WO2025087779A1

WO2025087779A1 - A measure of ageing based on epigenetics

Info

Publication number: WO2025087779A1
Application number: PCT/EP2024/079302
Authority: WO
Inventors: Jennifer BOURLAND; Sanjanaa NAGARAJAN; Suki ROY; Dhivya C DADLANI; Lingzhi Huang; Kit Yeng WONG; Florian Böhl
Original assignee: Evonik Operations GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2023-10-27
Filing date: 2024-10-17
Publication date: 2025-05-01
Anticipated expiration: 2026-04-27
Also published as: TW202536190A

Abstract

The present invention relates to an in vitro method of assessing the effect of at least one test compound on ageing in a mammalian cell, the method comprising: (a) contacting the test compound with a mammalian test cell; (b) determining the methylation status of at least one gene in a panel of genes from a DNA sample obtained from the test cell of (a); (c) comparing the test methylation status obtained from (b) to a control methylation status of the corresponding gene from a DNA sample obtained from the mammalian cell before contacting the cell with the test compound; wherein a significant similarity in the test methylation status of (b) compared to the control methylation status, is indicative of the test component having no effect on ageing in the mammalian cell; wherein a significant difference in the test methylation profile of (b) compared to the control methylation status, is indicative of the test component having an effect on ageing in the mammalian cell; wherein a measure of the significant difference in the test methylation profile of (b) compared to the control methylation status, is indicative of how much the test component has an effect on ageing in the mammalian cell test and wherein the gene in step (b) is selected from the group consisting of COL6A2, COL27A1 and KRT13 and the regulatory regions of the same.

Description

A MEASURE OF AGEING BASED ON EPIGENETICS

FIELD OF THE INVENTION

The present invention relates to a method for diagnosing a cell with ageing. In particular, the method is capable of identifying (qualitative) and measuring (quantitative) ageing in skin cell by determining differential methylation that takes place on CpG sites located on a particular gene body and/or the regulatory region of the specific gene.

BACKGROUND OF THE INVENTION

Skin is the largest organ of the human body and acts as a physical, chemical and biological barrier between the external environment and the organism. It plays a fundamental role in the defence against external factors such as UV and pollution but is also affected by individual lifestyle such as diet. The effect of exposome on skin is known to play a role in skin aging and to accelerate biological aging through multiple mechanisms.

Designing products and solutions to reduce the effect of exposome and lifestyle on skin aging is at the core of the cosmetic and pharmaceutical industry. In order to be able to assess and identify the most effective products, in vitro experiments based on either skin cells, skin tissue models or explants are key. They are often exposed to conditions mimicking aging or accelerated aging, however, there is no current way to correlate simulated aging under laboratory conditions to physiological aging in humans.

Further, the continuous exposure to air, radiation and UV rays, environmental pollutants, as well as physical and/or chemical agents (e.g., cosmetics) to human skin results in the ageing of the skin. Besides using the naked eye to observe ageing there is no specific means available to quantify ageing. There is thus a need in the art not only for a means to measure ageing, but an accurate and easy means of measuring ageing and to determine the effect of any component to reduce or halt the ageing process.

DESCRIPTION OF THE INVENTION

The present invention attempts to solve the problems above by providing at least one gene which when differentially methylated compared to a control cell, is capable of being used for detecting ageing in a cell. In particular, the ageing in the cell may be caused by different stresses (e.g. UV rays, environmental pollutants, physical and/or chemical agents (e.g., cosmetics)) and the differential methylation in the gene can be used to identify the ageing in the cell.

In particular, the present invention attempts to solve the problems above by providing a method of using a gene panel with at least two genes that are differentially methylated in a cell with ageing. In particular, at least two genes, where at least one of the genes is selected from the group consisting of COL6A2, COL27A 1 and KRT13, differential methylation of which, is capable of being used for detecting ageing in a cell.

Since environmental factors/ agents such as, air, radiation and UV rays, environmental pollutants, as well as physical and/or chemical agents and the like, may trigger ageing which can further induce an alteration in the promoter CpG methylation status by recruiting DNA methyltransferases (DNMTs) and TET enzymes to various promoters, a biomarker resulting in differential methylation in a cell exposing ageing is essential to overcome the problems mentioned above. In particular, an example of a biomarker for detecting ageing in a cell is differential methylation of at least one gene selected from the group consisting of collagen type VI alpha 2 chain (COL6A2), Collagen Type XXVII Alpha 1 Chain (COL27A ) and Keratin 13 (KRT13). The CpGs of COL6A2, COL27A 1 and KRT13 in a cell with ageing are differentially methylated (i.e. hypomethylated or hypermethylated) compared to the corresponding CpGs in a cell without ageing. In particular, the CpGs of at least one of the genes, COL6A2, COL27A 1 and KRT13 are differentially methylated because of ageing. Accordingly, any one of the three genes COL6A2, COL27A 1 and KRT13 may be effectively used to determine if a cell exhibits ageing. Even more in particular, the group of CpGs are one or more pre-selected CpG sites which are differentially methylated in cells with ageing. This is particularly advantageous as using epigenetics provides a means of predicting the onset of ageing in a cell, thus allowing ageing to be treated earlier before causing even more damage to the cell. Further, this method according to any aspect of the present invention may be used to determine the effect of at least one component on ageing in a cell by determining the methylation status of any one of the 3 genes before exposure to the component and after exposure to the component. An epigenetic marker is a long-term biomarker, that is to say it is inheritable and can be used to detect ageing in the next generation as well if need be.

