression of C2orf69 [SEQ ID NO: 1], comprising: a) contacting a plurality of fibroblasts in a test sample with a test compound, wherein the fibroblasts include a reporter gene inserted upstream of an endogenous C2orf69 [SEQ ID NO: 1] promoter; and b) determining reporter gene expression by measuring an amount of fluorescent protein produced by the fibroblasts; c) comparing the reporter gene expression of the test sample to a control; d) identifying the compound as able to modulate C2orf69 [SEQ ID NO: 1] expression when the amount of fluorescent protein of the test sample is significantly different from the control.