WO2020079248A1

WO2020079248A1 - Early detection of precursor of alzheimer's disease

Info

Publication number: WO2020079248A1
Application number: PCT/EP2019/078421
Authority: WO
Inventors: Gregory PENNER
Original assignee: Neoneuro
Current assignee: Neoneuro
Priority date: 2018-10-19
Filing date: 2019-10-18
Publication date: 2020-04-23
Anticipated expiration: 2021-04-19

Abstract

The present invention relates to the early detection of amyloid-beta (Αβ) accumulation and early diagnosis of associated diseases, in particular Alzheimer's disease. More specifically, the present invention relates to an enriched library of aptamers and uses thereof for the detection and diagnosis of Αβ and associated diseases.

Description

EARLY DETECTION OF PRECURSOR OF ALZHEIMER’S DISEASE

FIELD OF INVENTION

The present invention relates to the detection of amyloid-beta (Ab) brain lesion accumul ation and early diagnosis of associated diseases, in particular Alzheimer’ s disease through the analysis of blood.

More specifically, the present invention relates to an enriched library of aptamers and uses thereof for the detection and diagnosis of Ab brain lesions and associated diseases from blood analysis.

BACKGROUND OF INVENTION

Strimbu & Tavel (2010. Curr Opin HIV AIDS. 5(6):463-6) define biomarkers as “ objective indications of medical state observed from outside the patient - which can be measured accurately and reproducibly” . This definition does not require that the functional relationship between the biomarker and the medical state be understood, rather the definition only requires that the relationship between the measurement of the biomarker and the medical state be measured accurately and reproducibly.

Alzheimer’s disease (AD) is generally diagnosed by the loss of significant cognitive function. It is clear that, at this stage, a substantial portion of the brain has already been irreversibly damaged by the accumulation of Ab brain lesions, and hyperphosphorylated tau tangles. A description of the progression of the disease from Ab brain lesions to tau tangles and finally to neurological degeneration has become generally accepted as the A/T/N classification system (Jack et al. , 2016. Neurology. 87(5):539-547).

The accumulation of Ab brain lesions is an accepted risk factor for the eventual development of Alzheimer’s disease (AD). These brain lesions can be identified and quantified decades before symptoms of cognitive dysfunction become apparent through the use of positron emission tomography (PET) scans. Acceptable levels of predictability can also be obtained through the analysis ofbiomarkers in cerebrospinal fluid (CSF). This early diagnostic capacity is a necessary prerequisite for the identification of individuals at risk of developing AD for enrollment in clinical trials for testing potential treatments that may delay the onset of the disease. However, the cost and invasiveness of these analytical tools constrains their broad scale application. In addition, a significant proportion of individuals exhibiting clinically positive levels of Ab brain lesion accumulation as demonstrated by PET scans are not diagnosed with existing biomarkers in CSF. These individuals are described as“non-aligned” with the existing diagnostic platform. There is therefore a need for a reliable means of diagnosing the level of the accumulation of Ab brain lesions through an analysis of blood samples. The presence of Ab lesions can be determined by the introduction of a fluorescent probe followed by PET scans (Xu et al, 2016. Acta Pharmacol Sin. 37(6):719-30). Ab brain lesions can also be quantified through correlations with the ratio of Ab42 peptide to Ab40 peptide in CSF. These analyses show significant promise to become standard clinical tools, as a means of assessing risk factors for the development of AD. It is clear that the development of effective treatments will require diagnosis of subjects at this stage, preferably overcoming the cost and time associated with PET scans and/or lumbar punctures. There remains thus a clear need for diagnostic tests on blood. There are some encouraging results from the use of known biomarkers (the same biomarkers used for CSF analysis) on blood through the use of immunoprecipitation followed by mass-spectrophotometric analysis of the captured proteins and through the use of SIMOA technology. (McCaffrey, Aug. 24, 2018. With Sudden Progress, Blood Ab Rivals PET at Detecting Amyloid. Alzforum. Retrieved October 08, 2018, from https://www.alzforum.org/news/conference-coverage/sudden-progress-blood-av-rivals- pet-detecting-amyloid). These results are however biased by the inclusion of subjects that are exhibiting symptoms of cognitive dysfunction and thus exhibiting potentially higher levels of cognitive dysfunction, as well as potentially developing lesions in their brain- blood-barriers that are allowing more of these biomarkers to flow into blood.

The analysis of these biomarkers is time-consuming, and it remains unclear what the commercial cost of analysis will be. The level of diagnostic power exhibited by known biomarkers to date while encouraging, still provides considerable room for error. There remains thus a need for an efficient diagnostic process with the capacity for higher accuracy. It is clear that certain subjects are misdiagnosed consistently through the use of known biomarkers. There is thus a need for a diagnostic platform that can also be applied to the diagnosis of such cases that are non-aligned with traditional biomarkers. The use of aptamers meets these needs. Aptamers are short oligomers usually formed from nucleic acids (DNA, RNA, PNA or a mix thereof) that exhibit the capacity to bind to a specific epitope. A key advantage for aptamers over antibodies for the co-discovery of ligands and biomarkers is that the identification of aptamers is based on the re-iterative selection and counter-selection of very large random pools of potential ligands. Here, the Inventor has characterized a minimal set of aptamers that have been selected and counter-selected for their capacity to specifically bind to unknown epitopes in blood samples from individuals with levels of Ab brain lesions that are associated with a risk of developing AD. This set of aptamers has been used herein to predict the Ab brain lesion status across 70 human individuals previously diagnosed by PET scan analysis.

SUMMARY

The present invention relates to an enriched library of aptamers comprising at least two aptamers with oligonucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 44. In one embodiment, the enriched library of aptamers according to the present invention comprises:

a) one aptamer with oligonucleotide sequence set forth in SEQ ID NO: 38;

b) two aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 38 and 5; c) ten aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 38, 30, 5, 25, 33, 36, 42, 23, 4 and 35;

d) ten aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 to 10; e) thirteen aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 , 2 and 11 to 21; or f) twenty-two aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 5, 23, 20, 41, 39, 38, 30, 8, 13, 4, 25, 33, 36, 42, 24, 34, 29, 27, 35, 19, 16 and 9.

In one embodiment, the enriched library of aptamers according to the present invention comprises:

a) the twenty-one aptamers with oligonucleotide sequences set forth in SEQ ID NOs:

1 to 21; or

b) the forty-four aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44.

The present invention also relates to the use of an enriched library of aptamers according to the present invention, in the in vitro detection of amyloid-beta (Ab) brain lesions.

In one embodiment, the use in the in vitro detection of Ab brain lesions comprises contacting the enriched library of aptamers with a biological sample from a subject, quantifying the amount of aptamer bound onto said sample, and correlating said amount to the presence of Ab brain lesions. The present invention also relates to the use of an enriched library of aptamers according to the present invention, in the in vitro diagnosis of an amyloid-beta (Ab^bbqΰώίeά disease.

