In vitro use of a non-LTR RTE biomarker, such as line-1, to detect and diagnose the presence and risk of developing neurodegenerative diseases, particularly tauopathies, using a biological sample, method for detecting the biomarker and diagnostic reagent kit

Abstract

The invention discloses the in vitro use of a non-LTR RTE biomarker, such as LINE-1, to diagnose neurodegenerative diseases, particularly tauopathies. The diagnosis is based on identifying an increase in the number of LINE-1 copies in the biological fluids of an individual, and the difference in the number of copies of a non-LTR RTE, LINE-1, per ng of DNA in a biological sample, preferably a buffy coat, between healthy subjects (Control) and ill patients. The tauopathies are: Alzheimer's Disease (EA), Mild Cognitive Impairment (DCL), Progressive Supranuclear Palsy (PSP) / Corticobasal Syndrome (SCB) and Frontotemporal Dementia (DFT). The invention also relates to a kit comprising means for detecting the biomarker in a biological sample obtained from the individual and means for comparing the levels of the biomarker of interest in the sample with a control reference obtained from healthy individuals, and relates to an in vitro method for detecting a non-LTR RTE, such as LINE-1, in a biological sample.

Description

[0001] THE USE OF AN IN VITRO BIOAKER, RTE NO-LTR, AS LINE-1 TO DETECT AND DIAGNOSE, USING A BIOLOGICAL SAMPLE, THE PRESENCE AND RISK OF DEVELOPING NEURODEGENERATIVE DISEASES, PARTICULARLY TAUOPATHIES, A PROCEDURE FOR DETECTING THE BIOAKER AND A DIAGNOSTIC REAGENT KIT

[0002] OBJECT OF THE INVENTION

[0003] The objective of the present invention is the use of non-LTR retrotransposon measurement, for example, LINE-1, for the diagnosis of neurodegenerative diseases, particularly tauopathies, by means of a diagnostic reagent kit and a procedure for detecting such diseases. The diagnosis is based on the in vitro identification of an increase in the number of LINE-1 copies in an individual's biological fluids, which correlates with the presence or progression of these pathologies, thus improving diagnostic accuracy.

[0004] TECHNICAL SECTOR

[0005] The present invention relates, in general, to the field of neuroscience and neurodegenerative diseases. Application of biomarkers for tauopathies. Portable diagnostic system. Diagnostic kit for detecting biomarkers.

[0006] BACKGROUND OF THE INVENTION

[0007] Tauopathies are a group of neurodegenerative diseases characterized by the accumulation of abnormal aggregates of the tau protein in the brain. Examples include Alzheimer's disease (AD), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). The tau protein normally stabilizes neuronal microtubules, but in these diseases, it becomes hyperphosphorylated and forms neurofibrillary tangles that block neuronal transport, leading to nerve cell death. There are different types of tauopathies, some of which progress to dementia and others that do not. Among the tauopathies that develop into dementia are Alzheimer's disease (AD) and frontotemporal dementia (FTD), both of which primarily affect cognitive function.On the other hand, some tauopathies such as Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD) mainly affect motor control, and may present cognitive impairment, but do not always evolve into dementia.

[0008] The symptoms of tauopathies vary depending on the affected brain region, but often include behavioral changes such as apathy, disinhibition, and compulsive behaviors; motor difficulties such as rigidity, slowness of movement, and balance problems; and cognitive and language impairments, such as memory problems, difficulty speaking or finding words, and loss of interpersonal skills.

[0009] One of the best-known tauopathies is Alzheimer's disease (AD), characterized by the accumulation of beta-amyloid plaques and neurofibrillary tangles of tau protein in the brain, leading to neuronal dysfunction and death. This disease is the most common cause of dementia, accounting for approximately 60% of dementia cases worldwide. Dementia is defined as a progressive neurodegenerative disease that affects multiple cognitive functions, including memory, thinking, language, behavior, and emotional control.

[0010] Currently, early detection and accurate diagnosis of neurodegenerative diseases that progress to dementia are not easy and present significant clinical challenges due to several interrelated factors. First, initial symptoms are often subtle, such as minor forgetfulness, slight fluctuations in concentration, or mood changes, which are frequently mistaken for normal aging processes or transient conditions like stress, thus delaying early disease identification. Furthermore, the heterogeneity of dementia forms, including Alzheimer's disease, vascular dementia, and Lewy body dementia, among others, exacerbates diagnostic complexity, as each presents with a different progression and symptoms.In addition, the coexistence of common comorbidities in the older population, such as depression, anxiety, and cardiovascular disease, not only masks the symptoms of dementia but also makes it difficult to clinically differentiate between these conditions. Finally, social stigma and denial of initial symptoms by patients and their families further delay seeking appropriate medical care, negatively impacting early diagnosis and the timely implementation of therapeutic interventions.

[0011] The diagnosis of tauopathies is based on a combination of clinical evaluation and neuropsychological symptoms, along with brain imaging studies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) to detect abnormal deposits of tau protein. Cerebrospinal fluid analysis can also be used, and new blood biomarkers are currently being developed for more accurate and earlier diagnosis.

[0012] Neuroimaging techniques such as Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) are very expensive. MRI uses magnetic fields and radio waves to generate detailed images of the brain and identify brain atrophy, while PET uses radioactive tracers to assess brain metabolism and neurotransmitter activity, allowing for the differentiation of types of dementia by measuring the presence of tau neurofibrillary tangles or the accumulation of beta-amyloid plaques.

[0013] While the abnormal accumulation of tau protein has been well documented as a hallmark of neurodegenerative diseases such as Alzheimer's disease (AD) and other tauopathies, the use of biomarkers, primarily in blood, for early detection remains limited, although blood biomarkers for AD such as p-tau181, p-tau217, and p-tau231 are beginning to be documented (Ashton et al., 2024; Ossenkoppele et al., 2022). However, despite advances in understanding the pathologies of these diseases, no biomarkers exist for any type of tauopathy other than AD. The use of retrotransposons (RTEs) as an early risk marker for neurodegenerative diseases has not yet been widely explored, leaving a significant gap in current diagnostic methods.