According to one aspect of the present invention, there is provided an in vitro method of assessing the effect of at least one test compound on ageing in a mammalian cell, the method comprising:

(a) contacting the test compound with a mammalian test cell;

(b) determining the methylation status of at least one gene in a panel of genes from a DNA sample obtained from the test cell of (a);

(c) comparing the test methylation status obtained from (b) to a control methylation status of the corresponding gene from a DNA sample obtained from the mammalian cell before contacting the cell with the test compound; wherein a significant similarity in the test methylation status of (b) compared to the control methylation status, is indicative of the test component having no effect on ageing in the mammalian cell; wherein a significant difference in the test methylation profile of (b) compared to the control methylation status, is indicative of the test component having an effect on ageing in the mammalian cell; wherein a measure of the significant difference in the test methylation profile of (b) compared to the control methylation status, is indicative of how much the test component has an effect on ageing in the mammalian cell test and wherein the gene in step (b) is selected from the group consisting of COL6A2, COL27A 1 and KRT13 and the regulatory regions of the same.

The present invention is based on the finding that different components of the environment can change the epigenome of the cell through epigenetics. In particular, the capability to adapt to the environment and maintain the adapted biological pattern depends on epigenetic mechanisms, including DNA methylation. More in particular, the present invention is based on the finding that the environment may result in ageing and this also results in changes in epigenetic mechanisms of the cell, including DNA methylation patterns and these patterns may be passed down to the different products that may derive from the cell.

The inventors have unexpectedly found that this property can be utilized to identify "epigenetic fingerprints" on the genome of particularly the skin cell, that are specific to a component that may be used to stop, prevent, reduce or increase ageing of not just one cell being fed the component but possibly all the cells that are exposed to this component. Based on these findings, the present invention provides means to identify the specific effect short term and in the long run of any component on ageing in a cell In particular, the method according to any aspect of the present invention may be used to determine if a specific component has a positive or negative effect on ageing in a cell. For example, a component X when brought into contact with a test skin cell may stop, prevent and/or reduce ageing of the test cell in the short and/ or long run resulting in the cell having reduced ageing compared to a cell not brought into contact with the component X. In another example, a component Y may increase ageing or have no effect on ageing of the cell when the cell is brought into contact with the component. More in particular, the method according to any aspect of the present invention may be used to determine if a particular component of a cosmetic agent itself has a positive or negative effect on the ageing of the cell per se. In this way, the method according to any aspect of the present invention may then be used to accurately, reliably and quickly determine the specific effect of a component in a cosmetic agent on the cell and based on these results, it can be decided if the component should be included in the cosmetic agent of the cell or should be removed from the cosmetic agent in which the cell is exposed to.

In particular, differential methylation in the specific genes COL6A2, COL27A1 and KRT13 are more effective in being used as markers for detecting ageing compared to other markers (e.g. other genes) known in the art as the specific genes COL6A2, COL27A1 and KRT13 individually or in combination are able to identify ageing in a cell caused by more than one mechanism. In particular, ageing in a (test) cell may be caused by one or more mechanisms or stimuli such as environmental factors/ agents such as, air, radiation and UV rays, environmental pollutants, as well as physical and/or chemical agents and the like. The ageing in the (test) cell caused by one or more mechanisms can be detected effectively by measuring differential methylation in the specific genes COL6A2, COL27A1 and KRT13.

As used herein, the term "cell" refers to an intact live cell, naturally occurring or modified. The cell may be isolated from other cells, mixed with other cells in a culture, or within a tissue (partial or intact), or an organism. In particular, the cell may be a eukaryote cell. More in particular, the cell may be mammalian cell. The term "mammalian cell" refers to any cell derived from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. The cell may also have been genetically modified to express a recombinant protein and/or nucleic acid. The mammalian cell may be from humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters, and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. In particular, the subject is a mammal. More in particular, the mammal is selected from the group consisting of a mouse, a rat, a guinea pig, a dog, a mini-pig, a human being, a cow, a sheep, a pig, a goat, a horse, a donkey, and a mule. In particular, the mammalian cell may be a skin cell, or a cell derived therefrom. More in particular, the mammalian cell may be a skin cell.

As used herein, a “CpG site” or “methylation site” is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation either by natural occurring events in vivo or by an event instituted to chemically methylate the nucleotide in vitro. Some of these sites may be hypermethylated and some may be hypomethylated in a cell with ageing compared to a cell with no ageing.

As used herein, a “methylated nucleic acid molecule” refers to a nucleic acid molecule that contains one or more nucleotides that is/are methylated.

