WO2022258310A1 - Procédé épigénétique pour détecter une lésion cutanée induite par un rayonnement ultraviolet - Google Patents

Procédé épigénétique pour détecter une lésion cutanée induite par un rayonnement ultraviolet Download PDF

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Publication number
WO2022258310A1
WO2022258310A1 PCT/EP2022/063042 EP2022063042W WO2022258310A1 WO 2022258310 A1 WO2022258310 A1 WO 2022258310A1 EP 2022063042 W EP2022063042 W EP 2022063042W WO 2022258310 A1 WO2022258310 A1 WO 2022258310A1
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WO
WIPO (PCT)
Prior art keywords
uvr
cpg loci
skin
cpg
skin damage
Prior art date
Application number
PCT/EP2022/063042
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English (en)
Inventor
Arthika BAPPAL
Bethany May BARNES
David Andrew Gunn
John Chun Sing NIP
Sheila Alves ROCHA-GUSHIKEM
Rachel Elizabeth Beatrice WATSON
Original Assignee
Unilever Ip Holdings B.V.
Unilever Global Ip Limited
Conopco, Inc., D/B/A Unilever
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Application filed by Unilever Ip Holdings B.V., Unilever Global Ip Limited, Conopco, Inc., D/B/A Unilever filed Critical Unilever Ip Holdings B.V.
Publication of WO2022258310A1 publication Critical patent/WO2022258310A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/148—Screening for cosmetic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to an epigenetic method for indicating actual or potential ultraviolet radiation (UVR)-induced skin damage in an individual.
  • UVR ultraviolet radiation
  • UVR ultraviolet radiation
  • UVR ultraviolet radiation
  • WO 2019/238792 describes a specific set of CpG loci (aka CpG dinucleotides, CpG sites) that have enhanced efficacy in determining the effect of sun exposure on an individual and that can be used in an epigenetic method to assess the impact of sun exposure on the human skin.
  • WO 2019/238793 describes a specific set of CpG loci that have enhanced efficacy in determining the effect of use of sun protection products on an individual and that can be used in an epigenetic method to assess the impact of the use of sun protection products.
  • the CpG loci disclosed in WO 2019/238792 and WO 2019/238793 are excellent markers of sun exposure in older subjects. However, they were less effective in younger subjects, where changes occurred to a lesser extent both in number and in magnitude.
  • the present invention addresses this problem.
  • the present invention provides an epigenetic method for indicating actual or potential ultraviolet radiation (UVR)-induced skin damage in a human individual aged between 18 and 30 years; the method comprising:
  • the set of CpG loci is at least 2 of, preferably at least 10 of, more preferably at least 20 of, most preferably at least 30 of and ideally all the following 39 CpG loci: cg01079842, cg01665432, cg02078727, cg02737747, cg04812879, cg04857880, cg06090739, cg06636244, cg06823550, cg07198365, cg07482373, cg07869839, cg08464076, cg10488100, cg11148483, cg11607742, cg11920406, cg12092932, cgg
  • the invention also provides a kit comprising reagents and instructions for carrying out the epigenetic method as defined above.
  • epidermal as used herein means relating to, being, or involving a modification in gene expression that is not primarily through alterations of DNA sequence.
  • genomic DNA refers to DNA derived from the genetic material in the chromosomes of a human individual.
  • Cytosine methylation is an epigenetic modification of DNA in which cytosine is converted to 5- methylcytosine in a reaction that involves flipping a target cytosine out of an intact double helix and transfer of a methyl group from S-adenosylmethionine by a methyltransferase enzyme. Cytosine methylation in vertebrates occurs predominantly at dinucleotide sequences where the cytosine is followed by guanine (CpG). Accordingly, the term “cytosine methylation status” as used herein refers to the presence or absence of a methylated cytosine (5-methylcytosine) at a particular CpG locus in the genomic DNA.
  • CpG locus or “CpG loci” denote respectively, one, or a plurality, of the unique identifiers found in the lllumina® CpG loci database (as described in Technical Note: Epigenetics, CpG Loci Identification ⁇ 2010 lllumina® Inc.,
  • CpG loci There are more than 28 million CpG loci (aka CpG dinucleotides, CpG sites) in the human genome.
  • CpG sequences are symmetric on forward and reverse strands of any double- stranded DNA.
  • the lllumina® method consistently designates CpG loci based on the actual or contextual sequence of each individual CpG locus, taking advantage of sequences flanking a CpG locus to generate a unique CpG locus cluster ID. This method will consistently designate the same CpG locus identifier and orientation calls even if public databases and genome assemblies change.
  • UVR-induced skin damage refers to skin damage resulting from exposure to UVR in the A (320-400 nm), B (290-320 nm), or C ranges (200-290 nm) or combinations thereof.
  • UVR-induced skin damage include cosmetic damage (such as the visual and textural skin changes which are collectively termed photoaging) as well as skin pathologies such as melanoma and non-melanoma skin cancers.
  • Sources of UVR in the context of this invention include natural sources (such as the sun), and/or artificial sources (such as black lights, welding equipment, lasers, and tanning equipment).
  • Epidermal skin cells for use in the epigenetic method of the invention may be obtained by a variety of techniques such as punch biopsy, surgical excision, and non-invasive or minimally invasive skin sampling methods such as wet swabbing, tape lift, cotton tip swabbing, skin scraping or employing a small gauge needle (for example, 28 gauge) to collect micro-cores of skin tissue.