In one embodiment, the use in the in vitro diagnosis of an Ab-associated disease is for the early diagnosis of an Ab-associated disease. In one embodiment, the Ab-associated disease is selected from Alzheimer’s disease, cerebral amyloid angiopathy, dementia with Lewy bodies, chronic traumatic encephalopathy, inclusion body myositis and age-related macular degeneration.

In one embodiment, the Ab-associated disease is Alzheimer’s disease.

In one embodiment, the use in the in vitro diagnosis of an Ab-associated disease comprises contacting the enriched library of aptamers with a biological sample from a subject, quantifying the amount of aptamer bound onto said sample, and correlating said amount to the diagnosis of an Ab-associated disease. In one embodiment, contacting the enriched library of aptamers with a biological sample from a subject is performed through a single positive selection round or through multiple selection rounds, wherein multiple selection rounds include positive and/or negative selection rounds. In one embodiment, quantifying the amount of aptamer bound onto said sample is achieved by qPCR, next-generation sequencing and/or detection of a fluorescent label.

The present invention also relates to a method for the detection of amyloid-beta (Ab) brain lesions and/or for early diagnosing of an amyloid-beta (AP)-associated disease in a subject, comprising the steps of:

- providing a sample for said subject;

contacting the sample with an enriched library of aptamers according to the present invention;

quantifying the amount of aptamers bound onto said sample, preferably through qPCR, next-generation sequencing and/or detection of a fluorescent label;

- analyzing the frequency variation between aptamers bound onto said sample with the enriched library, preferably by partial least squares discriminant analysis thereby detecting amyloid-beta (Ab) brain lesions and/or diagnosing an amyloid-beta (Ab^bboeώίeά disease in the subject.

The present invention also relates to a diagnostic kit comprising:

i) an enriched library of aptamers according to the present invention;

ii) buffers and solutions for use; and

iii) instructions for use.

In one embodiment, the diagnostic kit further comprises:

a support comprising immobilized random nucleic acid sequences, preferably random 8-mer nucleic acid sequences, preferably the support is a chip;

a computed-implemented diagnosis system comprising an algorithm for the normalization and/or analysis of data;

kits and reagents for qPCR, next-generation sequencing (NGS), and/or detection of a signal; preferably for qPCR; preferably including oligonucleotide primers for aptamer amplification; preferably said oligonucleotide primers comprise pairs of forward and reverse primers, wherein the forward primer comprises an oligonucleotide sequence set forth in SEQ ID NO: 45 and the reverse primers comprise an oligonucleotide sequence selected from SEQ ID NOs: 46 to 89; and/or - PCR clean-up reagents.

DETAILED DESCRIPTION

In the present invention, the following terms have the following meanings:

“aptamer”, as used herein, refers to oligonucleotides that mimic antibodies in their ability to act as ligands and bind to analytes. In one embodiment, aptamers comprise natural DNA nucleotides, natural RNA nucleotides, modified DNA nucleotides, modified RNA nucleotides, or a combination thereof.

“library” or“enriched library”, as used herein, refer to a collection of aptamers that have been exposed to a target, where such a target may be present in a biological sample, through a process known in the art as“aptamer selection”, and where such an enriched library exhibits the characteristic that at least 0.01% of the sequences observed in a sample of one million sequences are observed again in a subsequent selection round against the same target.

“amyloid-beta” or“Ab”, as used herein, refers to peptides of 36-43 amino acids which are the main component of amyloid plaques found in the brains of Alzheimer’s disease (among others) patients. These peptides derive from the amyloid precursor protein (APP), which is cleaved by b-secretase and g-secretase to yield Ab. Ab molecules can aggregate to form flexible soluble oligomers which may exist in several forms. Among amyloid-beta peptides, one can cite Ab(1-40) and Ab(1-42) with SEQ ID NOs: 90 and 91, respectively.

The present invention relates to an enriched library of aptamers comprising oligonucleotide sequences which bind to at least one target in an amyloid-beta (Ab) brain lesions sample. It is indeed presumed that targets are enriched in samples of certain individuals with high levels of Ab brain lesion accumulation. The etiology of the accumulation of Ab brain lesions appears however to be complex and as such, it is presumed that different individuals with the same level of Ab brain lesions may have different levels of corresponding targets.

The present invention relates to an enriched library of aptamers comprising oligonucleotide sequences which specifically bind to at least one target in an amyloid- beta (AP)-associated disease sample.

In one embodiment, the enriched library of aptamers comprises oligonucleotide sequences which do not bind to targets in an Ab brain lesions-free sample.

In one embodiment, the enriched library of aptamers comprises oligonucleotide sequences which do not bind to targets in an Ab-associated disease-free sample.

As used herein, the term“amyloid-beta (Ab) brain lesions sample” refers to a sample collected from a subject diagnosed with or tested positive for Ab brain lesions. As used herein, the term“amyloid-beta (Ab) brain lesions-free sample” refers to a sample collected from a subject not diagnosed with or tested negative for Ab brain lesions. In the frame of the present invention, such subj ect will be hereafter referred to as“substantially healthy subject”. It is well-known in the art that the accumulation of Ab induces amyloid plagues to cause tissue damages, in particular brain lesions. Such brain lesions are a well- known risk factor for the onset of amyloid-beta (AP)-associated diseases. The presence of Ab brain lesions can be determined by means well-known to the skilled artisan, such as using radioisotopic probe and positron emission tomography (PET).

As used herein, the term“amyloid-beta (Ab^bboe bΰ disease sample” refers to a sample collected from a subject affected with or diagnosed with an Ab-associated disease. As used herein, the term“amyloid-beta (Ab^bboe eΰ disease-free sample” refers to a sample collected from a subject who is not affected with or has not been diagnosed with an Ab-associated disease. In the frame of the present invention, such subject will be hereafter referred to as“substantially healthy subject”.

As used herein, the term“Ab-associated disease” include any disease, disorder or condition induced or caused by the accumulation of Ab in any tissue of a subject. Examples of such diseases include, but are not limited to, Alzheimer’s disease, cerebral amyloid angiopathy, dementia with Lewy bodies, chronic traumatic encephalopathy, inclusion body myositis and age-related macular degeneration.

As used herein, the term“sample” refers to a biological sample from a subject, such as, without limitation, a bodily tissue or bodily fluid. Non-limitative examples of such biological samples include blood (including serum, plasma and whole blood), cerebrospinal fluid, peritoneal fluid, pericardial fluid, pleura fluid, synovial fluid, saliva, tear fluid, sweat, milk, nipple aspirates, vaginal fluid, urine, semen, fecal matter, interstitial fluid, mucous, pus, biopsied tissue ( e.g ., obtained by a surgical biopsy or a needle biopsy), swabs (such as buccal swabs), tissue samples (such as tissue sections and needle biopsies of tissues including, but not limited to, brain, heart, lungs, muscles, intestines, stomach, kidneys, skin, gonads, nerve cells, cornea, retina and other structures of the eye, tendons, bone, bone marrow, nail-base cells, cartilage, non-fetal maternity related tissues such as placenta, umbilical cord, foreskin, surfaces of internal lumens, vascular tissue, pancreas, spleen, adrenal gland, thyroid gland and pituitary glands), cell samples (for example, cytological smears), or any material that is derived from a first biological sample. In one embodiment, the sample was previously collected from a subject and is provided for the implementation of the uses and methods according to the present invention, i.e., any uses and methods described herein do not comprise an active step of collecting a sample in a subject.

