Telomeric repeat-containing RNA (TERRA) biomarker of biological aging

Abstract

This invention relates to a method of determining the biological age of an individual comprising determining the amount of Telomeric repeat-containing RNA (TERRA) in a sample of cells obtained from the individual, and determining the biological age of the individual from the amount of TERRA molecules in the biological sample. Methods of the invention may also be useful in predicting the severity of a disease, such as an age-related disease, in an individual and in identifying a compound that inhibits biological aging.

Description

[0001] Telomeric Repeat-containing RNA (TERRA) Biomarker of Biological Aging Field

[0002] The present invention relates to biomarkers of biological age and their use in predicting the risk of age-related diseases.

[0003] Aging affects people differently and some individuals develop age-related diseases much earlier than others. This is because biological age, which reflects the body's true aging process, does not always match chronological age.

[0004] Chronological and biological age do not directly coincide because the rate of cellular and tissue aging varies substantially among individuals due to molecular changes influenced not only by genetics, but also lifestyle and environmental factors. For example, alcohol and tobacco consumption, obesity and psychological stress are known to accelerate biological aging (and telomere shortening). As a result, individuals who are chronologically young but biologically older may develop age-related diseases earlier than the general population.

[0005] Also, biologically older individuals may experience more severe disease manifestations than biologically younger ones. These age-related diseases span a wide spectrum, including cardiovascular disease (CVD), metabolic disorders (e.g., type 2 diabetes), lung diseases, neurodegenerative conditions, and virus infections. Estimating biological age informs clinicians on the propensity of a patient to develop age-related diseases and co-morbidities as well as on how severely a disease (being a chronic disease, an infection, etc.) will develop in the patient.

[0006] Telomeres are protective structures at the ends of chromosomes that shorten as the patient ages. Short telomeres have been linked to aging and disease. However, telomere length alone is not a reliable marker of biological age because it varies greatly among healthy people and is influenced by genetic makeup and lifestyle. Moreover, telomeres can become dysfunctional and cause premature aging regardless of their length and current technologies that estimate biological aging through telomere length do not take into account patients with telomeres that are long but dysfunctional. Furthermore, since chronological and biological age do not directly coincide, molecular markers associated with chronological age cannot be reliably used to determine biological age.

[0007] To date, molecular markers of the biological age of a patient are scarce.

[0008] The present inventors have found that telomeric repeat-containing RNA (TERRA) is a robust biomarker of biological aging and may be useful for example in determining the risk of an age-related disease in a patient or predicting the severity of a disease in a patient.

[0009] A first aspect of the invention provides a method of determining the biological age of an individual comprising,determining the amount of Telomeric repeat-containing RNA (TERRA) in a sample of cells obtained from the individual, and

[0010] determining the biological age of the individual from the amount of TERRA molecules in the biological sample.

[0011] The determined biological age of the individual in methods of the first aspect may be different from the chronological age of the individual. For example, the determined biological age may be greater than the chronological age of the individual.

[0012] A second aspect of the invention provides a method of identifying an individual at risk of an age-related disease,

[0013] determining the amount of Telomeric repeat-containing RNA (TERRA) in a sample of cells obtained from the individual,

[0014] determining the biological age of the individual based on the amount of TERRA in the sample of cells, and

[0015] identifying the individual as being at risk of an age-related disease from their determined biological age.

[0016] In some embodiments of methods of the second aspect, the individual may be identified as being at risk in methods of the second aspect by:

[0017] comparing the determined biological age with the chronological age of the individual, and identifying an individual with a determined biological age greater than their chronological age as being at risk of an age-related disease.

[0018] In other embodiments of methods of the second aspect, the individual may be identified as being at risk in methods of the second aspect by:

[0019] comparing the determined biological age with a pre-determined threshold value, and identifying an individual with a determined biological age greater than the pre-determined threshold value as being at risk of an age-related disease.

[0020] A third aspect of the invention provides a method of predicting the severity of a disease in an individual comprising;

[0021] determining the amount of Telomeric repeat-containing RNA (TERRA) in a biological sample obtained from the individual having or suspected of having a disease, and

[0022] determining the severity of the disease in the individual based on the amount of TERRA in the biological sample.

[0023] A fourth aspect of the invention provides a method of identifying a compound that inhibits biological aging comprising:

[0024] determining the effect of a test compound on the amount of Telomeric repeat-containing RNA (TERRA) in a cell, and

[0025] identifying a test compound that reduces the amount of TERRA in the cell as a compound that inhibits biological aging.Other aspects and embodiments of the invention are described in more detail below.