[0014] The diagnosis of Alzheimer's disease (AD) is also combined with cerebrospinal fluid (CSF) biomarker analysis, such as total tau, phosphorylated tau, or the AP1-42 / AP1-40 ratio, among others (Alcolea, Pegueroles, et al., 2019). In the case of frontotemporal dementia (FTD), neurofilament light chain (NfL) levels are also measured, and these are elevated compared to AD. However, these procedures are not infallible.

[0015] In the prior art, patent application LIS2014 / 0377758 focuses on the detection of LINE-1 activity, understood as transcription (RNA) and translation (ORF1 / ORF2 proteins), to evaluate processes such as aging or cell damage. It is based on studies of cell lines, tissues, stem cells, and cell cultures, without reference to the use of samples accessible in real clinical settings. The publication primarily uses RT-PCR, reporter transfection, luciferase activity assays, and immunodetection techniques, aimed at detecting protein expression or activity.

[0016] In summary, the combination of clinical tools, neuroimaging, and biomarkers offers a comprehensive approach to dementia diagnosis. However, early detection remains a challenge, especially in the initial stages of the disease, highlighting the need to improve diagnostic methods and raise public awareness about the importance of early intervention, as early diagnosis represents the best opportunity for pharmaceutical intervention.

[0017] Transposable elements (TEs), also known as "jumping" genes or mobile elements, are large repeating DNA sequences that represent more than 50% of the human genome and have been self-amplifying in mammalian genomes throughout evolution (Burns & Boeke, 2012). These mobile elements are divided into two main groups: First, there are DNA transposons, discovered by Barbara McClintock, which are mobilized by the "cut and paste" mechanism, but are currently inactive in humans, despite having been very active during evolution. The other group is retrotransposons (RTEs), which are mobilized through the "copy and paste" mechanism using an RNA intermediate (a mechanism known as retrotransposition). Within the RTE group, there are two subgroups that differ based on the presence or absence of long terminal repeats (LTRs).Long-term retroviruses (LTR-RTEs) are endogenous retroviruses, while non-LTR-RTEs are composed of different endogenous retroviruses (ERs) grouped into the LINE, SINE, and SVA families (Misiak et al., 2019). Eight percent of the ERs active in the human genome belong to the non-LTR-RTE group. Activation of this type of ER leads to transposition, which can be detrimental to the cell's genomic stability and may compromise gene expression or produce cellular stress (Hancks & Kazazian, 2012; Kaer & Speek, 2013; Pace & Feschotte, 2007).

[0018] Numerous studies have linked oxidative stress and, consequently, DNA damage to neuronal death in Alzheimer's disease (AD) and other tauopathies. In animal models such as tau-transgenic mice and Drosophila, as well as in humans with AD, DNA damage has been observed to alter chromatin structure and cause epigenetic changes that contribute to tau-induced neurodegeneration (Frost et al., 2014). Several studies have described an increase in TE transcripts in tauopathy models (Guo et al., 2018; Ramirez et al., 2021; Sun et al., 2018; Vallés-Saiz et al., 2023). But despite these findings, previous studies have mainly focused on animal models or post-mortem human samples and in a more mechanistic way, that is, they focus more on looking for the underlying mechanisms of tau-induced TE activation (Ramirez et al., 2021), which in the use of RTEs as indicators of pathology or their use for early diagnosis. To date, no clinical method has been developed for the early detection of tauopathies by quantifying transposable elements, such as LINE-1, in biological fluids from living patients, the main objective of the present invention, which represents a novel and useful approach in the early diagnosis of these pathologies.

[0019] Patent application WO2021174217A1 describes methods and compositions related to the detection of polymorphic LINE-1 elements (pLIs) in the human genome through sequencing, focused on the diagnosis of diseases such as breast cancer, prostate cancer, and Alzheimer's disease by identifying specific LINE-1 genetic markers at known insertion sites. The patent protects the use of probes and electronic devices for the detection of these pLIs in genomic DNA, as well as detection kits focused on a subset of pLIs associated with different diseases. However, the invention described in WO2021174217A1 does not address or explore the use of quantifying the total number of LINE-1 elements as a biomarker in biological fluid samples such as serum or buffy coat for the early detection of tauopathies, such as Alzheimer's disease.Furthermore, WO2021174217A1 focuses on the detection of polymorphic LINE-1 vahant elements, while the present invention aims to quantify all LINE-1 elements in biological fluids, specifically circulating cell-free DNA (cfDNA), using digital droplet PCR (ddPCR). This approach provides a non-invasive, sensitive, less expensive, and specific diagnostic tool capable of detecting changes in LINE-1 activity in the early stages of tauopathies, when other conventional biomarkers are not yet detectable.

[0020] Japanese patent application JP7503558B2 protects a pharmaceutical composition containing either Sensavidine or Elvucitabine, antiretroviral drugs, and a pharmaceutically acceptable excipient. It is intended for the treatment or prevention of neurodegenerative and autoimmune diseases, focusing on describing LINE-1 in relation to the pathological mechanisms involved in these diseases, but not as a biomarker for early diagnosis, which is the primary objective of the present invention. The proposed use is geared towards evaluating cellular aging, genomic health, or the effects of treatments on genome stability, without mentioning specific neurodegenerative diseases. The primary techniques used are RT-PCR, reporter transfection, luciferase activity assays, and immunodetection, aimed at detecting protein expression or activity.

[0021] The scientific publication PROTASOVA MS et al. Quantitative analysis of L1- retrotransposons in Alzheimer's disease and aging. Biochemistry (Moscow), 2017, Vol. 82, Pages 962-971, reveals a procedure for quantifying the number of active copies of LINE-1 using RT-PCR in brain and peripheral blood samples from patients with Alzheimer's disease (AD), and in healthy control individuals without signs of dementia.

[0022] As a result of the analyses performed, no statistically significant differences were found in the number of copies of the RTE LINE-1 quantified in blood and frontal cortex samples from AD patients and a group of elderly control patients without signs of neuropathies.