A “CpG island” as used herein describes a segment of DNA sequence that comprises a functionally or structurally deviated CpG density. For example, Yamada et al. have described a set of standards for determining a CpG island: it must be at least 400 nucleotides in length, has a greater than 50% GC content, and an OCF/ECF ratio greater than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266). Others have defined a CpG island less stringently as a sequence at least 200 nucleotides in length, having a greater than 50% GC content, and an OCF/ECF ratio greater than 0.6 (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 3740-3745). In context of the present invention, the terms “methylation profile”, “methylation pattern”, “methylation state” or “methylation status,” are used herein to describe the state, situation or condition of methylation of a genomic sequence, and such terms refer to the characteristics of a DNA segment at a particular genomic locus in relation to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, location of methylated C residue(s), percentage of methylated C at any particular stretch of residues, and allelic differences in methylation due to, e.g., difference in the origin of the alleles.

The term "methylation status" refers to the status of a specific methylation site (i.e. methylated vs. nonmethylated) which means a residue or methylation site is methylated or not methylated. Then, based on the methylation status of one or more methylation sites, a methylation profile may be determined. Accordingly, the term "methylation profile" or also “methylation pattern” refers to the relative or absolute concentration of methylated C residues or unmethylated C residues at any particular stretch of residues in the genomic material of a biological sample. For example, if cytosine (C) residue(s) not typically methylated within a DNA sequence are methylated, it may be referred to as "hypermethylated"; whereas if cytosine (C) residue(s) typically methylated within a DNA sequence are not methylated, it may be referred to as "hypomethylated". Likewise, if the cytosine (C) residue(s) within a DNA sequence (e.g., the DNA from a sample nucleic acid from a test subject) are methylated as compared to another sequence from a different region or from a different individual (e.g., relative to normal nucleic acid or to the standard nucleic acid of the reference sequence), that sequence is considered hypermethylated compared to the other sequence. Alternatively, if the cytosine (C) residue(s) within a DNA sequence are not methylated as compared to another sequence from a different region or from a different individual, that sequence is considered hypomethylated compared to the other sequence. These sequences are said to be "differentially methylated". Measurement of the levels of differential methylation may be done by a variety of ways known to those skilled in the art. One method is to measure the methylation level of individual interrogated CpG sites determined by the bisulfite sequencing method, as a non-limiting example.

As used herein, a “methylated nucleotide” or a “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is usually not present in a recognized typical nucleotide base. For example, cytosine in its usual form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine in its usual form may not be considered a methylated nucleotide and 5- methylcytosine may be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at position 5 of its pyrimidine ring, however, for purposes herein, thymine may not be considered a methylated nucleotide when present in DNA. Typical nucleotide bases for DNA are thymine, adenine, cytosine and guanine. Typical bases for RNA are uracil, adenine, cytosine and guanine. Correspondingly a "methylation site" is the location in the target gene nucleic acid region where methylation has the possibility of occurring. For example, a location containing CpG is a methylation site wherein the cytosine may or may not be methylated. In particular, the term “methylated nucleotide” refers to nucleotides that carry a methyl group attached to a position of a nucleotide that is accessible for methylation. These methylated nucleotides are usually found in nature and to date, methylated cytosine that occurs mostly in the context of the dinucleotide CpG, but also in the context of CpNpG- and CpNpN- sequences may be considered the most common. In principle, other naturally occurring nucleotides may also be methylated but they will not be taken into consideration with regard to any aspect of the present invention.

In context of the present invention, the terms “methylation profile”, “methylation pattern”, “methylation state” or “methylation status,” are used herein to describe the state, situation or condition of methylation of a genomic sequence, and such terms refer to the characteristics of a DNA segment at a particular genomic locus in relation to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, location of methylated C residue(s), percentage of methylated C at any particular stretch of residues, and allelic differences in methylation due to, e.g., difference in the origin of the alleles.

The term “hypermethylation” refers to the average methylation state corresponding to an increased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample. In particular, control refers to a cell with no or non-measurable indication of ageing. The term “hypomethylation” refers to the average methylation state corresponding to a decreased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample. In particular, control refers to a cell with no or non-measurable indication of ageing.

In particular, when there is differential methylation detected in a test cell, that is to say that the cell displays absolute hypermethylation or hypomethylation or at least quantitative differential methylation at, at least one CpG site in comparison to the reference (i.e., from a cell without ageing), then the test cell also has no ageing. More in particular, when the CpG site displays the same methylation status in the test cell in comparison to the corresponding CpG site in the reference cell or reference methylation profile, the test cell has no ageing.

As used herein, the term “gene’ refers to the respective genomic DNA sequence, including any promoter and regulatory sequences of the gene (e.g., enhancers and other gene sequences involved in regulating expression of the gene), and/or the body of the gene in itself. A gene sequence may be an expressed sequence (e.g., expressed RNA, mRNA, cDNA). Further, where SNPs are known within genes the term shall be taken to include all sequence variants thereof.