  • a small gauge needle for example, 28 gauge
  • the set of CpG loci shows improved sensitivity in younger subjects, and so can be used to provide an indicator of actual or potential UVR-induced skin damage in younger subjects.
  • the term “younger subjects” in the context of this invention denotes human individuals aged from 18 to 30 years of age.
  • the information provided by the epigenetic method of the invention can thus be used to provide personalised advice and interventions which enable effective prevention of future skin damage and photoaging in younger subjects.
  • the set of CpG loci is at least 2 of, preferably at least 5 of, more preferably at least 10 of, most preferably at least 15 of and ideally all the following 19 CpG loci: cg01079842, cg01665432, cg02737747, cg04812879, cg06823550, cg07869839, cg11920406, cg12299478, cg13362436, cg13991350, cg14203437, cg15669600 cg15935526, cg21172464, cg22355498, cg25586364, cg25655489, cg26586287 and cg27579771.
  • the set of CpG loci is at least 2 of, preferably at least 5 of, more preferably at least 10 of and most preferably all the following 13 CpG loci: cg02078727, cg06636244, cg07198365, cg07482373, cg10488100, cg11148483, cg11607742, cg17381727, cg22247002, cg22384883, cg23090732, cg26164878 and cg26849382.
  • the cytosine methylation status of the set of CpG loci in the test sample provides an indicator of actual or potential UVR-induced skin damage.
  • the cytosine methylation status of the set of CpG loci in the test sample is compared to that of the same set of CpG loci in a control sample; and differences in the cytosine methylation status between the test sample and the control sample provides an indicator of actual or potential UVR-induced skin damage;
  • control sample in the context of this invention may include a skin sample obtained from a subject that has not been exposed to UVR, or whose exposure to UVR has been blocked or attenuated; or a skin sample obtained from the test subject at a body site that has not been exposed to UVR, or whose exposure to UVR has been blocked or attenuated.
  • control sample in the context of this invention may also include a standard reference value or range of values which may be determined empirically or historically from single or multiple skin samples from the same test subject, or from a reference subject or subjects, or a previously established range of values that represent baseline or normal values.
  • the epigenetic method of the invention as described above may also be used to measure the effectiveness of a test agent in reducing actual or potential ultraviolet radiation (UVR)- induced skin damage in a human individual.
  • UVR ultraviolet radiation
  • the source of UVR in the context of the above method may include natural sources (such as the sun), and/or artificial sources (such as a simulated solar radiation (SSR) source).
  • natural sources such as the sun
  • artificial sources such as a simulated solar radiation (SSR) source
  • a kit comprising reagents and instructions for carrying out the epigenetic methods as described above may suitably comprise primers or probes which specifically bind to the set of CpG loci as defined above; and a reagent used in: a genomic DNA polymerization process; a genomic DNA hybridization process; a genomic DNA direct sequencing process; a genomic DNA bisulfite conversion process; or a genomic DNA pyrosequencing process.
  • primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions for example, buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase.
  • the length of the primer in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides.
  • probe refers to a surface-immobilized molecule that can be recognized by a particular target.
  • the term "specifically bind” as used herein denotes a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times the background molecular association under physiological conditions.
  • kits comprising reagents and instructions for carrying out the epigenetic methods as described above may comprise reagents used in direct sequencing methods which measure the methylation status of the CpG loci as defined above.
  • Direct sequencing methods measure the nucleotide sequence of DNA, including the presence of DNA methylation, directly without the need of primers or probes (targeted or untargeted).
  • An example of a direct sequencing method is nanopore sequencing, which works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore.
  • SSR solar-simulating radiation
  • the minimal erythemal dose i.e. , the amount of SSR needed to produce reddening of the skin, was determined for each test subject.
  • test skin site was irradiated with a total of 5 suberythemogenic doses of SSR (80% of the subject’s MED per dose), with each dose administered to the test skin site on separate days over a week.
  • irradiated skin from the test skin site and non-irradiated skin from a control skin site was biopsied and quadrisected to allow DNA methylation analysis.
  • Epidermis was disassociated with dispase, and the methylation of the extracted DNA was interrogated using the lllumina® Infinium MethylationEPIC BeadChip. Differential methylation was determined by the limma package (Nucleic Acids Research, 2015, Vol. 43, No. 1 e47).
  • the methylation of the extracted DNA was interrogated using the lllumina® MethylationEPIC (850k) microarray, to measure the differential methylation between irradiated test samples and non-irradiated control samples.
  • the results showed that across the 3 separate ex-vivo experiments (i) to (iii), the methylation status of 39 CpG sites as shown in Table 1 also changed in the same expected direction in response to SSR.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
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Abstract