[0023] Real-time PCR analysis of the number of copies of active aL1 repeat families in frontal cortex DNA samples from Alzheimer's disease patients and healthy individuals without neuropathological signs was performed using two methods: TaqMan probes [14, 20, 22] or SYBR Green dye. Previous studies [14, 20, 22] have determined the number of aL1 copies in individual DNA samples in relation to the number of 5S rDNA copies. However, it has recently been determined that the number of 5S rDNA repeat copies also exhibits interindividual variability [27, 28]. In the first stage of the TaqMan probe analysis, the number of 5S rDNA copies is estimated in relation to the single-copy ALB gene. It is shown that in brain DNA samples (n = 26), the number of 5S rDNA copies exhibits considerable variability (from 52 to 93) (Fig.1), which corresponds to previously published data [27, 28]. Therefore, due to the differences revealed in the number of 5S rDNA repeat copies in human genomes and the observation of a possible increase in copies, given the number of 5S rDNA in patients with AD, the use of these repeats as an endogenous control would be incorrect ([29, 30] and the unpublished data. Therefore, for the quantitative assessment of the number of copies of active aL1 repeat families, both in patients with AD and in healthy individuals, it is essential to use an invariable copy number in the genome as a control, for example, the ALB gene [25, 31, 33]. This gene was used as an endogenous control in the experiment with the SYBR Green dye.

[0024] Therefore, in addition to studying the total copy number changes of aL1 in the genome of patients with Alzheimer's disease (AD), an important focus for future studies will be the direct determination of specific loci, primarily in somatic cells, where retrotransposition of mobile elements occurs. It is concluded that a significant change in aL1 activity can lead to severe pathologies, and therefore, during normal aging, changes in the aL1 copy number in the genome are negligible. It should be noted that insertions or deletions of aL1 repeats in certain parts of the genome affect the expression of nearby genes. Therefore, the absence of notable quantitative changes in aL1 in AD patients does not exclude possible functional changes in the genome associated with certain aL1 rearrangements, which could contribute to a pathological process.

[0025] For all the reasons mentioned above, there is a technical challenge in the early diagnosis of neurodegenerative diseases, such as tauopathies, particularly Alzheimer's disease (AD), mild cognitive impairment (MCI), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), and corticobasal syndrome (CBS), among others. Currently, the clinical diagnostic accuracy for these diseases ranges from 65% to 90%, being higher in centers specializing in memory disorders and lower in primary care. Current methods, such as imaging studies and cognitive assessments, do not always allow for the detection of the disease in its initial stages, when interventions could be most effective. For example, mild cognitive impairment (MCI) can be a prodromal phase of AD. If diagnosed early, starting therapeutic interventions in MCI offers a significant opportunity to achieve greater efficacy of current treatments.Furthermore, the most accurate diagnostic tests, such as lumbar punctures to detect biomarkers in cerebrospinal fluid (CSF), are invasive and not ideal for population screening. Therefore, there is a clear need to develop a non-invasive, highly sensitive, and specific diagnostic method that allows for the identification of patients at risk of developing dementia in its early stages and democratizes the diagnosis of these diseases.

[0026] The present invention solves this problem by using non-LTR RTE, such as LINE-1, in vitro as a biomarker in isolated human biological fluid samples for the early detection of tauopathy-related dementias. Because it is a non-invasive method, unlike techniques requiring cerebrospinal fluid (CSF) extraction (lumbar puncture), the use of serum samples offers a much less invasive solution for patients, facilitating its application in clinical or population screening settings.

[0027] The diagnosis is based on identifying an increased number of LINE-1 copies in an individual's biological fluids. Biological samples from the individual may be selected from: serum, buffy coat, plasma, blood, cerebrospinal fluid (CSF), saliva, tears, and urine.

[0028] In a preferred embodiment, the technique employed in the present invention uses DNA extracted from serum or buffy coat (the leukocyte and platelet layer remaining between the plasma (above) and the erythrocytes (below) after centrifuging anticoagulated blood), a leukocyte-rich fraction that is more representative of the cellular genomic content, which can significantly influence the detected signal. The methodology employed allows for the in vitro detection of significant and reproducible differences, enabling the discrimination of patients with tauopathies from controls, with diagnostic and screening potential. The invention quantifies DNA copies per ng, providing a standardized, comparable, and clinically useful result. The invention focuses on tauopathies in general and demonstrates diagnostic utility in several of them, including Alzheimer's disease (AD), mild cognitive impairment (MCI), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), and corticobasal syndrome (CBS).This reinforces the applicability and novelty of the biomarker as a cross-cutting tool in the early diagnosis of tau-related neurodegenerative diseases.

[0029] In the present invention, the term "biomarker" refers to a biological characteristic that can be measured and evaluated as an indicator of normal biological processes, pathogenic processes, or responses to therapeutic interventions. Biomarkers may be molecules, genes, gene products, hormones, or observable physical characteristics that reflect the state of health or disease in an organism. The term biomarker as used herein refers to a non-LTR RTE, such as LINE-1, quantifiable in biological fluid samples from living individuals.

[0030] As used in the invention, the term “individual” refers to any human patient.

[0031] The terms “biological fluid” and “biological sample” are used interchangeably in the present invention, referring to any substance that originates within the body of an organism and contains components such as cells, molecules, or metabolic products. These fluids reflect the physiological state of the body and are commonly used in medical diagnostics to detect diseases or study biological processes.

[0032] A “Point of Care (PoC) device,” in the context of the present invention, is a portable and easy-to-use diagnostic system designed for the rapid and accurate detection of the biomarker of interest in biological samples obtained from patients. This device is intended for direct use at the point of healthcare, such as in clinics, hospitals, doctor's offices, or population screening settings, without the need to send samples to a centralized laboratory. The PoC allows for the analysis of minimally invasive biological samples such as serum, plasma, buffy coat, blood, or saliva, and provides immediate results.

[0033] DESCRIPTION OF THE DRAWINGS

[0034] To complement the description being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of its practical embodiment, a set of drawings is included as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0035] FIG. 1: Comparison of the number of LINE-1 copies per ng of DNA from a buffy coat sample between healthy subjects (Control) and patients with Alzheimer's Disease (AD). (A) AD patients have a significantly higher number of LINE-1 copies compared to controls, with a statistically significant difference (p < 0.05). The graph shows the ± SEM value. (B) The graph shows that the residuals follow a normal distribution. (C) The estimation plot shows the difference in means between the two groups, with a confidence interval, indicating that the increase in LINE-1 copies in AD compared to controls is robust.