The term ‘epigenetic change’ as used herein refers to a chemical (e.g., methylation) change or protein (e.g., histones) change that takes place to a gene body or a promoter thereof. Through epigenetic changes, environmental factors like, diet, stress and prenatal nutrition can make an imprint on genes passed from one generation to the next.

As used herein, the term “significantly similar” refers to in particular in context with the comparison of methylation profiles (such as the comparison between test profiles (from test subject(s) and reference profiles) a similarity observed by statistical means (i.e. by using bioinformatics) and/or also by observation using the eye. A significant similarity is observed for example if a test profile overlaps with a reference profile that is defined by multiple training samples through multivariate statistical methods, such as Principal Component analysis or Multi-Dimensional Scaling. In particular, a test profile is significantly similar to the pre-determined reference profile if more than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 % of the methylation pattern/ profile overlaps with that of the reference profile. A similarity of a test profile to more than one, such as two, three or even all reference profile reduces the significance of the similarity.

As used herein, the term “genomic material” refers to nucleic acid molecules or fragments of the genome of the subject or group of subjects. In particular, such nucleic acid molecules or fragments are DNA or RNA or hybrids thereof, and most preferably are molecules of the DNA genome of a subject or group of subjects.

As used herein, the “promoter” or “gene promoter” used interchangeably with the term ‘regulatory region’ or ‘regulatory sequence’ refers to the respective contiguous gene DNA sequence extending from 1 .5 kb upstream to 1 .5 kb downstream relative to the transcription start site (TSS), or contiguous portions thereof. In particular, ‘regulatory region’ refers to the respective contiguous gene DNA sequence extending from 1 .5 kb upstream to 0.5 kb downstream relative to the TSS. In some examples, ‘regulatory region’ refers to the respective contiguous gene DNA sequence extending from 1 .5 kb upstream to the downstream edge of a CpG island that overlaps with the region from 1 .5 kb upstream to 1 .5 kb downstream from TSS (and is such cases, my thus extend even further beyond 1 .5 kb downstream), and contiguous portions thereof. In particular, with respect to COL6A2, COL27A1 and/or KRT13, any CpG dinucleotide of the gene that is coordinately methylated with the ‘regulatory region’ of the gene, has substantial diagnostic/classification utility as disclosed herein.

As used herein, the “DNA sample” refers to the DNA extracted from the cell according to any aspect of the present invention using known methods in the art.

In particular, when there is differential methylation detected in a test cell, that is to say that the cell displays hypermethylation or hypomethylation at, at least one CpG site in comparison to the control (i.e. , a cell without indication of ageing), then the test cell has ageing.

As used herein, the term “pre-selected methylation sites” refers to methylation sites that were selected from genes or regions that showed the highest degree of methylation variation during the training of the method and fulfils certain quality criteria such as a minimum sequencing coverage of >5x were considered and for >5 qualified CpG sites. Additionally, genes that have an average methylation level <0.1 or an average methylation level >0.9 can be excluded due to their limited dynamic range. In particular, the pre-selected methylation sites may be located in at least one of the three genes COL6A2, COL27A1 and KRT13.

In particular, in the method according to any aspect of the present invention, in step (a) the methylation status of at least 1 , 2, or 3, genes are determined. More in particular, in step (a) the methylation status of at least 2 or 3 genes are determined.

In one example, the methylation status of at least 3 genes are determined in step (a). The 3 genes are COL6A2, COL27A1 and/or KRT13.

In another example, the methylation status of at least 2 genes are determined in step (a). The 2 genes are COL6A2 and COL27A1 or COL6A2 and KRT13 or COL27A1 and KRT13.

The term “test” used in conjunction with the term cell herein refers to an entity that is subjected to the method according to any aspect of the present invention and is the basis for an analysis application of the present invention. A “test cell” or a “test profile” is therefore a cell being tested according to the invention or a profile being obtained or generated in this context. Conversely, the term “reference” shall denote, mostly predetermined, entities which are used for a comparison with the test entity. For example, the term ‘reference cell refers to a cell used for comparison or as a control in reference to the ‘test cell. Similarly, the term ‘sample’ and/or ‘test cell DNA sample’ used in accordance with any aspect of the present invention refers to an entity that may be subject to the method according to any aspect of the present invention. In particular, a sample may be any DNA sample obtained from a test cell that may be subject to the method according to any aspect of the present invention to determine the effect of a selected component of the cell on ageing of the cell by first determining the DNA methylation profile and then comparing this test methylation profile with a control (reference methylation profiles from control cells showing or not showing ageing). In another example the methylation profile of a control is the DNA methylation profile of the cell before it was exposed to the selected component.

The test component according to any aspect of the present invention may be a compound or substance that may be found in cosmetic products or agents that may be used to stop, prevent or reduce ageing. The component may be selected from the group consisting of surfactants, moisturizing agents, antioxidants, emollients, preservatives, humectants, viscosity modifiers, and a mixture thereof.

