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1. WO2020136192 - METHOD FOR MEASURING TELOMERE ASSOCIATED VARIABLES AND USES THEREOF FOR THE DIAGNOSIS AND/OR PROGNOSIS OF TELOMERIC-ASSOCIATED DISEASES

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CLAIMS

1. Method in vitro for measuring Telomere Associated Variables (TAVs) in an isolated biological test sample of a subject, wherein the method uses a set of standard isolated biological samples of known telomere length, and comprises the following steps:

a) providing a first set of intensity values of known telomere length from the set of standard isolated biological sample;

b) generating a standard curve correlating the first set of intensity values with the known telomere length from the set of standard isolated biological samples;

c) providing a second set of intensity values from the isolated biological test sample;

d) calculating the telomere lengths within the isolated biological test sample of a subject by interpolating the second set of intensity values into the standard curve; and

e) calculating, in the isolated biological test sample, at least one of the TAVs selecting from the list consisting of: telomere length percentiles from 1th to 99th, percentages of telomeric length values that can be specified to a particular bp range or number, percentages of cells with specific telomere median or average value that can be specified to a particular bp value, median absolute deviation of the intensities of a sample, difference between the 99th and 1 st percentile of the telomere length of the sample, interquartile of the telomere length of a sample, standard deviation of the median telomere length values, or any combinations thereof.

2. The method in vitro according to claim 1 , wherein the step a) of providing the first set of intensity values of known telomere length, in turn, comprises the steps of:

(i). performing high-resolution microscopy to the standard isolated biological sample of known telomere length, obtaining a first set of image data; and

(ii). processing the first set of image data obtained, extracting a first set of intensity values.

3. The method in vitro according any of claims 1 to 2, wherein the step c) of providing the second set of intensity values from the isolated biological test sample, in turn, comprises the steps of:

(i). performing high-resolution microscopy to the isolated biological test sample of a subject, obtaining a second set of image data; and

(ii). processing the second set image data obtained in step d), extracting a second set of intensity values;

4. The method in vitro according to claim 1 , wherein the percentage of telomeric length value, preferably the percentage of long telomeres is determined by the formula:

TLU = 100 - TSU

wherein the percentage of short telomeres TSU in the isolated biological test sample Mt , for a threshold length u is determined as:

TSU = 100


wherein

Mt = {Ti, T2 ... Tn} is the complete set of the telomeres measured in the isolated biological test sample Mt,

n is the number of telomeres in Mt, and,

k {{ti. ti, , tn)) is the telomeric length of the telomere Tt {{Tlr T2, - , Tn}).

5. The method in vitro according to claim 1 , wherein the percentage of cells with specific telomere length value, preferably percentage of cells having short telomeres ( CSU ) in the isolated biological test sample M for a threshold length u is determined by the formula:

CSU = 100


wherein:

cr is the length of Cr e Mc calculated as: cr = avg(tri),

n is the number of cells in Mc,

tri is the telomeric length of the telomere Tri

Kr = {Trl, Tr2, ... , Trn} is the set of telomeres analyzed in a cell Cr; and Mc = [Cri> cn> > Cm) is the complete set of cells in the isolated test biological sample M.

6. The method in vitro according to any of claims 1 to 5, wherein the threshold length u ranges from 500 to 40,000 pb in increments of 100 pb.

7. The method in vitro according to any of claims 1 to 6, wherein the TAVs are determined by HT Q-FISH.

8. The method in vitro according to any of claims 1 to 7, wherein further comprises the measurement of the MAD I2 of the telomeric length.

9. The method in vitro according to claim 8 wherein the MAD-I2 of the telomeric length is determined by the formula:

MAD = median(\m— fh\)

wherein

m is the set of fluorescence intensities corresponding to the telomeres in the test sample Mt and in is the median of m,


Mt = { Ti, T2 > , TJI } is the complete set of telomeres measured in the isolate biological test sample; and

tj ({tlr t2, ... , tn}) is the fluorescence intensity of the telomere Tt ({Tlr T2, ... , Tn }).

10. The method in vitro according to any of claims 1 to 9, wherein further comprises the measurement of the telomerase activity in the isolated biological sample.

1 1 . The method in vitro according to claim 10 wherein the telomerase activity is determined by Q-TRAP.

12. A method in vitro for the diagnosis, prognosis, identification of a subgroups of subjects who may benefit for a treatment, and/or monitoring of the evolution of a telomeric-associated disease, in an isolated biological test sample of a subject, wherein the method comprising:

a) measuring and/or quantifying at least one of the TAVs in the isolated biological test sample by the method according to any of claims 1 to 9; b) measuring and/or quantifying at least one of the same TAVs of the step a) in an isolated biological reference or control sample, by the steps c) to e) of the method according to any of claims 1 and 3 to 9; c) comparing the value of the at least one of the TAVs detected in step (a) with the value of at least one of the same TAVs obtained in a reference or control sample,

d) identifying a significant difference between the TAVs comparison of step b); and

e) correlating the significant difference of step (c) with the diagnosis, prognosis identification of a subgroups of subject who may benefit for a treatment, and/or the evolution of a telomeric-associated disease in the subject.

13. A method in vitro of screening, testing or validating drug candidates useful for the treatment of telomeric-associated diseases in an isolated biological test sample of a subject, wherein the method comprising:

a) contacting the drug candidate with an isolated biological test sample of a subject suffering from a telomeric-associated disease,

b) measuring and/or quantifying at least one of the TAVs by the method according to any of claims 1 to 9, and

c) select the drug candidate of step (a) having the ability to maintain or increase the telomere length regarding to the value of the telomere length obtained in a reference or control sample.

14. A method in vitro of response to a drug treatment in an isolated biological test sample of a subject with a telomeric-associated disease, wherein the method comprising:

a) measure and/or quantified at least one of the TAVs by the method according to any of claims 1 to 9; and

b) compare the value obtained in step a) with the value of at least one of the same TAVs obtained from responsive and non-responsive patients who showed, respectively, responsiveness to the treatment and those who showed non-responsiveness thereto or alternatively, compare the value obtained in step a) with the value of at least one of the same T AVs obtained from a reference sample, wherein the maintenance of the telomere length or a longer telomere length correlates with better drug efficacy.

15. The method in vitro according to any of claims 1 to 14 wherein the isolated biological sample is selected from: cells, tissues and/or biological fluid.

16. The method in vitro according to any of claims 1 to 15 wherein the isolated biological sample is selected from the list consisting of: plasma, serum, blood, lymphatic fluid, cerebrospinal fluid, synovial fluid, urine, saliva, mucous, phlegm and sputum, preferably plasma, serum or blood.

17. The method in vitro according to any of the claim 1 to 16 wherein the telomeric- associated disease is selected from the list consisting of: cancer, cardiovascular disease, dyskeratosis congenita, pulmonary fibrosis, aplastic anaemia, interstitial pneumonia, diabetes, infertility, haematological diseases, central nervous system diseases, liver diseases and metabolic diseases.

18. The method in vitro according to claim 17 wherein the cancer is selected from the list consisting of: prostate, lung, colon, gastric, melanoma, ovarian, breast and haematological neoplasms.