[0036] FIG. 2: Comparison of the number of LINE-1 copies per ng of DNA from a buffy coat sample between healthy subjects (Control) and patients with MCI (Mild Cognitive Impairment). (A) MCI patients have a significantly higher number of LINE-1 copies compared to controls, with a statistically significant difference (p < 0.05). The graph shows the ± SEM value. (B) The graph shows that the residuals follow a normal distribution. (C) The estimation plot shows the difference in means between the two groups, with a confidence interval, indicating that the increase in LINE-1 copies in MCI compared to controls is statistically significant.

[0037] FIG. 3: Comparison of the number of LINE-1 copies per ng of DNA from a buffy coat sample between healthy subjects (Control), MCI (Mild Cognitive Impairment), and patients with AD (Alzheimer's Disease). Control patients showed a significantly lower number of LINE-1 copies compared to patients with MCI and AD. No differences were found between patients with MCI and AD. The graph shows the ± SEM value.

[0038] ****P value < 0.0001 ; *P value < 0.045 (All standard: 0.05)

[0039] FIG. 4: Comparison of the number of LINE-1 copies per ng of DNA from a buffy coat sample between healthy subjects (Control) and patients with PSP / SCB (Progressive Supranuclear Palsy / Corticobasal Syndrome). (A) PSP / SCB patients have a significantly higher number of LINE-1 copies compared to controls, with a statistically significant difference (p < 0.001). The graph shows the ± SEM value. (B) The graph shows that the residuals follow an approximately normal distribution. (C) The estimation plot shows the difference in means between the two groups, with a confidence interval, indicating that the increase in LINE-1 copies in PSP compared to controls is robust.

[0040] FIG. 5: Comparison of the number of LINE-1 copies per ng of DNA from a buffy coat sample between healthy subjects (Control) and patients with FTD (Frontotemporal Dementia). (A) FTD patients have a significantly higher number of LINE-1 copies compared to controls, with a statistically significant difference (p < 0.01). The graph shows the ± SEM value. (B) The graph shows that the residuals follow an approximately normal distribution. (C) The estimation plot shows the difference in means between the two groups, with a confidence interval, indicating that the increase in LINE-1 copies in FTD compared to controls is robust.

[0041] FIG. 6: Shows the ROC curve, EA vs Control, for patients with Alzheimer's Disease (AD).

[0042] FIG. 7: Shows the ROC curve, MCI vs Control, for patients with Mild Cognitive Impairment (MCI). FIG. 8: Shows the ROC curve, PSP / SCB vs Control, for patients with Progressive Supranuclear Palsy / Corticobasal Syndrome (PSP / SCB).

[0043] FIG. 9: Shows the ROC curve, FTD vs Control, for patients with Frontotemporal Dementia (FTD).

[0044] PREFERRED EMBODIMENT OF THE INVENTION

[0045] In general terms, the procedure of the invention for detecting non-LTR RTE in vitro, as LINE-1, comprises the following steps:

[0046] 1. Obtaining the human biological fluid sample: Isolated biological samples are obtained, allowing for external diagnostic procedures. The individual's biological samples can be selected from: serum, buffy coat, plasma, blood, cerebrospinal fluid (CSF), saliva, tears, and urine. In a preferred embodiment, the biological sample is buffy coat. The sample can be processed immediately or stored at low temperatures (-80°C) until analysis.

[0047] 2. Genomic DNA Extraction: DNA is extracted from the biological sample, preferably buffy coat, using conventional nucleic acid extraction methods, such as commercial high-purity DNA kits. This step can be optionally omitted.

[0048] 3. Quantification of non-LTR RTEs: This can be carried out using various techniques that allow for their identification and quantification: a. Digital droplet PCR (ddPCR): This technique allows for the absolute quantification of DNA molecules by dividing the sample into thousands of individual droplets. Each droplet is amplified by PCR, allowing for counting how many contain the biomarker of interest. It is a very accurate technique for detecting low levels of DNA and small variations in concentration. b. Quantitative real-time PCR (qPCR): This is a widely used standard molecular technique that uses fluorescent probes to quantify the amount of RTEs. It is useful for detecting the presence of DNA and measuring its amount relative to a reference sample. c. Next-generation sequencing (NGS): This allows for a more in-depth and comprehensive quantification of RTEs.It can sequence all the DNA or RNA present in a sample and, from there, identify and quantify the amount of the biomarker of the invention present. d. CRISPR-based assays: With the recent advancement of CRISPR technology, techniques have been developed for the specific detection of DNA sequences. e. ELISA assay adapted for nucleic acid detection: Although ELISA is generally used to detect proteins, it can be adapted to detect DNA or RNA of the biomarker of interest by incorporating specific antibodies that bind to modified nucleic acid sequences, as in the case of aptamer techniques. f. DNA microarrays: Microarrays allow for the simultaneous analysis of many DNA sequences in a sample. They can be designed to detect and quantify the biomarker of the invention along with other relevant genetic elements. g.NGS (Next-Generation Sequencing, applied to DNA and multi-omics): Massive parallel sequencing technology that allows the order of nucleotides in large segments of DNA or RNA to be determined quickly and efficiently. Its application to DNA allows the detection of various genomic alterations, while in the multi-omics field it makes it possible to simultaneously analyze DNA, RNA (transchaptome) and other molecular profiles to obtain a comprehensive view of a patient or a biological system.

[0049] 4. Analysis of results: Non-LTR RTE levels in the patient's biological sample are compared with reference values ​​obtained from healthy individuals (controls) of similar ages. The analysis of results includes the use of positive and negative controls to ensure the validity of the diagnosis. A significant increase in the number of copies of the biomarker of interest indicates a higher risk of developing tauopathy-related dementias.

[0050] The method of the invention is suitable for implementation in clinical settings or in Point of Care (PoC) devices, facilitating early detection in at-risk populations, improving patient prognosis through earlier interventions.