As used herein, the term “surfactant” is a substance which decreases the surface tension of a composition with respect to the same composition in the absence of said component and furthermore facilitates the uniform distribution of the composition when it is used. Examples of surfactants suitable are lauryl isoquinolinium bromide and isopropyl alcohol, polysorbate 20, steareth-2 (polyethylene glycol ether (2 units) and stearyl alcohol), oleth-2 (polyethylene glycol ether (2 units) and oleyl alcohol), PEG-8 caprylic/capric glycerides (ethoxylated with 8 units of polyethylene glycol), sodium cocoamphoacetate, coconut oil esters - polyglycerol 6, almond oil esters- PEG-8, ammonium cocosulfate and avocado oil esters PEG-11 , and mixtures thereof.

As used herein, the term “moisturizing agent” refers to a substance which increases the water content of the skin or hair and helps to keep it soft. Examples of moisturizing agents are vitis vinifera seed oil, ceramide, glucosylceramide, grape oil esters - PEG-8, glyceryl esters - cocoa butter, shea butter cetyl esters, shea butter glyceride, lauryl cocoate, and mixtures thereof.

As used herein, the term “antioxidant” refers to a substance which inhibits or reduces reactions promoted by oxygen, thereby preventing oxidation and rancidity. Examples of antioxidants are tocopherol, sodium tocopheryl phosphate, 3-glyceryl ascorbate, acetylcysteine, aloe vera plant extract, ascorbic acid, ascorbyl dipalmitate, ascorbic acid polypeptide, acetyl trihexylcitrate, ascorbyl linoleate, 2- acetylhydroquinone, apo-lactoferrin, ascorbyl glucoside, ascorbyl lactoside, and mixtures thereof.

As used herein, the term “emollient” refers to a substance which softens the skin. Examples of emollients are apo-lactoferrin, acacia dealbata flower wax, acetylarginine, acetylproline, acetylhydroxyproline, acetylated glycol stearate, algae extract, almond oil esters and propylene glycol, aminopropyltocopheryl phosphate, 1 ,2,6-hexanetriol, and mixtures thereof.

As used herein, the term “preservative” refers to a substance which inhibits the development of microorganisms in the composition. Examples of preservatives are phenoxyethanol; a mixture of caprylyl glycol, glyceryl caprylate, glycerin, and phenylpropanol; a mixture of benzyl alcohol, glyceryl caprylate, and glyceryl undecylenate; a mixture of 2,2-hexanediol and caprylyl glycol; a mixture of phenethyl alcohol, and ethylhexylglycerin; a mixture of pentylene glycol, caprylyl glycol, and ethylhexylglycerin.

As used herein, the term “humectant” refers to a substance which retains humidity. Examples of humectants are 3-glyceryl ascorbate, acetylcyclodextrin, propanediol, algae extract, 2,3-butanediol, 3- ethylhexylglyceryl ascorbate, 3-laury Iglycery I ascorbate, and capryl 3-glyceryl ascorbate.

As used herein, the term “viscosity modifier” refers to a substance which increases the viscosity of a composition, particularly an aqueous composition. Examples of viscosity modifiers are carbomer, sodium carbomer, dextran sulfate sodium, carboxymethyl chitosan, propanediol, carboxymethyl dextran, steareth- 30, steareth-40, steareth-50, sodium poly polynaphthalene sulfonate, croscarmelose, sodium glycereth- polyphosphate, and mixtures thereof.

The methylation profile of the control and/or the test sample may be determined using is a DNA-based array, particularly a DNA-methylation based array. Arrays allow for a high-throughput and robust method to determine semi-quantitative/quantitative DNA-methylation information through a small sample of extracted DNA of interest. These custom designed arrays may use Illumina iScan and Infinium platform technology or an equivalent thereof, which allows on each chip for example 100,000 different bead types that covalently bind DNA-methylation probes. Each probe represents one CpG Methylation site at the end of the probe sequence. DNA samples undergo bisulfite conversion, amplification, fragmentation, precipitation and resuspension steps before hybridization on an array chip. Once on the chip the DNA hybridizes to the beads for each CpG site so that methylation changes at each site can be detected specifically through single nucleotide extension. This is especially advantageous as the array-based method is simple and the results of the array are accurate and reproducible.

Further, compared to traditional sequencing which can take weeks to generate data, the array technology has a much shorter turn-around time. The volume and complexity of data generated is lesser compared to sequencing making it computationally less intensive. This allows for quicker computation to achieve interpretable results from experimental groups. Overall microarray technology is roughly 10x faster and 10x cheaper than traditional sequencing while still quantifiable for the methylation level at specific CpG sites.

The term “array” as used herein refers to an intentionally created collection of probe molecules which can be prepared either synthetically or biosynthetically. The probe molecules in the array can be identical or different from each other. The array can assume a variety of formats, for example, libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.