[0026] Brief Description of the Figures

[0027] Figure 1 shows the precise and quantitative measurement of TERRA produced from all chromosome ends. (A) Schematic representation of the improved TERRA ONTseq pipeline (TERRA ONTseqV2). (B) Comparison of the number of uniquely mapped TERRA reads from all chromosome ends obtained using the original (TERRA ONTseqVI) and improved TERRA ONTseq methods (C) Examples of TERRA ONTseqV2 read coverage at three chromosome ends (7q, 16p, and 20p) in parental HCT116 cells and derivative cells knocked outfor DNMTI and DNMT3b enzymes (DKO cells). (D) TERRA metaprofiles for HCT116 and DKO cells from experiments as shown in (C).

[0028] Figure 2 shows that TERRA ONTseqV2 detects increased TERRA transcripts in primary human lung fibroblasts (HLF) (A), HeLa cells transfected with siRNAs targeting the shelterin protein TRF2 (siTRF2) or control siRNAs (siCtrl) (B) and PBMCs from a 21 -donor and a 90-year-old donor (C).

[0029] Detailed Description

[0030] This invention relates to methods of assessing the biological age of an individual by determining the amount of Telomeric repeat-containing RNA (TERRA) in a sample of cells obtained from the individual. The biological age of the individual may be assessed from the level of TERRA in the sample.

[0031] Telomeric repeat-containing RNA (TERRA) are long non-coding RNA molecules that are transcribed from the end regions (i.e. telomeres and subtelomeres) of chromosomes.

[0032] A TERRA molecule may be 100 to 10000 bases in length.

[0033] A TERRA molecule may comprise sequence transcribed from a telomere of a chromosome. Suitable sequences may comprise multiple repeats of the sequence 5’-UUAGGG-3’. For example, a TERRA may comprise the sequence [UUAGGG]n, wherein n is 2 to 1000 or more.

[0034] A TERRA molecule may further comprise sequence transcribed from sub-telomeric regions of a chromosome. This may be useful for example in determining the chromosome or chromosome end from which a TERRA is transcribed. For example, a method may comprise determining the amount of TERRA from a target chromosome end in the sample of cells, Suitable target chromosome ends may include the 17p chromosome end.

[0035] The transcription of TERRA may be activated in a cell by dysfunctional or unstable telomeres. For example TERRA transcription may be activated by telomere shortening or telomeric DNA damage, for example damage induced by genetic / epigenetic alterations, exogenous agents, such a UV light, oxidative stress or chemotherapy, and lifestyle.

[0036] The amount or level of TERRA expression may be determined in a sample of cells from the individual. The sample of cells may be a population of cells of the same cell type from the individual.The sample may conveniently be a sample of blood cells, such as human peripheral blood mononuclear cells (PBMCs).

[0037] The methods described herein may be used to determine the biological age of any suitable individual.

[0038] Suitable individuals may include individuals being assessed for personalized therapy. The biological age of the individual may determine or influence the selection of the personalized therapy for the individual. Other suitable individuals may include individuals being assessed for having a disease condition or being at risk of having a disease condition, for example, an age related condition; such as cardiovascular disease, diabetes or a neurodegenerative condition, such as Parkinson’s disease or Alzheimer’s disease.

[0039] The methods described herein may be useful in identifying individuals with a mismatch between their chronological and biological ages. In some embodiments, the individual may have a chronological age that does not place the individual at risk of an age-related disease; or places the individual at low risk of an age-related disease. For example, the individual may have a chronological age of 60 years or lower. 55 years or lower or 50 years or lower.

[0040] A suitable method may further comprise assessing the life-style, familial history and general health of the individual.

[0041] TERRA may be isolated from cells in the sample. For example, total RNA may be isolated from the cells. TERRA may be isolated from the isolated total RNA.

[0042] In some embodiments, isolated TERRA may be reverse transcribed into cDNA molecules, which may then be amplified and sequenced. The amplified cDNA molecules may be sequenced using standard sequencing techniques. Suitable techniques include using any convenient low or high throughput sequencing technique or platform, including Sanger sequencing, Solexa-lllumina sequencing (for example Hiseq™, MiSeq™ or NextSeq™), ligation-based sequencing (SOLiD™; Life technologies), pyrosequencing (e.g. 454 Sequencing; Roche 454); single molecule real-time sequencing (SMRT™; PacBioscience); or nanopore sequencing (Oxford Nanopore e.g. MinlON™, GridlON™, PromethlON™).

[0043] In some embodiments, duplicate reads, low quality sequence reads and reads arising only from sequencing adaptors may be removed.

