WO2021141733A1 - Méthode permettant de moduler une pathologie fibrotique - Google Patents

Méthode permettant de moduler une pathologie fibrotique Download PDF

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WO2021141733A1
WO2021141733A1 PCT/US2020/065002 US2020065002W WO2021141733A1 WO 2021141733 A1 WO2021141733 A1 WO 2021141733A1 US 2020065002 W US2020065002 W US 2020065002W WO 2021141733 A1 WO2021141733 A1 WO 2021141733A1
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seq
c2orf69
expression
fibrosis
compound
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PCT/US2020/065002
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Robert Joseph Isfort
Frederic Bard
Hui Hui WONG
Sze Hwee SEET
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The Procter & Gamble Company
Agency For Science, Technology And Research (A*Star)
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Publication of WO2021141733A1 publication Critical patent/WO2021141733A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types

Definitions

  • 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.
  • 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.
  • 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.
  • Collagen is the primary component in the extracellular matrix (ECM) and makes up approximately one-third of the protein in the human body.
  • ECM extracellular matrix
  • 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.
  • 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.
  • C2orf69 Chromosome 2 Open Reading Frame 69
  • a method of modulating a fibrotic process 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.
  • C2orf69 Chromosome 2 Open Reading Frame 69
  • 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.
  • 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.
  • C2orf69 Chromosome 2 Open Reading Frame 69
  • 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).
  • a subject gene signature e.g., in the form of gene identifiers and direction of regulation
  • other optional components described herein e.g., metadata
  • “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.
  • the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
  • “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.
  • GRAS Generally Recognized as Safe
  • cosmetic agents may be incorporated in a cosmetic composition comprising a dermatologically acceptable carrier suitable for topical application to skin.
  • 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.
  • 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.
  • Safety 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).
  • collagen synthesizing genes 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.
  • 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.
  • 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.
  • 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.
  • C2orf69 [SEQ ID NO: 1] 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.
  • 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.
  • 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]
  • 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.
  • 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.
  • a treatment compound that interferes with the activity of a gene expression product of C2orf69 [SEQ ID NO: 1] 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.
  • 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.
  • the reporter gene may encode for a protein that emits at a defined fluorescent wavelength when excited by a specific wavelength range.
  • 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”).
  • RFPs are mCherry, mStrawberry, mOrange, and dTomato.
  • 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.
  • TALEN transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • transcriptomic analysis may be desirable to use transcriptomic analysis to measure gene activity of cells contacted with a test compound relative to a control.
  • 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]
  • transcriptomic analysis 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • Western blot mass spectrometry
  • UY absorption a bicinchoninic acid
  • a Bradford assay a Kjeldahl assay
  • a Folin-Lowry assay a conventional 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 Syn
  • 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).
  • 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.
  • 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.
  • 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
  • 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
  • the treatment compound is mixed with a suitable acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compositions herein may be administered in accordance with conventional methods of administering compositions of the type.
  • the compositions may be administered intravenously, intradermally, subcutaneously, orally (e.g., via inhalation or ingestion), topically, and/or transmucosally.
  • the present compositions may be administered topically by applying an effective amount of the composition (e.g., 0.1 mg/cm 2 to about 20 mg/cm 2 ) to a target area of skin where treatment is needed or desired.
  • an effective amount of the composition e.g., 0.1 mg/cm 2 to about 20 mg/cm 2
  • 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.
  • 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.
  • 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.
  • Example 1 Identification of C2orf69 [SEQ ID NO: 1] as a novel collagen inhibitor.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • C2orf69 [SEQ ED NO: 1] 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.
  • 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).
  • Example 4 The role of secreted C2orf69 protein [SEQ ID NO: 2] in the control of collagen synthesis.
  • C2orf69 [SEQ ID NO: 1] revealed the presence of a signal peptide, suggesting that it may function as a secreted protein.
  • C2orf69 protein [SEQ ID NO: 2] was expressed in bacteria and the resulting recombinant protein purified by Nickel-affinity chromatography.
  • 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.
  • 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.
  • 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.
  • Example 7 C2orf69 protein [SEQ ID NO: 2] blocks TGFb-mediated collagen synthesis.
  • T ⁇ Rb transforming growth factor beta
  • 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).
  • 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.
  • 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.
  • 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

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Abstract

Une méthode permettant de moduler la fibrose dans un tissu d'un sujet par augmentation ou diminution de l'expression de C2orf69. Il a été trouvé que l'expression de C2orf69 inhibe l'expression de gènes impliqués dans la construction, la réparation et l'entretien de la matrice extracellulaire. En particulier, C2orf69 inhibe la synthèse du collagène de COL1A1. En modulant l'expression de C2orf69, la synthèse du collagène peut être augmentée ou diminuée, ce qui peut procurer divers avantages cosmétiques et médicaux.
PCT/US2020/065002 2020-01-07 2020-12-15 Méthode permettant de moduler une pathologie fibrotique WO2021141733A1 (fr)

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