In particular, an array provides a convenient platform for simultaneous analysis of large numbers of CpG sites, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, 10,000, 100,000 or more sites or loci. In particular, the array comprises a plurality of different probe molecules that can be attached to a substrate or otherwise spatially distinguished in an array. Examples of arrays that may be used according to any aspect of the present invention include slide arrays, silicon wafer arrays, liquid arrays, bead-based arrays and the like. In one example, array technology used according to any aspect of the present invention combines a miniaturized array platform, a high level of assay multiplexing, and scalable automation for sample handling and data processing.

In particular, the array according to any aspect of the present invention may be an array of arrays, also referred to as a composite array, having a plurality of individual arrays that is configured to allow processing of multiple samples simultaneously. Examples of composite arrays and the technology behind them are disclosed at least in US 6,429,027 and US 2002/0102578. A substrate of a composite array may include a plurality of individual array locations, each having a plurality of probes, and each physically separated from other assay locations on the same substrate such that a fluid contacting one array location is prevented from contacting another array location. Each array location can have a plurality of different probe molecules that are directly attached to the substrate or that are attached to the substrate via rigid particles in wells (also referred to herein as beads in wells).

In one example, an array substrate can be a fibre optical bundle or array of bundles as described in US6,023,540, US6,200,737 and/or US6,327,410. An optical fibre bundle or array of bundles can have probes attached directly to the fibres or via beads. A skilled person would be able to easily determine which substrate will be most suitable for the array according to any aspect of the present invention. W020041 10246 further discloses other substrates and methods of attaching beads to the substrates that may be used in the array according to any aspect of the present invention.

In one example, a surface of the substrate may have physical alterations to enable the attachment of probes or produce array locations. For example, the surface of a substrate can be modified to contain chemically modified sites that are useful for attaching, either-covalently or non-covalently, probe molecules or particles having attached probe molecules. Probes may be attached using any of a variety of methods known in the art including, an ink-jet printing method, a spotting technique, a photolithographic synthesis method, or printing method utilizing a mask. W02004110246 discloses these techniques in more detail.

In one example, the array according to any aspect of the present invention may be a bead-based array, where the beads are associated with a solid support such as those commercially available from Illumina, Inc. (San Diego, Calif.). An array of beads useful according to any aspect of the present invention can also be in a fluid format such as a fluid stream of a flow cytometer or similar device. Commercially available fluid formats for distinguishing beads include, for example, those used in XMAP(TM) technologies from Luminex or MPSS(TM) methods from Lynx Therapeutics.

The term “solid support”, “support”, and “substrate” as used herein are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many examples, at least one surface of the solid support will be substantially flat, although in some examples it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like.

The array or microarray according to any aspect of the present invention may be a very high-density array, for example, those having from about 10,000,000 probes/cm² to about 2,000,000,000 probes/cm² or from about 100,000,000 probes/cm² to about 1 ,000,000,000 probes/cm². High density arrays are especially useful according to any aspect of the present invention for including the multitude of CpG sites on the array.

The array according to any aspect of the present invention may be used to analyse or evaluate such pluralities of loci simultaneously or sequentially as desired. In one example, a plurality of different probe molecules can be attached to a substrate or otherwise spatially distinguished in an array. Each probe is typically specific for a particular locus and can be used to distinguish methylation state of the locus.

The term “probe molecules” or ‘probes’ as used interchangeably herein refers to a surface-immobilized molecule that can be recognized by a particular target. Probes used in the array can be specific for the methylated allele of a CpG site, the non-methylated allele of the CpG site or both or for the methylated allele of a non-CpG site, the non-methylated allele of the non-CpG site or both.

The term “target” as used herein refers to a molecule that has an affinity for a given probe molecule. Targets may be naturally occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed according to any aspect of the present invention are methylated and non-methylated CpG sites. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended.

The term “complementary” as used herein refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Perfectly complementary refers to 100% complementarity over the length of a sequence. For example, a 25-base probe is perfectly complementary to a target when all 25 bases of the probe are complementary to a contiguous 25 base sequence of the target with no mismatches between the probe and the target over the length of the probe.

The method according to any aspect of the present invention, further comprises the step of: (i) performing bisulfite modification to the DNA sample before step (a).

‘Bisulfite treatment’ of genomic DNA used interchangeably with the term ‘bisulfite modification’, refers to the treatment of the genomic DNA with a deaminating agent such as a bisulfite that may be used to treat all DNA, methylated or not. In particular, the term “bisulfite” as used herein encompasses any suitable type of bisulfite, such as sodium bisulfite, or other chemical agents that are capable of chemically converting a cytosine (C) to an uracil (U) without chemically modifying a methylated cytosine and therefore can be used to differentially modify a DNA sequence based on the methylation status of the DNA, e.g., U.S. Pat. Pub. US 2010/0112595. As used herein, a reagent that "differentially modifies" methylated or non-methylated DNA encompasses any reagent that modifies methylated and/or unmethylated DNA in a process through which distinguishable products result from methylated and nonmethylated DNA, thereby allowing the identification of the DNA methylation status. Such processes may include, but are not limited to, chemical reactions (such as a C to U conversion by bisulfite) and enzymatic treatment (such as cleavage by a methylation-dependent endonuclease). Thus, an enzyme that preferentially cleaves or digests methylated DNA is one capable of cleaving or digesting a DNA molecule at a much higher efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves or digests unmethylated DNA exhibits a significantly higher efficiency when the DNA is not methylated.