[0051] Thus, in another aspect, the invention also protects the development of diagnostic kits and devices based on the in vitro detection of a non-LTR RTE, such as LINE-1, in various biological samples from patients. This biomarker is useful for the early diagnosis of neurodegenerative diseases, such as tauopathies, ALS, and MS, among others.

[0052] Materials and methods

[0053] Example: Detection of LINE-1 as a biomarker in buffy coat.

[0054] The patient groups analyzed were diagnosed according to criteria described in (Alcolea, Clarimón, et al., 2019; Alcolea, Pegueroles, et al., 2019; Armstrong et al., 2013; Boxer et al., 2017). For this study, control patient groups, patients with Alzheimer's Disease (AD), Mild Cognitive Impairment (MCI), Frontotemporal Dementia (FTD), and patients with Progressive Supranuclear Palsy (PSP) and Corticobasal Syndrome (CBS) were used. Since a definitive distinction between PSP and CBS is only possible post-mortem, both were operationally grouped within the same clinical category. Age groups were homogeneous. Data relating to the samples are summarized in the table below.

[0055] For buffy coat extraction, blood was drawn from patients without prior fasting and centrifuged within the first two hours after collection. The Qiagen DNeasy Blood & Tissue kit was used for total DNA extraction from the blood samples. The DNA analyzed is extracted from the buffy coat (the leukocyte and platelet layer remaining between the plasma (above) and the erythrocytes (below) after centrifuging anticoagulated blood), as it is a leukocyte-rich fraction and more representative of the cellular genomic content, which can significantly influence the detected signal.

[0056] The detection and quantification of the LINE-1 element was performed by droplet digital PCR (ddPCR) using the Bio-Rad QX200 Droplet Digital PCR platform. The LINE-1-specific primers and probe were designed based on the publicly available reference sequence deposited in the GenBank database under accession number NM_019079.5. PCR reactions were performed using the ddPCR probe supermix (Bio-Rad, CN 1863010) with the FAM fluorophore-labeled probe. Samples were analyzed with 2 ng of undigested DNA per well. Droplet generation was performed according to the manufacturer's instructions using the Bio-Rad QX200 droplet generator. Subsequently, the droplet emulsions were subjected to the following PCR thermal conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds and 54°C for 2 minutes, and a final step at 98°C for 10 minutes.Droplet readings were performed using the QX200 droplet reader, and data analysis was conducted using Poisson's Law with QuantaSoft v1.7.4.0917 software (Bio-Rad), which determined the target DNA concentration by quantifying the positive and negative drops for each sample. For the calculation to be reliable, the following three conditions had to be met simultaneously: at least 10,000 drops had to be accepted; the number of negative drops had to be greater than one-third of the total drops; and a well was only considered positive if it contained fewer than three positive drops. All samples with a coefficient of variation greater than 10% were discarded.

[0057] Results

[0058] The present invention provides experimental data demonstrating the applicability of the proposed method for the in vitro detection of the presence, risk, or severity of tauopathies. Data normality analysis and confidence intervals were performed to verify comparability, as shown in sections B and C of Figures 1, 2, 4, and 5.

[0059] For Alzheimer's Disease (AD) patients, comparing the number of LINE-1 copies per ng of DNA from a buffy coat sample between non-disease subjects (Control) and AD patients shows a significantly higher number of LINE-1 copies compared to controls, approximately greater than 300, with a significant difference (p < 0.05).

[0060] Patients with Mild Cognitive Impairment (MCI) have a significantly higher number of LINE-1 copies compared to controls, approximately greater than 180, with a significant difference (p < 0.05).

[0061] Patients with Progressive Supranuclear Palsy / Corticobasal Syndrome (PSP / CBS) have a significantly higher number of LINE-1 copies compared to controls, approximately greater than 400, with a significant difference (p < 0.001).

[0062] Patients with Frontotemporal Dementia (FTD) have a significantly higher number of LINE-1 copies compared to controls, approximately greater than 300, with a significant difference (p < 0.01).

[0063] In the analyses performed on the tauopathies, we observed a higher number of LINE-1 copies per ng of DNA sample in the buffy coat, even with significant differences between the control groups and the groups of patients with AD (p<0.05 (Fig. 1), patients with PSP / SCB with p<0.001 (Fig. 4), and patients with FTD with p<0.01 (Fig. 5). However, the most relevant finding of the present invention is how we found significant differences (p<0.05) between the control group and the group with MCI (Fig. 6).

[0064] 2), a level of sensitivity that current biomarkers do not reach, demonstrating that the LINE-1 biomarker is a promising candidate to improve early diagnosis in these pathologies.

[0065] To evaluate the performance of a binary classification model, a Receiver Operating Characteristic (ROC) curve is used. This curve shows the relationship between the true positive rate (sensitivity) and the false positive rate (specificity) for different model cutoff points. Sensitivity indicates the true positive rate, while specificity indicates the false positive rate. In this case, the quantization could have different configurations (different cutoff points). The ROC curve shows how sensitivity and specificity change when these configurations are altered.

[0066] ROC curves can be used to see which classifier performs best in terms of sensitivity and specificity. To choose the best cutoff point, the ROC curve can be used to find the cutoff point that provides high sensitivity.

[0067] When choosing between two different diagnostic tests, ROC curves are used because they provide a comprehensive measure independent of the cutoff point. For this reason, in the healthcare field, ROC curves are also called diagnostic performance curves.

[0068] The choice is made by comparing the area under the curve (AUC) of both tests. This area ranges from 0.5 to 1, where 1 represents a perfect diagnostic value and 0.5 represents a test with no diagnostic discriminatory capacity. In other words, if the AUC for a diagnostic test is 0.8, it means there is an 80% probability that the diagnosis made for a sick person will be more accurate than that of a randomly chosen healthy person. Therefore, the diagnostic test with the larger area under the curve is always chosen.

[0069] The area under the ROC curve (AUC) is a number that summarizes the overall performance of the classifier. An AUC of 1 indicates a perfect classifier, while an AUC of 0.5 indicates a random classifier.