A“subject”, as used herein, refers to a warm-blooded animal, preferably a human, a pet or livestock. As used herein, the terms“pet” and“livestock” include, but are not limited to, dogs, cats, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses and poultry. In some embodiments, the subject is a male or female subject. In some embodiments, the subject is an adult (for example, a subject above the age of 18 (in human years) or a subject after reproductive capacity has been attained). In another embodiment, the subject is a child (for example, a subject below the age of 18 (in human years) or a subject before reproductive capacity has been atained). In one embodiment, the subject is above the age of 20, preferably above the age of 30, 40, 50, 60, 70, 80, 90 years old or more. In one embodiment, the subject is from 30 to 90 years old, preferably from 40 to 90 years old, more preferably from 50 to 90 years old, even more preferably from 60 to 90 years old, even more preferably from 70 to 90 years old. In some embodiments, the subject may be a“patient”, i.e., a subject who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical or diagnostic procedure according to the uses and methods of the present invention, or is monitored for the development of a disease.

In one embodiment, the subject was/is/will be the object of a diagnostic procedure according to the uses and methods of the present invention. In one embodiment, the subject is at risk of developing or being affected with Ab brain lesions. In one embodiment, the subject is at risk of developing or being affected with an Ab-associated disease.

In one embodiment, the enriched library of aptamers comprises oligonucleotide sequences selected by methods well-known in the art, including, but not limited to, SELEX or FRELEX. “SELEX” (Systematic Evolution of Ligands by Exponential enrichment) is described in US patent application 07/536,428 and US patents 5,475,096 and 5,270,163. The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid- target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.

“FRELEX” refers to an improved SELEX method described in International patent application WO2017035666 and Lecocq et al. (2018. PLoS One. 13(l):e0190212), which enables the partitioning of aptamers that bind to a target from aptamers that do not bind, without immobilization of the aptamers nor of the target. The selection process proceeds in two phases: in phase 1, aptamers are selected for their capacity to bind to random oligonucleotides immobilized on a surface. In phase 2, the selected aptamers are allowed to incubate with a target analyte. FRELEX also introduces one or several counter-target analytes as a means of counter-selection.

In one embodiment, the enriched library of aptamers comprises oligonucleotide sequences selected by re-iteration of selection rounds against at least one target comprised in an Ab brain lesions sample, and counter-selection rounds against at least one target in an Ab brain lesions-free sample.

In one embodiment, the enriched library of aptamers comprises oligonucleotide sequences selected by re-iteration of selection rounds against at least one target comprised in an Ab-associated disease sample, and counter-selection rounds against at least one target in an Ab-associated disease sample-free sample. Examples of selection processes are further described in the Example section below.

In one embodiment, the enriched library of aptamers comprises oligonucleotide sequences at a concentration of about 0.1 pmoles, 0.5 pmoles, 1 pmoles, 2.5 pmoles, 5 pmoles, 7.5 pmoles, 10 pmoles, 15 pmoles, 20 pmoles, 25 pmoles, 30 pmoles, 35 pmoles, 40 pmoles, 45 pmoles, 50 pmoles, 55 pM, 60 pmoles, 65 pmoles, 70 pmoles, 75 pmoles, 80 pmoles, 85 pmoles, 90 pmoles, 95 pmoles, 100 pmoles, 125 pmoles, 150 pmoles, 175 pmoles, 200 pmoles.

In one embodiment, the enriched library of aptamers comprises at least one aptamer selected from the group comprising or consisting of oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21. In one embodiment, the enriched library of aptamers comprises at least one aptamer selected from the group comprising or consisting of oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44.

In one embodiment, the enriched library of aptamers comprises at least 1 , preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, more preferably 21 oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21. In one embodiment, the enriched library of aptamers comprises at least 1 , preferably at least 2, more preferably at least 3, even more preferably at least 4 oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21, chosen among those with the highest weight in each relevant dimension.

In one embodiment, the enriched library of aptamers comprises at least 1 , preferably at least 2, more preferably at least 3, even more preferably at least 4 oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21, chosen among those with the highest weight in at least one dimension.

The term“highest weight”, as used herein, means at least > j0.3j, at least > j0.29j, at least > j0.28j, at least > j0.27j, at least > j0.26j, at least > j0.25j, at least > j0.24j, at least > j0.23j, at least > J0.22J, at least > j0.21 j, at least > j0.2j.

In one embodiment, the aptamer among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21 having the highest weight in dimension 1 is SEQ ID NO: 10.

In one embodiment, the aptamer among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21 having the highest weight in dimension 2 is SEQ ID NO: 1 In one embodiment, the aptamers among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21 having the highest weight in dimension 3 are SEQ ID NO: 5 and 15.

In one embodiment, the enriched library of aptamers comprises at least 1, preferably 2, more preferably at least 3, even more preferably at least 4 oligonucleotide sequences set forth in SEQ ID NOs: 10, 1, 5 and 15.

In one embodiment, the enriched library of aptamers comprises at least 2, preferably at least 3, 4, 5, 6, 7, 8, 9, more preferably at least 10 oligonucleotide sequences set forth in SEQ ID NOs: 1-10.

In one embodiment, the enriched library of aptamers comprises at least 2, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, more preferably at least 11 oligonucleotide sequences set forth in SEQ ID NOs: 1-10 and 15.

In an alternative embodiment, the enriched library of aptamers comprises at least 2, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, more preferably at least 13 oligonucleotide sequences set forth in SEQ ID NOs: 1-2 and 11-21. In one embodiment, the enriched library of aptamers comprises at least 2, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, more preferably at least 15 oligonucleotide sequences set forth in SEQ ID NOs: 1-2, 5 and 10-21.

In one embodiment, the enriched library of aptamers comprises 21 oligonucleotide sequences set forth in SEQ 1D NOs: 1 to 21. In one embodiment, the enriched library of aptamers comprises at least 1 , preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, more preferably 44 oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44.

In one embodiment, the enriched library of aptamers comprises at least 1 , preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44, chosen among those with the highest weight in each relevant dimension. In one embodiment, the enriched library of aptamers comprises at least 1 , preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44, chosen among those with the highest weight in at least one dimension.

In one embodiment, the aptamer among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44 having the highest weight in dimension 1 is SEQ ID NO: 38. In one embodiment, the aptamers among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44 having the highest weight in dimension 1 are SEQ ID NOs: 38, 30 and 5.

In one embodiment, the aptamer among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44 having the highest weight in dimension 2 is SEQ ID NO: 25. In one embodiment, the aptamers among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44 having the highest weight in dimension 2 are SEQ ID NOs: 25, 33, 36 and 42.