Accordingly, before step (a) according to any aspect of the present invention is carried out, the genomic DNA contained/ obtained or extracted from the cell, is first bisulfite treated.

An alternative method available in the art may be used instead of bisulfite treatment. A skilled person will understand which other methods to use. In one example, TET-assisted pyridine borane sequencing (TAPS) may be used for detection of 5mC and 5hmC (Yibin Liu, et al., Nature Biotechnology, 37: 424- 429 (2019).

According to a further aspect of the present invention, there is provided a method of identifying ageing in a test mammalian cell, the method comprising:

(a) determining the methylation status of at least one gene in a DNA sample obtained from the test cell;

(b) comparing the methylation status of the gene from step (a) to the methylation status of the corresponding gene in a control without ageing, wherein a difference in the methylation status of the gene in the test cell compared to the corresponding gene in the control is indicative of the cell exhibiting ageing; and wherein the gene in step (a) is selected from the group consisting of COL6A2, COL27A1 and KRT 13 and the regulatory regions of the same.

According to yet another aspect of the present invention, there is provided a use of a DNA-methylation based array in a method for determining the effect of at least one test compound on ageing in a mammalian cell according to any aspect of the present invention.

According to one other aspect of the present invention, there is provided a DNA methylation-based array for determining the effect of at least one test compound on ageing in a mammalian cell, wherein the array comprising probes that are complementary to CpG sites found on genes COL6A2, COL27A1 and KRT13 and the regulatory regions of the same.

Unless stated otherwise, all percentages (%) given are percentages by mass.

BRIEF DESCRIPTION OF FIGURES

Figure 1 is graph showing the top 50 overlapping genes for different treatments in Example 1 .

The examples adduced hereinafter describe the present invention by way of example, without any intention that the invention, the scope of application of which is apparent from the entirety of the description and the claims, be restricted to the embodiments specified in the examples.

EXAMPLES

Example 1

Photoaging on Human Tissue with UV light rays

Photoaging was induced in the cell culture system and skin tissue model to analyze the methylation status of promoters.

T-Skin models were obtained from Episkin SA, France which is composed of reconstructed human skin. Each skin model consists of a dermal equivalent overlaid by a stratified, well-differentiated epidermis derived from normal human keratinocytes. Upon receiving the skin models, it was recovered by incubating in T-Skin culture medium overnight at 37°C in a 5% CO2 incubator.

To induce photoaging on a human tissue system, skin models (5x replicates) were exposed to UV radiation (UVA 24 J/cm² + UVB 50mJ/cm²) (high UV) and cultured for 24 hrs.

In another group, skin models (5x replicates) were exposed to UV radiation (UVA 12 J/cm² + UVB 25mJ/cm² daily) (low UV) and cultured for 24 hrs.

Exposure to UV radiation leads to the generation of ROS which finally results in the development of a photoaging phenotype within the cells. A control set of skin models (5x replicates) were maintained for 24hrs without any exposure to UV radiation. Followed by the treatment, skin models were collected, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDrop™ 2000.

The genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ Kit (Zymo Research). The methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution. Oxidative Stress on Human Tissue with H2O2

Another way to mimic aging is by directly inducing oxidative stress on a human tissue system is through Hydrogen peroxide treatment which leads to the generation of ROS within the cells. The skin models (5x replicates) were treated with two different concentrations of Hydrogen peroxide [100pM (low) and 200pM (high)] for 2hrs and were maintained for 24hrs. A control set of skin models (5x replicates) were maintained for 24hrs without any treatment with Hydrogen peroxide. Followed by the treatment, skin models were collected, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDrop™ 2000.

The genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ Kit (Zymo Research). The methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.

Exposome-induced premature aging on Human Tissue with Particulate Matter 2.5

Another way to induce premature aging on a human tissue system is through exposure to pollutants such as Particulate Matter 2.5 (PM2.5) which leads to the generation of ROS within the cells by its chemical components and metals triggering stress and aging mechanisms. The skin models (5x replicates) were treated with two different concentrations of PM2.5 [15 pg/cm² (low) and 30 pg/cm² (high)] and were maintained for 24hrs. A control set of skin models (5x replicates) were maintained for 24hrs without any treatment with PM2.5. Followed by the treatment, skin models were collected, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDrop™ 2000.

Glycation-induced aging on Human Tissue with Glyoxal

Another way to induce premature aging on a human tissue system is through the production of advanced glycation end products, which can be generated by a treatment with glyoxal which triggers oxidative stress by increasing the level of ROS within the cells by producing advanced glycation end-products and induces premature aging pathways. The skin models (5x replicates) were treated with two different concentrations of glyoxal [0.5 mM (low) and 1 mM (high)] and were maintained for 24hrs. A control set of skin models (5x replicates) were maintained for 24hrs without any treatment with glyoxal. Followed by the treatment, skin models were collected, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDrop™ 2000. The genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ Kit (Zymo Research). The methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution. Cell-culture induced ageing of Human Tissue

Another way to mimic aging in vitro of a human tissue system is through long term culture which leads to the generation of Reactive oxygen and nitrogen species (RONS) as well as triggers senescence within the cells.