[0044] Aset of sequence reads may be generated by sequencing. For example 1000 or more, 10,000 or more, 100,000 or more, 1000,000, 10,000,000 or more, or 100,000,000 or more, or 1000, 000,000 or more sequence reads may be generated. The sequence reads may be analysed, for example by bioinformatic techniques, and TERRA sequences reads may be identified within the set. The TERRA sequence reads may be mapped to individual telomeric regions or chromosome ends. This may be useful in identifying a sequence read as a TERRA sequence read.

[0045] The number of TERRA sequence reads may be indicative of the amount of TERRA in the sample.Other suitable techniques for determining the amount of TERRA in a sample may include RT-qPCR.

[0046] Biological age is a parameter that reflects the extent of aging-driven biological changes, such as molecular and cellular degradation, that accrue in an individual over time (Moqri, M. et al. Cell 186, 3758-3775 (2023)). Biological age may be determined from the amount of TERRA molecules in the biological sample.

[0047] Chronological age is the time elapsed since the birth of the individual. Biological age is distinct from chronological age and the two parameters may not display a linear correlation. The biological age of an individual may be greater than their chronological age (positive age acceleration); the biological age of an individual may be lower than their chronological age (negative age acceleration); or the biological age of an individual may be the same as their chronological age. A high biological age may be indicative that the individual may have an increased risk of an age-related disease compared to control individuals or a control population of individuals of the same chronological age.

[0048] Biological age may be expressed in any convenient format, including numerical scales or adjective scales. In some embodiments, biological age may be expressed in a binary form, such as “young” and “old”.

[0049] Individuals identified as “old” may have an increased risk of an age-related disease compared to control individuals or a control population of individuals of the same chronological age. In some embodiments, biological age may be expressed as difference in the risk of the individual having an age-related disease compared to the risk in control individuals or a control population of individuals of the same chronological age.

[0050] Biological age is determined as described herein from the amount of TERRA or level of TERRA expression in the sample of cells. In some embodiments, biological age may be determined from the amount of TERRA molecules in the biological sample in combination with one or more other parameters, such as telomere length and / or the chronological age.

[0051] The biological age of an individual determined by a method described herein may be predictive of onset and / or severity of age-related diseases or co-morbidities. For example, a method of identifying an individual at risk of an age-related disease; or determining the risk of age-related disease in an individual may comprise;

[0052] determining the amount of Telomeric repeat-containing RNA (TERRA) in a biological sample obtained from the individual,

[0053] determining the biological age of the individual based on the amount of TERRA in the biological sample, and

[0054] identifying an individual as being at risk of an age-related disease from their determined biological age.

[0055] In some embodiments, a method may comprise identifying the individual as having a determined biological age greater than a predetermined threshold. For example, a method may comprise

[0056] determining the amount of Telomeric repeat-containing RNA (TERRA) in a biological sample obtained from the individual,determining the biological age of the individual based on the amount of TERRA in the biological sample,

[0057] comparing the determined biological age with a predetermined threshold value, and

[0058] identifying an individual with a determined biological age greater than the predetermined threshold value as being risk of an age-related disease.

[0059] In some embodiments, a method may comprise identifying the individual as having a determined biological age greater than their chronological age. For example, a method may comprise

[0060] determining the amount of Telomeric repeat-containing RNA (TERRA) in a biological sample obtained from the individual,

[0061] determining the biological age of the individual based on the amount of TERRA in the biological sample,

[0062] comparing the determined biological age with the chronological age of the individual, and identifying an individual with a determined biological age greater than their chronological age as being at risk of an age-related disease.

[0063] A suitable predetermined threshold value may be determined by measuring the amount of TERRA in a sample of the same type of cells in a population of patients of different chronological ages with and without age-related diseases

[0064] Age-related diseases are diseases whose onset and / or severity is associated with aging. Age related diseases may be selected from lung diseases, such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease; metabolic diseases, such as type 2 diabetes, osteoporosis, and sarcopenia; cardiovascular disease, such as hypertension, atherosclerosis, coronary artery disease, heart failure, and stroke; neurodegenerative diseases, such as Parkinson’s disease, Huntingdon’s disease and Alzheimer’s disease; cancer, such as lung cancer, colorectal cancer, breast cancer, prostate cancer, leukaemia and lymphoma; immune system disorders, such as increased susceptibility to infections; autoimmune diseases, such as rheumatoid arthritis, and chronic inflammation; chronic kidney and liver diseases, such as chronic kidney disease, non-alcoholic fatty liver disease, liver fibrosis and cirrhosis; musculoskeletal disorders, such as osteoarthritis, rheumatoid arthritis, and degenerative disc disease; haematological and vascular disorders, such as anaemia of aging, deep vein thrombosis, and aneurysms.