In one embodiment, the aptamers among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44 having the highest weight in dimension 3 are SEQ ID NOs: 25, 16 and 23. In one embodiment, the aptamers among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44 having the highest weight in dimension 3 are SEQ ID NOs: 23, 4 and 35. In one embodiment, the aptamers among oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44 having the highest weight in dimension 3 are SEQ ID NOs: 25, 16, 23, 9, 4 and 35.

In one embodiment, the enriched library of aptamers comprises at least 1, preferably 2 oligonucleotide sequences set forth in SEQ ID NOs: 38 and 5.

In one embodiment, the enriched library of aptamers comprises at least 1, preferably 2, more preferably at least 3, even more preferably at least 4 oligonucleotide sequences set forth in SEQ ID NOs: 38, 25, 16 and 23.

In one embodiment, the enriched library of aptamers comprises at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, more preferably at least 10 oligonucleotide sequences set forth in SEQ ID

NOs: 38, 30, 5, 25, 33, 36, 42, 23, 4 and 35. In one embodiment, the enriched library of aptamers comprises at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, more preferably at least 12 oligonucleotide sequences set forth in SEQ ID NOs: 38, 30, 5, 25, 33, 36, 42, 16, 23, 9, 4 and 35.

In one embodiment, the enriched library of aptamers comprises at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, more preferably at least 22 oligonucleotide sequences set forth in SEQ ID NOs: 5, 23, 20, 41, 39, 38, 30, 8, 13, 4, 25, 33, 36, 42, 24, 34, 29, 27, 35, 19, 16 and 9.

In one embodiment, the enriched library of aptamers comprises 44 oligonucleotide sequences set forth in SEQ ID NOs: 1 to 44. In one embodiment, the enriched library of aptamers is labeled, i.e., the aptamers in the enriched library comprise a label or a detection moiety.

As used herein, the terms“label” or“detection moiety” refer to one or more reagents that can be used to detect interactions involving a target and an aptamer. A detection moiety or label is capable ofbeing detected directly or indirectly. In general, any reporter molecule that is detectable can be a label.

Labels include, but are not limited to, reporter molecules that can be detected directly by virtue of generating a signal, specific binding pair members that can be detected indirectly by subsequent binding to a cognate that contains a reporter molecule, mass tags detectable by mass spectrometry, and oligonucleotide sequences that can provide a template for amplification or ligation.

The reporter molecule can be a catalyst, such as an enzyme, dye, fluorescent molecule, quantum dot, chemiluminescent molecule, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, a mass tag that alters the weight of the molecule to which it is conjugated for mass spectrometry purposes, and the like. The label can be selected from electromagnetic or electrochemical materials. In one embodiment, the detectable label is a fluorescent dye such as Cy-3 or Cy-5. Other labels and labeling schemes will be evident to one skilled in the art based on the disclosure herein.

The detection moiety can be detected by emission of a fluorescent signal, a chemiluminescent signal, or any other detectable signal that is dependent upon the identity of the moiety. In the case where the detectable moiety is an enzyme (for example, alkaline phosphatase), th e signal can be generated in the presence of the enzyme substrate and any additional factors necessary for enzyme activity. In the case where the detectable moiety is an enzyme substrate, the signal can be generated in the presence of the enzyme and any additional factors necessary for enzyme activity. Suitable reagent configurations for attaching the detectable moiety to a target molecule include covalent attachment of the detectable moiety to the target molecule, non-covalent association of the detectable moiety with another labeling agent component that is covalently attached to the target molecule, and covalent attachment of the detectable moiety to a labeling agent component that is non-covalently associated with the target molecule. In one embodiment, the detection moiety is a molecular switch based on a FRET pair, e.g., an Alloswitch such as described in US patent applications 20060216692 and 20060029933.

In one embodiment, the aptamer sequences comprise oligonucleotide sequences for amplification. In one embodiment, the aptamer sequences are flanked on both ends by oligonucleotide sequences for amplification. In one embodiment, oligonucleotide sequences for amplification may be identical throughout all aptamers; or may be different for each aptamer sequence. In one embodiment, oligonucleotide sequences may be identical throughout all aptamers on one end of the aptamer sequences; and be different for each aptamer sequence on the other end of the aptamer sequences.

The present invention further relates to the use of an enriched library of aptamers according to the present invention, in the detection of Ab brain lesions in a subject.

The present invention further relates to the use of an enriched library of aptamers according to the present invention, in the diagnosis of a Ab-associated disease in a subject. In one embodiment, the Ab-associated disease is selected from the group comprising or consisting of Alzheimer’s disease, cerebral amyloid angiopathy, dementia with Lewy bodies, chronic traumatic encephalopathy, inclusion body myositis and age-related macular degeneration. In a preferred embodiment, the Ab-associated disease is Alzheimer’s disease.

In one embodiment, the uses according to the present invention are in vitro uses.

In one embodiment, the uses according to the present invention are for the early detection and/or early diagnosis of Ab brain lesions and/or of a Ab-associated disease in a subject.

As used herein, the terms“early detection” and“early diagnosis” refer to the detection or the diagnosis of a state before the appearance of symptoms. In the frame of the present invention, symptoms of Ab brain lesions and/or of Ab-associated diseases include, but are not limited to, loss of cognitive functions, memory loss, forgetfulness, time and/or space confusion, apathy, anxiety, agitation, mood changes, depression. As used herein, the term“cognitive function” is used to define any mental process that involves symbolic operations such as perception, memory, creation of imagery, thinking, awareness and capacity for judgment.

It is an aim of the present invention to detect or diagnose Ab brain lesions and/or Ab- associated diseases in a subject before the appearance of symptoms, to offer treatment before the onset of the disease or, at least, at the earliest stage of the development of the disease. It is not the aim of the present invention to offer treatments of Ab brain lesions and/or Ab-associated diseases, which are well-known from the practitioners and physicians who are in position and able to choose the best treatment depending on the disease to be treated and other factors such as the age or sex of the subject, the stage of the disease, possible drug interactions, and various other factors alike. In one embodiment, the uses according to the present invention comprise a step of contacting the enriched library of aptamers described above with a sample from a subject.

In one embodiment, the sample was previously collected from the subject. In one embodiment, the sample is blood. In one embodiment, the sample is blood serum, blood plasma or whole blood.

In one embodiment, the enriched library of aptamers is allowed to incubate with a sample from a subject. In one embodiment, the enriched library of aptamers is contacted with a sample from a subject once, in a single positive selection round. In other words, after contacting, aptamers which do not bind to any target in the sample from a subject are discarded, while aptamers which bind are recovered for further analysis.

In one embodiment, the enriched library of aptamers is contacted with a sample from a subject at least twice, 3 times, 4 times, 5 times or more, in positive selection rounds. In other words, after contacting, aptamers which do not bind to any target in the sample from a subject are discarded, while aptamers which bind are recovered for re-iteration of the contacting step.