[0070] In this case, the ROC curve is shown in Figure 6, Alzheimer's Disease (AD) vs Control; the values ​​are:

[0071] The ROC curve in Figure 7, Mild Cognitive Impairment (MCI) vs Control, the values ​​are:

[0072] Metric Value

[0073] AUC 0.709

[0074] Sensitivity 46.15%

[0075] Specificity 100%

[0076] Positive predictive value (PPV) 100%

[0077] Negative predictive value (NPV) 66.67%

[0078] Accuracy 74.07%

[0079] The ROC curve in Figure 8, Progressive Supranuclear Palsy / Corticobasal Syndrome (PSP / CBS) vs. Control, the values ​​are: Metric Value

[0080] AUC 0.9184

[0081] Sensitivity 85.71%

[0082] Specificity 100%

[0083] Positive predictive value (PPV) 100%

[0084] Negative predictive value (NPV) 93.33%

[0085] Accuracy 95.24%

[0086] The ROC curve in Figure 9, Frontotemporal Dementia (FTD) vs Control, the values ​​are:

[0087] Metric Value

[0088] AUC 0.8175

[0089] Sensitivity 66.67%

[0090] Specificity 100%

[0091] Positive predictive value (PPV) 100%

[0092] Negative predictive value (NPV) 82.35%

[0093] Accuracy 86.96%

[0094] Unlike techniques that require CSF extraction (lumbar puncture), the use of samples such as buffy coat offers a much less invasive solution for patients, making it easier to apply in a clinical or population screening context.

[0095] In the preferred embodiment, the procedure for detecting non-LTR RTE, such as LINE-1 in vitro, comprises the following steps:

[0096] 1. Obtaining the human biological fluid sample: Isolated biological samples are obtained, and this diagnosis is carried out in vitro. The biological samples from the individual can be selected from: serum, plasma, blood, buffy coat, cerebrospinal fluid (CSF), saliva, tears, and urine. In a preferred embodiment, the biological sample is buffy coat. The sample can be processed immediately or stored at low temperatures (-80°C) until analysis.

[0097] 2. Genomic DNA Extraction: DNA is extracted from serum or buffy coat using conventional nucleic acid extraction methods, such as commercial high-purity DNA kits. The DNA analyzed is extracted from the buffy coat (the leukocyte and platelet layer remaining between the plasma (above) and the erythrocytes (below) after centrifuging anticoagulated blood), as it is a leukocyte-rich fraction and more representative of the cellular genomic content, which can significantly influence the detected signal.

[0098] 3. Quantification of non-LTRs: This can be carried out using various techniques that allow for their identification and quantification: a. Digital droplet PCR (ddPCR): This technique allows for the absolute quantification of DNA molecules by dividing the sample into thousands of individual droplets. Each droplet is amplified by PCR, allowing the number of droplets containing the biomarker of interest to be counted. It is a very accurate technique for detecting low levels of DNA and small variations in concentration. b. Quantitative real-time PCR (qPCR): This is a widely used standard molecular technique that uses fluorescent or intercalating probes to quantify the amount of TTRs. It is useful for detecting the presence of DNA / RNA and measuring its relative or absolute quantity compared to a reference sample. c. Next-generation sequencing (NGS): This allows for a more in-depth and comprehensive quantification of TTRs.It can sequence all the DNA or RNA present in a sample and, from there, identify and quantify the amount of the biomarker of the invention present. d. CRISPR-based assays: With the recent advancement of CRISPR technology, techniques have been developed for the specific detection of DNA sequences. e. ELISA assay adapted for nucleic acid detection: Although ELISA is generally used to detect proteins, it can be adapted to detect DNA or RNA of the biomarker of interest by incorporating specific antibodies that bind to modified nucleic acid sequences, as in the case of aptamer techniques. f. DNA microarrays: Microarrays allow for the simultaneous analysis of many DNA sequences in a sample. They can be designed to detect and quantify the biomarker of the invention along with other relevant genetic elements. g.NGS (Next-Generation Sequencing, applied to DNA and multi-omics variables) is a massive parallel sequencing technology that allows the order of nucleotides in large segments of DNA or RNA to be determined quickly and efficiently. Its application to DNA allows the detection of various genomic alterations, while in the multi-omics field it makes it possible to simultaneously analyze DNA, RNA (transcriptome) and other molecular profiles to obtain a comprehensive view of a patient or a biological system.

[0099] 4. Analysis of the results: Non-LTR RTE levels in the patient's buffy coat sample were compared with reference values ​​obtained from healthy (control) individuals of similar ages. A significant increase in the number of copies of the biomarker of interest indicates a higher risk of developing tauopathy-related dementias.

[0100] The method of the invention is suitable for implementation in clinical settings or integration into Point of Care (PoC) devices, facilitating early detection in at-risk populations, improving patient prognosis through earlier interventions.

[0101] As can be seen, the method has been initially validated for the detection of AD, PSP / SCB and FTD, and can also be applied to other tauopathies or neurodegenerative diseases where there is aberrant genetic activity of these RTEs such as neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS) and Multiple Sclerosis (MS), which significantly expands its clinical utility.

[0102] One aspect of the invention relates to a non-invasive detection technique.

[0103] Thus, in another aspect, the invention also protects the development of reagent kits and diagnostic devices based on the detection of a non-LTR RTE, such as LINE-1, in various biological samples from patients. This biomarker is useful for the early diagnosis of neurodegenerative diseases, such as tauopathies, ALS, and MS, among others.

[0104] In a preferred embodiment, the kit or device of the invention comprises:

[0105] (A) Means for detecting the biomarker of the invention in a biological fluid obtained from the individual, wherein the means may include probes or reagents specific for the quantification of the biomarker, using technologies such as: (a) ddPCR (droplet digital PCR)

[0106] 1. Forward and reverse oligonucleotides targeting specific regions of the LINE-1 biomarker.

[0107] 2. TaqMan type probe: Fluorophore (FAM, HEX, etc.) and quencher (BHQ, TAMRA).

[0108] 3. MasterMix specific for ddPCR: Contains Taq DNA polymerase, dNTPs, MgCI2, and optimized buffers.