[0065] An individual at risk of an age-related disease may have an increased risk relative to a control population of individuals of the same chronological age.

[0066] A method of predicting the severity of a disease or determining the prognosis for a disease in an individual may comprise;

[0067] determining the amount of Telomeric repeat-containing RNA (TERRA) in a biological sample obtained from the individual having or suspected of having a disease, and

[0068] determining the likely severity of the disease in the individual based on the amount of TERRA in the biological sample.The disease may be an infection, chronic disease; or an age-related disease, for example an age-related disease set out above.

[0069] An individual identified as at risk of an age-related disease or determined to have a poor prognosis on the basis of biological age determined as described herein may be subjected to a monitoring and / or treatment regimen adapted for high-risk individuals. For example, the individual may be subjected to increased clinical monitoring relative to control individuals with the same chronological age and disease. The individual may be subjected to a treatment regimen with an appropriate risk profile, for example a treatment regimen with a lower risk of side-effects than a regimen suitable for individuals not identified as at risk or determined to have a poor prognosis.

[0070] An individual identified as at risk of an age-related disease or determined to have a poor prognosis on the basis of biological age determined as described herein may be subjected to lifestyle advice, such as diet and / or exercise advice to promote lowering of biological age.

[0071] Other aspects of the invention relate to methods of screening for compounds that inhibit or reduce biological aging. A method of identifying a compound that inhibits biological aging may comprise:

[0072] determining the effect of a test compound on the amount of Telomeric repeat-containing RNA (TERRA) molecules in a cell.

[0073] A test compound that decreases the amount of TERRA in the cell, blocks the increase of TERRA in the cell or reduces the rate of increase of TERRA in the cell may be identified as a candidate compound for inhibiting biological aging. Suitable compounds may for example reduce telomere dysfunction or instability.

[0074] The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments.

[0075] A test compound may be an isolated molecule or may be comprised in a sample, mixture or extract, for example, a biological sample. Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used

[0076] Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate TERRA levels in cells. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances

[0077] The amount of test compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100pM, e.g.0.1 to 50 pM, such as about 10 pM. Even a compound which has a weak effect may be a useful lead compound for further investigation and development.

[0078] A test compound identified as inhibiting biological aging in may be investigated further using one or more secondary screens. A secondary screen may involve testing for a biological function or activity in vitro and / or in vivo, e.g. in an animal model. For example, the ability of a test compound to inhibit TERRA levels in an animal model may be determined.

[0079] Following identification of a compound that is potentially useful in inhibiting biological aging as described herein, a method may further comprise modifying the compound to optimise its pharmaceutical properties. Suitable methods of optimisation, for example by structural modelling, are well known in the art. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.

[0080] A test compound identified as inhibiting biological aging may be isolated and / or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis.

[0081] Furthermore, it may be manufactured and / or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition ordrug. Methods described herein may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application.

[0082] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of’.

[0083] It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0084] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

[0085] All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

[0086] “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.TERRA ONTseqV2 enables precise and quantitative measurement of TERRA produced from all chromosome ends. (A) Schematic representation of the improved TERRA ONTseq pipeline (TERRA ONTseqV2).

[0087] TERRA transcripts were affinity-purified from total RNA using a biotinylated DNA oligonucleotide comprising five CCCTAA repeats, complementary to the UUAGGG tracts within TERRA molecules (Figure 1 A). The biotinylated oligonucleotides were 3' end-blocked with dideoxycytosine to prevent interference with subsequent reverse transcription. TERRA-enriched RNA was reverse-transcribed with an oligonucleotide comprising a unique sequence followed by a repetitive sequence complementary to the UUAGGG repeats. This was followed by template-switching, second-strand synthesis and PCR amplification using oligonculeotides that bind to the unique sequence added during reverse transcription and the templateswitching primer. PCR products were barcoded and ligated to ONT adapters and sequenced using a MinlON apparatus. Reads were demultiplexed during base-calling using the MinKNOW software and raw read quality control was extracted from the MinKNOW sequencing summary file and the barcode summary file.

[0088] Demultiplexed reads were then strand-oriented and filtered for full-length reads using Pychopper. Finally, the processed reads were aligned to a human subtelomeric reference sequence derived from the T2T-CHM13v2.0 / hs1 build of the human genome, comprising 25 kb sequences terminating at the fourth telomeric TTAGGG repeat on each chromosome end. Only full-length reads with an orientation consistent with TERRA (subtelomere toward the chromosome end) were included in the final analysis.