In one embodiment, the enriched library of aptamers is contacted with a sample from a subject multiple time, in positive and negative selection rounds. In other words, the contacting step comprises positive selection rounds wherein aptamers which do not bind to any target in the sample from a subject are discarded, while aptamers which bind are recovered for re-iteration of the contacting step; and negative selection rounds wherein aptamers which bind to any target in a reference sample from a substantially healthy subject are discarded, while aptamers which do not bind are recovered for re -iteration of the contacting step.

For illustrative but non-limiting purposes, the“contacting step” could be implemented as follows: an enriched library comprising 5 pmoles of each aptamer selected from oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21 or SEQ ID NOs: 1 to 44 in a selection buffer (20 mM Tris, 5 mM MgCh, 5 mM KC1, 120 mM Nad) is applied, in a positive selection round, on a blood sample from a subject in which the detection of Ab brain lesions and/or the diagnosis of a Ab-associated disease is to be assessed. The enriched library is incubated on the blood sample for 15 minutes at room temperature. This mixture is then applied for another 15 minutes on a gold chip previously coated with random 8-mer nucleic acid sequences. Aptamers from the enriched library which have not bound to any target in the blood sample bind to the random 8-mer nucleic acid sequences, thereby depleting the supernatant from unspecific aptamers. The supernatant is taken off the chip, and kept for further analysis. The chip is then washed twice with the selection buffer. These washes are pooled with the supernatant, then cleaned through PCR cleanup-up and eluted in 100 pL (Gene Jet PCR purification kit, thermo).

In one embodiment, the uses according to the present invention comprise a step of quantifying the amount of aptamer bound onto the sample from the subject.

In one embodiment, the amount of aptamer bound onto the sample is quantified using techniques well-known in the art, including, but not limited to, qPCR, next-generation sequencing (NGS), and/or detection of a signal (such as fluorescence, chemiluminescence, or any other detectable signal that is dependent upon the identity of the label or detection moiety used, as defined hereinabove).

For illustrative but non-limiting purposes, the“quantifying step” could be implemented as follows: after each single selection, aptamers are analyzed through real-time PCR (RT- PCR) using primers complementary to oligonucleotide sequences for amplification flanking the aptamers. The primers are designed to limit complementarity to each other, in order to decrease non-specific amplification of self-dimerizing primers. Quantitative PCR is performed on an Mx3000P thermocycler (Stratagene, AH Diagnostics, Aarhus, Denmark) with 10X Sybr green master mix according to the manufacturer's instructions (Bio-Rad). 20 mT reactions include 5 mT of template from selection and 250 nM of oligonucleotide primers. PCR is performed as follows: 95°C for 2 minutes; 30 cycles [95°C for 10 seconds; 62°C for 15 seconds; 72°C for 30 seconds].

In one embodiment, each aptamer is flanked on both end with oligonucleotide sequences, wherein said oligonucleotide sequences are identical throughout all aptamers on one end and different for each aptamer sequence on the other end of the aptamer sequences.

According to this embodiment, the step of“step of quantifying the amount of aptamer bound onto the sample from the subject” can be carried out using oligonucleotide primers complementary to the oligonucleotide sequences flanking the aptamers. For illustrative but non-limiting purposes, the oligonucleotide primers can comprise pairs of forward and reverse primers, wherein the forward primers comprise an oligonucleotide sequence set forth in SEQ ID NO: 45 and the reverse primers comprise an oligonucleotide sequence set forth in SEQ ID NOs: 46-66 or SEQ ID NOs: 46-89.

In one embodiment, the uses according to the present invention comprise a step of diagnosis of Ab brain lesion status and/or of an Ab-associated disease in the subject.

In one embodiment, the diagnosis is based on the variation of the aptamer sequences previously quantified with the initial content of the enriched library. In one embodiment, the diagnosis is based on the frequency variation between aptamer sequences previously quantified with the initial enriched library.

For illustrative but non-limiting purposes, the“diagnosis step” could be implemented as follows: for data normalization, Ct values at a threshold at 1000 and a threshold at 12000 are used to study the ranking of the aptamers in terms of frequency relative to each other.

In other terms, this describes how much aptamer“A” there is compared to aptamer“B”. These values are normalized for potential experimental effect by determining the average for all aptamers for each sample from multiple subjects. These average values are averaged, and each sample average is divided by this grand average. The normalized quantitation values are then divided by this correction factor. For analysis, the normalized data is further normalized to a mean of zero and a standard deviation of unity. This is achieved by dividing the normalized aptamer values by average aptamer value across all samples for that aptamer, and dividing this value by the standard deviation of this average. This data is called the final aptamer data. Ab brain lesion status is categorized into two classes (+/-) based on a threshold value. Partial least squares discriminant analysis (PLS- DA) is then used to ascribe variation in the final aptamer data to the explanation of variation in the Ab brain lesion status across indi viduals and/or the diagnosis of an Ab- associated disease. A vector that provides different loading values to each aptamer value is derived through least squares goodness of fit. This vector is applied to explain variation in Ab brain lesion status is one dimension. The remaining variation is applied to the derivation of a second vector of loading values. This vector explain variation in Ab brain lesion status in a second dimension that is orthogonal to the first dimension. This process continues for as many dimensions as are meaningful. The application of this process can be validated either with a training and testing set of samples, or through cross-validation. In cross-validation analysis of the data is simulated reiteratively with a gi ven number of samples removed in each iteration. Cross-validation is favored when sample size is small.

In one embodiment, normalization and analysis is computer-implemented with a diagnostic system.

The present invention further relates to a method for the detection of Ab brain lesions in a subject.

The present invention further relates to a method for the diagnosis of an Ab-associated disease in a subject. The present invention further relates to a method for recruiting a subject affected with an Ab-associated disease into a clinical trial.

In one embodiment, the Ab-associated disease is selected from the group comprising or consisting of Alzheimer’s disease, cerebral amyloid angiopathy, dementia with Lewy bodies, chronic traumatic encephalopathy, inclusion body myositis and age-related macular degeneration.

In a preferred embodiment, the Ab-associated disease is Alzheimer’s disease.

In one embodiment, the methods according to the present invention are in vitro methods.

In one embodiment, the methods according to the present invention are for the early detection and/or early diagnosis of Ab brain lesions and/or of a Ab-associated disease in a subject. In one embodiment, the methods according to the present invention comprise a step of providing a sample from a subject. In one embodiment, the sample was collected from the subject beforehand and this step does not comprise the collection of the sample itself.

In one embodiment, the methods according to the present invention comprise a step of contacting the enriched library of aptamers described above with the sample from the subject.

In one embodiment, the methods according to the present invention comprise a step of quantifying the amount of aptamer bound onto the sample from the subject.

In one embodiment, the methods according to the present invention comprise a step of diagnosis of Ab brain lesions and/or of an Ab-associated disease in the subject. In one embodiment, the diagnosis is based on the variation of the aptamer sequences previously quantified with the initial content of the enriched library. In one embodiment, the diagnosis is based on the frequency variation between aptamer sequences previously quantified with the initial enriched library. In one embodiment, the subject is recruited into the clinical trial if said subject is diagnosed with Ab brain lesions and/or an Ab-associated disease.