The skin models were maintained in the deep well plate with media for 7 days (4x replicates), 14 days (4x replicates) and 21 days (4x replicates) respectively to induce ageing in the skin tissue. Skin models were collected after 7 days, 14 days and 21 days respectively, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDrop™ 2000.

Quality control and data processing A total of 103 samples from 16 different treatments and their respective controls were analyzed. The treatments were grouped into two batches. Batch 1 consisted of all 24 hour treatments, at high and low concentrations. All treatments in batch 1 shared the same control group. Table 1 shows the sample information for batch 1 .

Table 1. Batch 1 samples

Methylation EPIC array data processing was performed in R version 4.2.2 (2021 -11-10 r83330) using the minfi version 1.42.0. The raw intensity data (IDAT) were imported into R (4.2.2) and processed using the minfi (1 .42.0) Bioconductor package. Quality check on samples were performed to keep probes that had a detection P-value <0.01 in one or more samples or had a mean detection P-value <0.05 in all samples. The samples were then normalized using functional normalization (implemented by preprocesssFunnorm function in minfi) for type-bias correction and background correction.

Prior to differential methylation analysis, the probes with non-specific binding, cross reactive probes, probes affected by common SNPs, and probes annotated to the X,Y chromosomes were also filtered out. Beta-value and M-value of normalized and filtered samples were calculated using getBeta and getM function respectively, the samples were then subjected to further downstream analysis.

Differential methylation analysis

Pair-wise differential methylation analysis (total of 16 pairs) was performed using the limma package version 3.52.4 . The batch 1 samples were analyzed together, while for the batch 2 samples, the UV light and aged samples were analyzed separately. Contrast matrix was set up by comparing each corresponding treatment and control group and empirical Bayesian algorithm was used to fit the M- values based on the design and contrast model. Probes with adjusted P-value lower than 0.05 were considered as differentially methylation positions (DMPs). Annotation was performed using HluminaHumanMethylationEPICanno.ilm10b2.hg19. Genes that overlapped at all treatments were then identified. Figure 1 shows the top 50 genes seen in all 16 comparisons.

Out of these 50 genes, three genes COL6A2, COL27A1 and KRT13 which are related to collagen and keratin were selected to be further analysed. These genes were found to have the following CPG sites provided in Table 2.

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Table 2. CpG sites for genes COL6A, COL27A1 and KRT13

Claims

1 . An in vitro method of assessing the effect of at least one test compound on ageing in a mammalian cell, the method comprising:

(a) contacting the test compound with a mammalian test cell;

2. The method according to claim 1 , wherein in step (b) the methylation status of at least two genes is determined.

3. The method according to either claim 1 or 2, wherein in step (b) the methylation status of at least three genes is determined.

4. The method according to any one of the preceding claims, wherein the test and control methylation status of the gene in steps (b) and (c) are determined using DNA array.

5. The method according to any one of the preceding claims, further comprising the step of:

(i) performing bisulfite modification to the DNA sample before step (b).

6. The method according to any one of the preceding claims, wherein the cell is a skin cell or a cell derived therefrom.

7. The method according to any one of the preceding claims, wherein the DNA methylation- based array is a bead-based array

8. A method of identifying ageing in a test mammalian cell, the method comprising:

(b) comparing the methylation status of the gene from step (a) to the methylation status of the corresponding gene in a control without ageing, wherein a difference in the methylation status of the gene in the test cell compared to the corresponding gene in the control is indicative of the cell exhibiting ageing; and wherein the gene in step (a) is selected from the group consisting of COL6A2, COL27A1 and KRT13 and the regulatory regions of the same.

9. The method according to claim 8, wherein in step (b) the methylation status of at least two genes is determined.

10. The method according to either claim 8 or 9, wherein in step (b) the methylation status of at least three genes is determined.

11. The method according to any one of the claims 8 to 10, wherein the methylation status of the gene in steps (a) and (b) is determined using DNA array.

12. The method according to any one of the claims 8 to 11 , wherein the cell is a skin cell or a cell derived therefrom.

13. The method according to any one of the preceding claims, wherein the mammal is selected from the group consisting of a mouse, a rat, a guinea pig, a dog, a mini-pig, a human being, a cow, a sheep, a pig, a goat, a horse, a donkey, and a mule.

14. Use of a DNA-methylation based array in a method for determining the effect of at least one test compound on ageing in a mammalian cell according to any one of the claims 1 to 7.

15. A DNA methylation-based array for determining the effect of at least one test compound on ageing in a mammalian cell, wherein the array comprising probes that are complementary to CpG sites found on genes COL6A2, COL27A1 and KRT13 and the regulatory regions of the same.