[0109] 4. Partitioning oil or reagent

[0110] 5. PCR plate or specific cartridge for ddPCR

[0111] 6. Positive and negative controls (genomic DNA with a known amount of LINE-1 and blank without a target)

[0112] 7. Pattern curve

[0113] (b) qPCR (quantitative PCR, including qPCR with intercalators and TaqMan type qPCR)

[0114] 1. Specific oligonucleotides (forward and reverse) targeting specific regions of the LINE-1 biomarker. (Compatible with detection using fluorescent intercalating agent or probe.)

[0115] 2. Labeled probe (TaqMan or equivalent) (only in qPCR TaqMan)

[0116] • Fluorophore (FAM, VIC, etc.) and quencher (TAMRA, BHQ).

[0117] 3. Intercalating dye (optional for probeless qPCR)

[0118] • SYBR Green or other double-chain bonding dyes.

[0119] 4. Specific MasterMix for qPCR: Contains

[0120] • Taq DNA polymerase

[0121] ■ Thermostable, with or without 5'-3' exonuclease activity (depending on the type of qPCR).

[0122] ■ TaqMan type qPCR requires exonuclease activity.

[0123] • 10X reaction buffer

[0124] • Magnesium chloride (MgCl2)

[0125] • Mixture of dNTPs (dATP, dTTP, dCTP, dGTP)

[0126] • Enzymatic stabilizers

[0127] • UNG (uracil-N-glycosylase, optional)

[0128] • Passive stain (ROX, optional) 5. Positive and negative controls (genomic DNA with a known amount of

[0129] LINE-1 and white without target)

[0130] 6. Pattern curve

[0131] (c) NGS (Next-Generation Sequencing, applied to DNA)

[0132] 1. Specific primers for LINE-1 (DNA)

[0133] 2. High-fidelity enzyme (HiFi DNA polymerase)

[0134] 3. Adapters and indexes

[0135] 4. Buffers and MgCI2

[0136] 5. DNA purification system (SPRI beads or columns)

[0137] 6. Positive and negative DNA controls

[0138] 7. Bioinformatics guide (optional): Suggested pipeline for analysis of variations in LINE-1 from genomic DNA.

[0139] (d) CRISPR assays (DNA detection using Cas12a or other nucleases)

[0140] 1. Guide RNA (crRNA or sgRNA) specific for LINE-1 DNA sequences

[0141] 2. Enzyme Casi 2a or Cas9 (DNA-targeting CRISPR effector)

[0142] 3. Reaction buffer with MgCl2

[0143] 4. Reporting probe (reporter) with fluorophore / quencher

[0144] 5. Pre-amplification system (optional): Isothermal amplification of DNA (RPA or PCR) to increase sensitivity.

[0145] 6. Positive and negative controls

[0146] 7. Lateral-flow format or portable reader (optional for PoC): With strips that detect reporter release.

[0147] (e) DNA microarrays

[0148] 1. Capture probes immobilized on solid support: Oligonucleotides complementary to specific LINE-1 sequences.

[0149] 2. Labeled genomic DNA of the patient

[0150] • Previously fragmented and labeled with fluorophore (Cy3, Cy5...) or biotin.

[0151] • Prepared by titration or amplification reactions with modified nucleotides.

[0152] 3. Internal and external control probes: These include known nonspecific and positive sequences. 4. Hybridization and wash buffers

[0153] 5. Compatible detection systems

[0154] 6. Analysis software (optional)

[0155] (f) ELISA adapted for nucleic acids (DNA detection)

[0156] 1. Functionalized microtiter plate

[0157] • Coated with capture oligonucleotides complementary to LINE-

[0158] 1.

[0159] • They can be bound by biotin, amine or thiols depending on the immobilization chemistry.

[0160] 2. Previously denatured patient target DNA

[0161] 3. Labeled detection probe: Oligo complementary to another region of LINE-1, labeled with biotin, digoxigenin or fluorescein.

[0162] 4. Enzymatic development system

[0163] • Streptavidin-HRP or anti-digoxigenin-HRP antibody.

[0164] • Chromogenic substrate (TMB, ABTS...) for colorimetric detection.

[0165] 5. Specific buffers for hybridization, washing, and blocking

[0166] 6. Positive and negative controls: DNA with high and low LINE-1 charge to establish response curve or cutoff value g) NGS (Next-Generation Sequencing, applied to DNA and multi-omics vahant)

[0167] 1. Specific primers for LINE-1 (DNA).

[0168] 2. High fidelity enzyme (Hi Fi DNA polymerase).

[0169] 3. Sequencing adapters and indices.

[0170] 4. Buffers and MgCI2.

[0171] 5. DNA purification system (SPRI beads or columns).

[0172] 6. Positive and negative DNA controls.

[0173] With compatibilities and extensions:

[0174] 7. Bioinformatics guide (reference pipeline for LINE-1 analysis from genomic DNA).

[0175] 8. Multi-omics sequencing platforms capable of simultaneously identifying the nucleotide sequence and epigenetic modifications (including, but not limited to, 5-methylcytosine and 5-hydroxymethylcytosine) in a single experiment. (B) Means for comparing the levels of the biomarker of interest in the sample with a control reference obtained from healthy individuals.

[0176] In one particular embodiment, the kit is designed to work with biological samples such as serum, plasma, blood, buffy coat, CSF, saliva, tears, and urine, with buffy coat being the preferred option as it is a less invasive and more accessible sample for use in clinical or population screening settings.

[0177] The described kit is compatible with digital PCR technologies, quantitative PCR, next-generation sequencing, multi-omics sequencing platforms (capable of detecting bases and epigenetic modifications), CRISPR assays, microarrays, and nucleic acid-adapted ELISAs.

[0178] As someone skilled in the art would understand, the kit may comprise any component useful for implementing the present invention, including primers, probes, antibodies, buffer solutions, delivery vehicles, material carriers, components for positive and / or negative controls, among others. This kit may also include elements for performing a rapid diagnosis, facilitating the immediate detection of the biomarker of interest in a clinical setting. In addition to the aforementioned components, the kit may include instructions for carrying out the diagnostic technique, which may be presented in different formats, such as information printed on a suitable medium (e.g., inserts in the kit packaging or a sheet of paper) or in digital format (e.g., a link to a website or a QR code providing access to online information).