The present invention further relates to a method of treating a subject affected with or likely to be affected with Ab brain lesions and/or with an Ab-associated disease.

In one embodiment, the method of treating comprises a step of diagnosing the subject according to the methods described hereinabove.

In one embodiment, the method of treating comprises a further step of treating the subject if diagnosed with Ab brain lesions and/or with an Ab-associated disease.

Suitable treatments for Ab brain lesions and/or Ab-associated diseases are well-known to the one skilled in the art and include, but are not limited to, acetylcholinesterase inhibitors (such as, e.g., tacrine, rivastigmine, galantamine, and donepezil), N-methyl-D-aspartate (NMD A) receptor antagonists (such as, e.g. , memantine), secretase 1 (BACE1) inhibitors (such as, e.g., AZD3293, CTS-21166, E-2609, JNJ-54861911, LY3314814, LY2886721, verubecestat, solanezumab, and lanabecestat), anti-amyloid beta antibodies (such as, e.g., crenezumab, solanezumab, bapineuzumab, aducanumab, gantenerumab, and BAN- 2401), anti-Tau antibodies (such as, e.g., BIIB092, BMS-986168, ABBV-8E12, and C2N-8E12), Tau inhibitors (such as, e.g., methylthioninium, LMTX, Rember™, PBT2, and PTI-51-CH3), beta-amyloid aggregation inhibitors (such as, e.g., ELND-005, tramiprosate, and PTI-80), and general misfolding inhibitor (such as, e.g., NPT088).

The present invention further relates to a diagnostic kit comprising:

the enriched library of aptamers according to the present invention;

buffers and solutions for use, and

- instructions for use.

In one embodiment, buffers and solutions of use may include a selection buffer (such as, e.g., the selection buffer described hereinabove but not limited thereto); kits and reagents for qPCR, next-generation sequencing (NGS), and/or detection of a signal; PCR clean-up reagents; and the like. In one embodiment, the diagnostic kit further comprises a solid support comprising immobilized random nucleic acid sequences.

In one embodiment, immobilized nucleic acid sequences are 8-mer nucleic acid sequences.

As used herein, the term“solid support” refers to any substrate having a surface to which molecules, preferably aptamers, can be attached, directly or indirectly, through either covalent or non-covalent bonds. The substrate materials can be naturally occurring, synthetic, or a modification of a naturally occurring material. Solid support materials include silicon, graphite, mirrored surfaces, laminates, ceramics, plastics (including polymers such as, e.g., poly( vinyl chloride), cyclo-olefin copolymers, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, polyethylene terephthalate, polytetrafluoroethylene [PTFE or Teflon^®], nylon, poly(vinyl butyrate)), germanium, gallium arsenide, gold, silver, etc., either used by themselves or in conjunction with other materials. Additional rigid materials can be considered, such as glass, which includes silica. Other materials that can be employed include porous materials, such as, for example, controlled pore glass beads. Any other materials known in the art that are capable of having one or more functional groups, such as any of an amino, carboxyl, thiol, or hydroxyl functional group, for example, incorporated on its surface, are also contemplated. The solid support can take any of a variety of configurations ranging from simple to complex and can have any one of a number of shapes, including a strip, plate, disk, rod, particle, including bead, tube, well, and the like. The surface can be relatively planar (e.g., a slide), spherical ( e.g ., a bead), cylindrical (e.g., a column), or grooved. Exemplary solid supports include, but are not limited to, microtiter wells, microscope slides, membranes, paramagnetic beads, charged paper, filters, gels, Langmuir-Blodgett films, silicon wafer chips, flow-through chips, microarray chips, microbeads and magnetic beads.

In one embodiment, the solid support is a chip.

In one embodiment, the diagnostic kit further comprises a computed-implemented diagnosis system comprising an algorithm for the normalization and/or analysis of data.

In one embodiment, the computed-implemented diagnosis system provides a diagnosis of Ab brain lesion status and/or of Ab-associated disease in the subject.

The present invention relates to an enriched library of aptamers comprising at least one, preferably at least 2, 3 or 4 aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 10, 1, 5 and 15.

In one embodiment, the enriched library of aptamers comprises the twenty-one aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21.

The present invention further relates to the use of the enriched library of aptamers according to the present invention, in the in vitro detection of amyloid-beta (Ab) brain lesions.

In one embodiment, the use according to the present invention comprises contacting the enriched library of aptamers with a biological sample from a subject, quantifying the amount of aptamer bound onto said sample, and correlating said amount to the presence of Ab brain lesions.

The present invention further relates to the use of the enriched library of aptamers according to the present invention, in the in vitro diagnosis of an amyloid-beta (Ab)- associated disease.

In one embodiment, the use according to the present invention is for the early diagnosis of an amyloid-beta (Ab^bboeώΐeά disease.

In one embodiment, the Ab-associated disease is selected from Alzheimer’s disease, cerebral amyloid angiopathy, dementia with Lewy bodies, chronic traumatic encephalopathy, inclusion body myositis and age-related macular degeneration.

In one embodiment, the Ab-associated disease is Alzheimer’s disease.

In one embodiment, the use according to the present invention comprises contacting the enriched library of aptamers with a biological sample from a subject, quantifying the amount of aptamer bound onto said sample, and correlating said amount to the diagnosis of an Ab-associated disease.

In one embodiment, contacting the enriched library of aptamers with a biological sample from a subject is performed through a single positive selection round or through multiple selection rounds, wherein multiple selection rounds include positive and/or negative selection rounds. In one embodiment, quantifying the amount of aptamer bound onto said sample is achieved by qPCR, next-generation sequencing and/or detection of a fluorescent label.

The present invention further relates to a method for the detection of amyloid-beta (Ab) brain lesions and/or for early diagnosing of an amyloid-beta (Ab)-associated disease in a subject, comprising the steps of:

- providing a sample for said subject;

contacting the sample with an enriched library of aptamers comprising at least 1, 2, 3 or 4 aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 10, 1, 5 and 15; preferably the twenty-one aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21;

- analyzing the frequency variation between aptamers bound onto said sample with the enriched library, preferably by partial least squares discriminant analysis

thereby detecting amyloid-beta (Ab) brain lesions and/or diagnosing an amyloid-beta (AP)-associated disease in the subject.

The present invention further relates to a diagnostic kit comprising:

at least 1, 2, 3 or 4 aptamers with oligonucleotide sequences set forth in SEQ ID NOs:

10, 1, 5 and 15; preferably the twenty-one aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21;

buffers and solutions for use; and

instructions for use. In one embodiment, the diagnostic kit according to the present invention further comprises:

kits and reagents for qPCR, next-generation sequencing (NGS), and/or detection of a signal; preferably for qPCR; preferably including oligonucleotide primers for aptamer amplification; preferably said oligonucleotide primers comprise pairs of forward and reverse primers, wherein the forward primers comprise an oligonucleotide sequence set forth in SEQ ID NO: 24 and the reverse primers comprise an oligonucleotide sequence set forth in SEQ ID NOs: 25-45; and/or

PCR clean-up reagents. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a graphical view of the classification of Ab brain lesion status based on 10 aptamer frequencies in two dimensions as applied to blood samples from 22 individuals. Figure 2 shows the ROC-AUC for 10 aptamer frequencies in a two-dimensional weighted model.