La présente invention procure un procédé épigénétique pour indiquer les dommages cutanés réels ou potentiels induits par le rayonnement ultraviolet (UVR) chez un individu humain ; le procédé comprenant les étapes suivantes : obtention d'un échantillon de test d'ADN génomique à partir de cellules épidermiques de la peau prélevées sur l'individu ; dans l'échantillon de test, la détermination de l'état de méthylation de la cytosine d'un ensemble de loci CpG dans l'ADN génomique, l'état de méthylation de la cytosine ainsi déterminé fournissant un indicateur de lésions cutanées réelles ou potentielles induites par les UVR, et caractérisé en ce que l'ensemble de loci CpG est au moins 2 des 39 loci CpG suivants, de préférence au moins 10, plus préférentiellement au moins 20, le plus préférentiellement au moins 30, et idéalement tous : cg01079842, cg01665432, cg02078727, cg02737747, cg04812879, cg04857880, cg06090739, cg06636244, cg06823550, cg07198365, cg07482373, cg07869839, cg08464076, cg10488100, cg11148483, cg11607742, cg11920406, cg12092932, cg12299478, cg13027321, cg13362436, cg13991350, cg14203437, cg15669600, cg15935526, cg17381727, cg21004104, cg21172464, cg22247002, cg22355498, cg22384883, cg23090732, cg23919385, cg25586364, cg25655489, cg26164878, cg26586287, cg26849382 et cg27579771.
PCT/EP2022/063042 2021-06-07 2022-05-13 Procédé épigénétique pour détecter une lésion cutanée induite par un rayonnement ultraviolet WO2022258310A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180078870A (ko) * 2016-12-30 2018-07-10 의료법인 성광의료재단 피부노화 특이적 후성유전자 마커 및 이를 이용한 피부노화의 검출
WO2019238793A1 (fr) 2018-06-15 2019-12-19 Unilever Plc Procédé épigénétique d'évaluation de la protection contre le soleil
WO2019238792A1 (fr) 2018-06-15 2019-12-19 Unilever Plc Procédé épigénétique pour évaluer l'exposition au soleil

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180078870A (ko) * 2016-12-30 2018-07-10 의료법인 성광의료재단 피부노화 특이적 후성유전자 마커 및 이를 이용한 피부노화의 검출
WO2019238793A1 (fr) 2018-06-15 2019-12-19 Unilever Plc Procédé épigénétique d'évaluation de la protection contre le soleil
WO2019238792A1 (fr) 2018-06-15 2019-12-19 Unilever Plc Procédé épigénétique pour évaluer l'exposition au soleil

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
AMY R VANDIVER ET AL: "Age and sun exposure-related widespread genomic blocks of hypomethylation in nonmalignant skin", GENOME BIOLOGY, vol. 16, no. 1, 16 April 2015 (2015-04-16), pages 80, XP021221763, ISSN: 1465-6906, DOI: 10.1186/S13059-015-0644-Y *
ANONYMOUS: "Data Sheet: Epigenetics Infinium MethylationEPIC BeadChip", 19 October 2015 (2015-10-19), XP055833502, Retrieved from the Internet <URL:https://filgen.jp/Product/Bioscience/Methyl/humanmethylationepic-data-sheet-1070-2015-008.pdf> [retrieved on 20210819] *
DATABASE EMBASE [online] ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL; 1 March 2020 (2020-03-01), ROCHA S ET AL: "Short-term, incidental sun-exposure causes epigenetic drift, which is significantly reduced through daily spf use", XP002804808, Database accession no. EMB-634430343 *
NUCLEIC ACIDS RESEARCH, vol. 43, no. 7, 2015, pages e47
SATHYANARAYANA ET AL: "Sun exposure related methylation in malignant and non-malignant skin lesions", CANCER LETTERS, NEW YORK, NY, US, vol. 245, no. 1-2, 22 December 2006 (2006-12-22), pages 112 - 120, XP005813550, ISSN: 0304-3835, DOI: 10.1016/J.CANLET.2005.12.042 *
VANDIVER AMY ET AL: "Table S1: Donor Demographics - Supplementary material for "Age and sun exposure-related widespread genomic blocks of hypomethylation in nonmalignant skin"", GENOME BIOLOGY, 16 April 2015 (2015-04-16), XP055949316, Retrieved from the Internet <URL:https://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0644-y#Sec23> [retrieved on 20220805] *
VANDIVER AMY R ET AL: "Table S3 - online supplementary material for "Age and sun exposure-related widespread genomic blocks of hypomethylation in nonmalignant skin"", GENOME BIOLOGY, 16 April 2015 (2015-04-16), XP055863749, Retrieved from the Internet <URL:https://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0644-y#Sec23> [retrieved on 20211119] *

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