Claims

REIVINDICACIONES MODIFICADAS recibidas por la oficina Internacional el 07 de abril de 2026 (07.04.2026) 1. In vitro use of a non-LTR RTE such as LINE-1 biomarker to detect and diagnose by means of a biological sample the presence and risk of developing neurodegenerativo diseases, in particular tauopathies which in that risk, detection and diagnosis is determined with a biological sample from the subject to be diagnosed by quantification of the non-LTR RTE LINE-1 and the difference in number of LINE-1 copies per ng of DNA from a sample between non-diseased subjects (Control) and in diseased patients, which is characterized by the fact that the risk of suffering from tauopathy is determined for each of the diseases by the following conditions: - Alzheimer's disease (AD) when the difference for a number of LINE-1 copies per ng of DNA between the control group and the AD patient groups is approximately greater than 300; - Mild Cognitive Impairment (MCI) when the difference for a number of LINE-1 copies per ng of DNA between the control group and the MCI patient groups is approximately greater than 180; - Progressive Supranuclear Palsy (PSP) / Basal Cortical Syndrome (BCS) when the difference for a number of LINE-1 copies per ng of DNA between the control group and the PSP patient groups is approximately greater than 400; and - Frontotemporal Dementia (FTD) when the difference for a number of LINE-1 copies per ng of DNA between the control group and the FTD patient groups is approximately greater than 300.

2. The in vitro use of a non-LTR RTE such as LINE-1 biomarker, according to claim 1 characterised in that the tauopathy diagnosed is Mild Cognitive Impairment (MCI) when the difference for a number of LINE-1 copies per ng of DNA between the control group and the MCI patient groups is approximately greater than 180.

3. The in vitro use of a non-LTR RTE such as LINE-1 biomarker, according to claim 1 characterised in that the tauopathy diagnosed is Progressive Supranuclear Palsy (PSP) / Basal Cortical Syndrome (BCS) when the difference for a number of LINE-1 copies per ng of DNA between the control group and the PSP patient groups is approximately greater than 400.

4. The in vitro use of a non-LTR RTE such as LINE-1 biomarker, according to claim 1 characterised in that the tauopathy diagnosed is Frontotemporal Dementia (FTD) when the difference for a number of LINE-1 copies per ng of DNA between the control group and the FTD patient groups is approximately greater than 300.

5. The in vitro use of a non-LTR RTE such as LINE-1 biomarker, according to claim 1 characterised in that the biological samples of the subject to be diagnosed are selected from: serum, plasma, blood, buffy coat, cerebrospinal fluid (CSF), saliva, tears and urine.

6. The in vitro use of the non-LTR RTE such as LINE-1 biomarker, according to claim 6 characterised in that the DNA is extracted from serum using conventional nucleic acid extraction methods, such as the use of commercial kits for high purity DNA.

7. The in vitro use of the non-LTR RTE such as LINE-1 biomarker, according to claim 6 characterised in that the DNA is extracted from the buffy coat using conventional nucleic acid extraction methods, such as the use of commercial kits for high purity DNA.

8. The in vitro use of a non-LTR RTE biomarker, such as LINE-1 according to claim 1 characterised in that the quantification of non-LTR RTE, such as LINE- 1 is selected from (a) droplet digital PCR (ddPCR) techniques; b) real-time quantitative PCR (qPCR); c) Next Generation Sequencing (NGS); d) CRISPR- based assays; e) ELISA adapted for detecting nucleic acids; f) DNA microarrays; and g) NGS (Next-Generation Sequencing, applied to DNA and multi-omic variants).

9. A method to detect non-LTR RTE, such as LINE-1 in vitro comprising the following steps:

1. Obtaining the sample of human biological fluids, wherein the biological samples are selected from: serum, plasma, blood, buffy coat, cerebrospinal fluid (CSF), saliva, tears and urine; 2. Genomic DNA extraction, wherein DNA is extracted from the sample in step 1 ; 3. Quantification of non-LTR RTE, wherein quantification is performed by selecting the following techniques: a. Droplet digital PCR (ddPCR); b. Real-time quantitative PCR (qPCR); c. Next generation sequencing (NGS); d. CRISPR-based assays; e. Adapted ELISA assay for detecting nucleic acids; f. DNA microarrays; g. NGS (Next-Generation Sequencing, applied to DNA and multi-omic variants); and 4. Analysis of the results, wherein the levels of non-LTR RTE, such as LINE 1 in the biological sample from the patient are compared with reference values obtained from healthy (control) individuals of similar age.

10. The method according to claim 10, wherein the analysis of the results includes the use of positive and negative controls to ensure the validity of the diagnosis.

11. A diagnostic reagent kit for the in vitro detection of the non-LTR RTE, such as LINE-1 biomarker which is characterised in that it comprises: (A) Means for detecting the biomarker of the invention in a biological sample obtained from the individual, wherein the means may include specific probes or reagents for the quantification of the biomarker and using technologies such as: (a)ddPCR (droplet digital PCR); (b)qPCR (quantitative PCR, including qPCR with intercalants and TaqMan qPCR); (c)NGS (Next-Generation Sequencing, applied to DNA); (d) CRISPR assays (detection of DNA by Cas12a or other nucleases); (e) DNA microarrays; (f) Adapted ELISA for nucleic acids (DNA detection); and g) NGS (Next-Generation Sequencing, applied to DNA and multi-omic variants) (B) Means to compare the levels of the biomarker of interest in the sample with a control reference obtained from healthy individuals.

12. The diagnostic reagent kit for the in vitro detection of the non-LTR RTE such as LINE-1 biomarker according to claim 12 wherein the biological samples are selected from serum, plasma, blood, buffy coat, CSF, saliva, tears and urine.

13. The diagnostic reagent kit for the in vitro detection of the non-LTR RTE such as LINE-1 biomarker according to claim 12 characterised in that the kit is compatible with digital PCR technologies, quantitative PCR, next generation sequencing, multi-omics sequencing platforms (capable of detecting bases and epigenetic modifications), CRISPR assays, microarrays and ELISAs adapted to nucleic acids.

14. The diagnostic reagent kit for the in vitro detection of the non-LTR RTE such as LINE-1 biomarker, according to claim 12, characterised in that it can be implemented in clinical environments or integrated into Point of Care (PoC) devices.

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