Figure 3 provi des a graphical view of the classification of Ab brain lesion status based on 10 aptamer frequencies in two dimensions as applied to blood samples from 42 individuals. Figure 4 shows the ROC-AUC following cross-validation analysis for two dimensions and 10 aptamer frequencies across 42 samples with 10 folds and 100 replications.

Figure 5 illustrates the class separation in two dimensions with the combination of the two aptamer subsets 1 and 2 (21 aptamer sequences in total, 23 aptamer frequencies) as applied to blood samples from 42 individuals.

Figure 5 A: dimension 1 versus dimension 2.

Figure 5B: dimension 2 versus dimension 3.

Figure 6 shows the error level with different threshold methods and dimensions with the set of 23 aptamers.

Figure 7 shows the ROC-AUC following cross-validation analysis for 21 aptamer frequencies across 42 samples with 10 folds and 100 replications.

Figure 8 shows the weighting of each aptamer following cross-validation analysis for 21 aptamer frequencies across 42 samples with 10 folds and 100 replications.

Figure 9 illustrates the class separation in two dimensions with the combination of the three aptamer subsets 1 , 2 and 3 (44 aptamer sequences in total, 46 aptamer frequencies) as applied to blood samples from 70 individuals. Figure 10 shows the ROC-AUC following cross-validation analysis for 44 aptamer frequencies across 70 samples with 5 fold and 100 replications.

EXAMPLES The present invention is further illustrated by the following examples.

Example 1

We had access to blood samples from the IN SIGHT -preAD cohort. This cohort was established as a pre -Alzheimer’s disease (AD) cohort with the purpose of aiding in the earlier detection of the disease. These samples were subject to the ethical guidelines regarding collection and use.

Subset 1

In a first instance, we applied a statistical package compiled in the computer language‘R’ named sparse partial least squares - discriminant analysis (sPLS-DA) described by Le Cao et al. (2011. BMC Bioinformatics . 12:253). A selected library was applied to blood serum from 22 individuals enrolled in the INSIGHT -Pre AD cohort, for a single round of positive selection. Eleven of these subjects exhibited positive brain amyloid levels (Ab brain lesions above 0.79 SUVR) and eleven exhibited negative levels of brain amyloid (Ab brain lesions below 0.79 SUVR). These values were determined with a radioisotopic probe and positron emission tomography (PET). Negative and positive designations are based on comparison to a threshold value from an average of total amyloid across all portions of the brain. All of the subjects were cognitively normal (CN).

Through a reiterative process of next-generation sequencing, sPLS-DA analysis, model building and cross-validation, we identified a set of 10 aptamers that provided clear differentiation of the cl asses over three dimensions. Figure 1 illustrates the distribution of the amyloid classes based on these ten aptamers frequencies in two dimensions. When this model with 10 aptamers is applied in three dimensions we obtain full separation of classes (Figure 2).

The ten aptamers identified in this analysis are listed as SEQ ID NOs: 1 to 10.

This subset of ten aptamers was further applied in a single round of FRELEX selection to 42 samples from the INSIGHT -pre AD cohort (17 negative Ab brain lesion status, 25 positive Ab brain lesion status).

Figure 3 provides a graphical view of the separation of the Ab brain status classes in two dimensions. There is more overlap of the samples in this analysis than in the results illustrated in Figure 1. Figure 4 provides the ROC-AUC following cross-validation analysis with 10-folds and 100 replications over two dimensions. The application of this subset of aptamers as a diagnostic does provide a reasonable level of sensitivity but the specificity is not sufficient to be of value.

Sensitivity: 0.84

Specificity: 0.41

Accuracy: 0.67

Subset 2

To improve the performance of this test, we created an additional subset of 13 aptamers. This second subset contained two aptamers that were included in the original subset. The frequency values for each aptamer were affected by the presence of other aptamers in the subset, thus these two repeated aptamers were treated as novel data.

This subset of 13 aptamers corresponds to SEQ ID NOs: 1 , 2 and 1 1 to 21.

As detailed above for the first subset, this second subset of 13 aptamers was further applied in a single round of FRELEX selection and cross-validation analysis with 10- folds and 100 replications over two dimensions was carried out. The application of this second subset of aptamers as a diagnostic provides a reasonable level of sensitivity and a fair level of specificity.

Sensitivity: 0.84

Specificity: 0.71

Accuracy: 0.79 Subsets 1+2

Finally, we combined the data from both aptamer subsets into a comprehensive test involving 21 aptamers (subset 1 with 10 aptamers and subset 2 with 13 aptamers, with two aptamers being reproduced in both subsets) across the 42 samples from the INSIGHT-preAD cohort (17 negative Ab brain lesion status, 25 positive Ab brain lesion status). This data was analyzed as one combined data set in a manner identical to that of the two subsets.

Figure 5 illustrates the class separation of Ab brain lesion status based on 23 aptamer frequencies in two dimensions as applied to blood samples from 42 individuals (Figure 5 A: dimension 1 versus dimension 2; Figure 5B: dimension 2 versus dimension 3). The combination of the analysis of these two subsets of aptamers results in a clear separation of Ab brain lesion classes in two dimensions. We performed cross-validation analysis on this model with 100 replications and ten folds. Figure 6 provides a summary of error analysis showing that error was lowest for this combined dataset when four dimensions are considered. We chose however to use only the first three dimensions with our model. Following cross- validation with ten-folds and 100 replications, we obtain the ROC AUC shown in Figure 7, with the following specifications:

Sensitivity: 0.80

Specificity: 0.88

Accuracy: 0.83

Overall, the combination of the two aptamer data sets provided an improvement in predictive capacity as compared to the initial ten aptamer subset (SEQ ID NOs: 1 to 10).

A means of determining the most important aptamers in regard to diagnostic capacity, or prediction potential, is to determine the weight of each aptamer in the PLS-DA model. The weight of each aptamer is a direct function of the meaningfulness of the aptamer in the overall mathematical model. The weights of each of the aptamers in the combined model is provided in Figure 8. The key aptamers in terms of diagnostic capacity or prediction potential at those with the highest weight in each relevant dimension: SEQ ID NO: 10 in dimension 1; SEQ ID NO: 1 in dimension 2; and SEQ ID NO: 5 and 15 in dimension 3.

Subset 3

To provide a further improvement to the predictive power of this test, we added a third set of 23 aptamers (SEQ ID NOs: 22 to 44) to subsets 1 and 2 (i.e ., a total of 44 aptamer sequences and 46 aptamer frequencies, two aptamers being reproduced in subsets 1 and 2) and we extended the number of individual samples analyzed from 42 to 70. This additional aptamer set was analyzed as described for the previous two aptamer subsets.