[0089] The number of uniquely mapped TERRA reads from all chromosomes ends obtained using the original (TERRA ONTseqVI) and improved TERRA ONTseq methods were compared (Figure 1B). For TERRA ONTseqVI , the starting material was 750 and 250 pg of nuclear RNA for HeLa (low TERRA levels) and U2OS cells (high TERRA levels), respectively, whereas TERRA ONTseqV2 can use as low as 5 pg of total RNA. Despite starting with much less material, TERRA ONTseqV2 produced approximately four times more TERRA reads. TERRA ONTseqV2 read coverage was assessed at three chromosome ends (7q, 16p, and 20p) in parental HCT116 cells and DKO cells, a clonal derivative lacking expression of DNA methyltransferases DNMT1 and DNMT3b, previously shown to express more TERRA (Figure 1C). TERRA metaprofiles for HCT116 and DKO cells were produced (Figure 1D). Alignments span 1250 bp regions starting from the first telomeric repeat (position 0). Note that TERRA ONTseqV2 was found to retain its quantitative nature across these samples. TERRA ONTseqV2 was found to allow precise and quantitative measurement of TERRA produced from all chromosome ends.

[0090] TERRA metaprofiles were generated from total RNA isolated from primary human lung fibroblasts (HLF) with progressively shortened telomeres due to replicative aging. Cells were passaged every three days for 10 days (young), 44 days (middle-aged) and 66 days (senescent) (Figure 2A). TERRA metaprofiles were also generated from total RNA isolated from HeLa cells transfected with siRNAs targeting the shelterin protein TRF2 (siTRF2) or control siRNAs (siCtrl) (Figure 2B). Cells with shorter or dysfunctional telomeres were found to exhibit elevated TERRA levels compared to cells with longer, properly capped telomeres. TERRA metaprofiles were also generated from total RNA (10 pg of starting material) isolated from PBMCs from a 21-and a 90-year-old donor (Figure 2C). TERRA ONTseqV2 was found to readily detect TERRA from PBMCs. These experiments show that TERRA transcripts are increased in cells with short or dysfunctional telomeres.

Claims

Claims1 A method of determining the biological age of an individual comprising,determining the amount of Telomeric repeat-containing RNA (TERRA) in a sample of cells obtained from the individual, anddetermining the biological age of the individual from the amount of TERRA molecules in the biological sample.

2. A method according to claim 1 wherein the biological age of the individual is determined to be different to the chronological age of the individual.

3. A method of identifying an individual at risk of an age-related disease,determining the amount of Telomeric repeat-containing RNA (TERRA) in a sample of cells obtained from the individual,determining the biological age of the individual based on the amount of TERRA in the sample of cells, andidentifying the individual as being at risk of an age-related disease from their determined biological age.

4. A method according to claim 3 wherein the individual is identified as being at risk by:comparing the determined biological age with the chronological age of the individual, and identifying an individual with a determined biological age greater than their chronological age as being at risk of an age-related disease.

5. A method according to claim 3 wherein the individual is identified as being at risk by:comparing the determined biological age with a pre-determined threshold value, and identifying an individual with a determined biological age greater than the pre-determined threshold value as being at risk of an age-related disease.

6. A method according to any one of claims 3 to 5 wherein the identified individual has an increased risk of an age-related disease relative to control individuals of the same chronological age.

7. A method according to any one of claims 3 to 6 wherein the age-related disease is a metabolic disease, a lung disease, a cardiovascular disease or a neurodegenerative disease8. A method of predicting the severity of a disease in an individual comprising;determining the amount of Telomeric repeat-containing RNA (TERRA) in a sample of cells obtained from the individual having or suspected of having a disease, anddetermining the severity of the disease in the individual from the amount of TERRA in the sample.

9. A method according to any one of claims 3 to 8 wherein the individual has a chronological age associated with low risk of age-related disease.

10. A method according to any one of claims 3 to 9 wherein the sample of cells is a sample of blood cells.

11. A method according to claim 10 wherein the blood cells are PBMCs12. A method according to any one of claims 1 to 11 wherein the amount of Telomeric repeat-containing RNA (TERRA) in the sample is determined by isolating TERRA from the sample, converting the TERRA into cDNA molecules, amplifying the cDNA molecules and sequencing the amplified cDNA molecules to produce a set of sequence reads, wherein the number of TERRA sequence reads in the set is indicative of the amount of TERRA molecules in the biological sample.

13. A method of identifying a compound that inhibits biological aging comprising:determining the effect of a test compound on the amount of Telomeric repeat-containing RNA (TERRA) in a cell, andidentifying a test compound that reduces the amount of TERRA in the cell as a compound that inhibits biological aging.

Images