To improve the learning process with two well-characterized sample groups between “neg” and“pos”, we established our model for the dataset by using a training set that excluded all samples that had SUVr values between 0.75 and 0.85 (close to the threshold value). Finally, the predictive capacity of this model was evaluated on all 70 samples (including 15 samples excluded for training) by a Receiver Operator Characteristics (ROC) curve analysis using a 5 -fold cross-validation with 100 repetitions and 3 components for PLS-DA. Figure 9 show the predictive classification of samples in the first two components analysed for a SUVr of 0.79. The left and right sectors on both sides of the thick black line in Figure 9 correspond to the predicted positive and negative areas within these two components, respectively. Figure 10 provides the ROC - AUC analysis for 46 aptamer frequencies used to predict brain Ab brain deposition with five k-folds and 100 replications. A data set comprising 55 samples (excluding those near the threshold of 0.79) was used for training and the full dataset (70 samples) was used for testing.

For this analysis, the AUC was 0.9653, with the following specifications:

Sensitivity: 0.90

Specificity: 0.84

Accuracy: 0.87

Table 1 shows the weighting of each aptamer frequency following cross-validation analysis (with aptamers set forth as SEQ ID NO: 4 and 10 being reproduced twice).

Table 1

Table 2 shows the sequences with the heaviest weighting across the three dimensions.

Table 2 In conclusion, this example provides a demonstration that selected aptamer frequency can be used as a means of predicting risk factors for Alzheimer’s disease based through blood analysis, and provides 3 combinable subsets of aptamers demonstrating high sensitivity, specificity and accuracy for the diagnosis of Ab brain lesion status. Example 2

Based on the above-identified aptamer sequences, we carried out partial least squares - discriminant analyses to identify minimal subsets of aptamers suitable for the diagnosis of Ab brain lesion status.

These minimal subsets of aptamers were selected based on their weighting across the three dimensions, the heaviest ones being retained for these analyses.

Minimal subset 1

Minimal subset 1 consists of two aptamers sequences with nucleic acid sequence set forth in SEQ ID NOs: 38 and 5.

For this analysis, the specifications were as follows:

Sensitivity: 0.85

Specificity: 0.48

Accuracy: 0.69

This minimal subset remains of value even with a 0.48 specificity, in particular for the screening of subjects for enrollment in clinical trials. Minimal subset 2

Minimal subset 2 consists of ten aptamers sequences with nucleic acid sequence set forth in SEQ ID NOs: 38, 30, 5, 25, 33, 36, 42, 23, 4 and 35.

For this analysis, the specifications were as follows:

Sensitivity: 0.77

Specificity: 0.58

Accuracy: 0.69 Minimal subset 3

Minimal subset 3 consists of twenty-two aptamers sequences with nucleic acid sequence set forth in SEQ ID NOs: 5, 23, 20, 41, 39, 38, 30, 8, 13, 4, 25, 33, 36, 42, 24, 34, 29, 27, 35, 19, 16 and 9. For this analysis, the specifications were as follows:

Sensitivity: 0.79

Specificity: 0.71

Accuracy: 0.76

Claims

1. An enriched library of aptamers comprising at least two aptamers with oligonucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 44. 2. The enriched library of aptamers according to claim 1, comprising:

a) one aptamer with oligonucleotide sequence set forth in SEQ ID NO: 38; b) two aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 38 and 5;

c) ten aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 38, 30, 5, 25, 33, 36, 42, 23, 4 and 35;

d) ten aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 to

10;

e) thirteen aptamers with oligonucleotide sequences set forth in SEQ ID NOs:

1,

2 and 11 to 21; or

f) twenty-two aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 5, 23, 20, 41, 39, 38, 30, 8, 13, 4, 25, 33, 36, 42, 24, 34, 29, 27, 35, 19, 16 and 9.

3. The enriched library of aptamers according to claim 1 or 2, comprising:

a) the twenty-one aptamers with oligonucleotide sequences set forth in SEQ ID NOs: 1 to 21; or

4. Use of an enriched library of aptamers according to any one of claims 1 to 3, in the in vitro detection of amyloid-beta (Ab) brain lesions.

5. The use according to claim 4, comprising contacting the enriched library of aptamers with a biological sample from a subject, quantifying the amount of aptamer bound onto said sample, and correlating said amount to the presence of Ab brain lesions.

6. Use of an enriched library of aptamers according to any one of claims 1 to 3, in the in vitro diagnosis of an amyloid-beta (AP)-associated disease.

7. The use according to claim 6, for the early diagnosis of an amyloid-beta (Ab)- associated disease.

8. The use according to claim 6 or 7, wherein the Ab-associated disease is selected from Alzheimer’s disease, cerebral amyloid angiopathy, dementia with Lewy bodies, chronic traumatic encephalopathy, inclusion body myositis and age-related macular degeneration.

9. The use according to any one of claims 6 to 8, wherein the Ab-associated disease is Alzheimer’s disease.

10. The use according to any one of claims 6 to 9, comprising contacting the enriched library of aptamers with a biological sample from a subject, quantifying the amount of aptamer bound onto said sample, and correlating said amount to the diagnosis of an Ab-associated disease.

11. The use according to claim 5 or 10, wherein contacting the enriched library of aptamers with a biological sample from a subject is performed through a single positive selection round or through multiple selection rounds, wherein multiple selection rounds include positive and/or negative selection rounds.

12. The use according to claim 5 or 10, wherein quantifying the amount of aptamer bound onto said sample is achieved by qPCR, next-generation sequencing and/or detection of a fluorescent label.

13. A method for the detection of amyloid-beta (Ab) brain lesions and/or for early diagnosing of an amyloid-beta (Ab^bbqϋώΐeά disease in a subject, comprising the steps of:

- providing a sample for said subject;

contacting the sample with an enriched library of aptamers according to any one of claims 1 to 3; quantifying the amount of aptamers bound onto said sample, preferably through qPCR, next-generation sequencing and/or detection of a fluorescent label; analyzing the frequency variation between aptamers bound onto said sample with the enriched library, preferably by partial least squares discriminant analysis

thereby detecting amyloid-beta (Ab) brain lesions and/or diagnosing an amyloid- beta (AP)-associated disease in the subject.

14. A diagnostic kit comprising:

i) an enriched library of aptamers according to any one of claims 1 to 3;

ii) buffers and solutions for use; and

iii) instructions for use.

15. The diagnostic kit according to claim 14, further comprising:

kits and reagents for qPCR, next-generation sequencing (NGS), and/or detection of a signal; preferably for qPCR; preferably including oligonucleotide primers for aptamer amplification; preferably said oligonucleotide primers comprise pairs of forward and reverse primers, wherein the forward primer comprises an oligonucleotide sequence set forth in SEQ ID NO: 45 and the reverse primers comprise an oligonucleotide sequence selected from SEQ ID NOs: 46 to 89; and/or

PCR clean-up reagents.