KR20230096835A - Composition for diagnosing non -invasive dementia and diagnostic kits using the same - Google Patents

Abstract

The present invention relates to a composition for non-invasive diagnosis of dementia and a dementia diagnosis kit using the same.
The present invention can diagnose brain disease states without sampling brain tissue or imaging by using a combination of brain-derived ectosome-specific markers and expression patterns of multiple marker groups in blood. Specifically, it is possible to diagnose dementia non-invasively by isolating brain tissue-specific and brain region-specific expression ectosomes from blood, using specific discovery biomarkers, and comparing their profiles.

Description

Composition for diagnosing non-invasive dementia and diagnostic kits using the same}

The present invention relates to a composition for non-invasive diagnosis of dementia and a dementia diagnosis kit using the same.

In recent years, due to the rapid aging of society, the population with dementia, represented by Alzheimer's disease, is also increasing. However, in most cases, since dementia shares many of its clinical features with other diseases in the early stages of the disease, the appropriate treatment time is often missed. In particular, the brain is an organ for which biopsy is extremely limited, and indirect means to replace biopsy are being mobilized to improve the possibility of diagnosing each type of dementia. However, until now, tests for dementia have relied on cognitive function tests, neuropsychological tests, and imaging methods such as MRI and CT. In particular, although the specificity of diagnosis is increasing due to the recent development of imaging technology, the cost of the examination is high, so it becomes an economic burden for patients to perform the examination periodically and repeatedly.

On the other hand, an ectosome is an endoplasmic reticulum with a diameter of 0.1 to 1 μm, and is any endoplasmic reticulum formed by sprouting the cell membrane to the outside of the cell. Ectosomes are very heterogeneous and diverse in composition. Ectosomes are generally derived from the cell from which they are derived, and these characteristics distinguish them from ectosomes derived from other cell types. It is known that ectosomes are produced in abundance when cells are placed under stress. Ectosomes function as molecular carriers for various purposes and play an important role in cell-to-cell communication. The ectosome fuses with the cell membrane of the target cell and delivers various molecules such as proteins and RNA inside. Using the characteristic that ectosomes contain constituents of the derived cells, molecular fingerprinting for each tissue is identified, and only ectosomes having elements corresponding to the desired tissue are isolated. It is possible to purify only tissue-specific ectosomes from ectosomes, and studies are currently underway to indirectly diagnose the state of target tissues using the characteristics of ectosomes through a minimally invasive approach using blood.

Recent studies have shown that ectosomes can cross the blood brain barrier and be secreted into the blood. For this reason, a large amount of brain-derived ectosomes secreted from the brain are mixed in human blood, and research is being conducted to indirectly diagnose brain conditions by isolating these brain-derived ectosomes from blood. According to previous studies, brain-derived ectosomes are known to have a surface antibody called CD171, and it is known that only brain-derived ectosomes can be isolated by isolating ectosomes with CD171 surface antibodies from blood (Biology 9(9), 258, 2020). Through the establishment of molecular fingerprints of these brain-derived ectosome surface antibodies, only brain-derived molecules from blood can be isolated and the condition of the brain can be indirectly diagnosed using this.

As described above, the ectosome-based test method has the possibility of diagnosing brain diseases only with a minimally invasive blood test, and accordingly, for more accurate diagnosis, it is important to construct an ectosome profiling library in the blood of a diseased population. On the other hand, Korean Patent Publication No. 10-2020-0066249 suggests new biomarkers other than CD171 that can capture brain-derived ectosomes in blood, but the biomarkers, existing traditional blood dementia biomarkers, and newly discovered dementia biomarkers No studies have been conducted on complex diagnostic systems using markers.

Under this background, the present inventors completed the present invention by confirming that diagnosis or prediction of dementia is possible without brain tissue sampling or imaging approach using a biomarker specifically expressed in brain-derived ectosomes.

One object of the present invention is to measure the level of mRNA of one or more genes selected from the group consisting of amyloid beta precursor like protein 2 (APLP2), amyloid precursor protein (APP), and amyloid beta (Aβ) or a protein expressed therefrom. It is to provide a composition for diagnosing dementia, including an agent to be measured.

Another object of the present invention is to provide a kit for diagnosing dementia comprising the composition.

Another object of the present invention is (a) in a biological sample isolated from a subject suspected of having dementia, consisting of amyloid beta precursor like protein 2 (APLP2), amyloid precursor protein (APP), and amyloid beta (Aβ). Measuring the level of mRNA or protein expressed therefrom of one or more genes selected from the group; and (b) comparing the measured level with the level measured in a sample isolated from a normal individual.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object is an mRNA of one or more genes selected from the group consisting of APLP2 (amyloid beta precursor like protein 2), APP (amyloid precursor protein), and Aβ (amyloid beta), or mRNA thereof. It provides a composition for diagnosing dementia, including an agent for measuring the level of a protein expressed from.

The composition may include one gene or a combination of two or three genes among the three genes. Specifically, APLP2; APP; or Aβ; including each gene, or APLP2 and APP; or a combination of APLP2 and Aβ, but is not limited thereto.

The composition of the present invention can obtain useful information for the diagnosis of dementia by using the significant difference in expression of the gene in a patient with dementia compared to a normal individual. This has not been known so far, and is the first discovered by the present invention, and is significant in that it is possible to diagnose dementia with high sensitivity and specificity.

The term "APLP2" gene of the present invention is a gene encoding Amyloid Beta Precursor Like Protein 2, and belongs to the amyloid precursor protein (APP) gene family, and the expression of the gene is involved in the development of pancreatic cancer, colon cancer, breast cancer, prostate cancer, etc. (ONCOLOGY LETTERS 17, 508-513, 2019). However, no study has been conducted on a method for providing information for diagnosis of dementia by integrating information on the simultaneous expression of the gene and APP or Aβ in a biological sample including ectosomes. APLP2 information of the present invention is disclosed in, for example, NCBI gene ID 334 in humans, but is not limited thereto.

The term "APP" gene of the present invention is a gene encoding an amyloid precursor protein, and is known as one of the causative genes of familial Alzheimer's disease. However, no study has been conducted on a method for providing information for diagnosing dementia by integrating information on simultaneous expression of the gene and the APLP2 gene in a biological sample including ectosomes. APP information of the present invention is disclosed in, for example, NCBI gene ID 351 in humans, but is not limited thereto.

The term "Aβ" gene of the present invention is a gene encoding Amyloid Beta protein. Amyloid beta is a 36-43 amino acid peptide that is the main component of amyloid plaques found in the brains of Alzheimer's patients and is critically involved in Alzheimer's disease. It is known (Acta Pharmacologica Sinica 38, 1205-1235, 2017). However, no study has been conducted on a method for providing information for diagnosing dementia by integrating information on simultaneous expression of the gene and the APLP2 gene in a biological sample including ectosomes. Information on proteins encoded by Aβ of the present invention is disclosed, for example, in EMBL-EBI Pfam ID PF03494, but is not limited thereto.

The APLP2, APP or Aβ gene of the present invention may be specifically expressed in brain-derived ectosome. Specifically,

The term "ectosome" of the present invention is an endoplasmic reticulum having a diameter of 0.1 to 1 μm, and refers to all endoplasmic reticulum formed by budding of cell membranes to the outside of the cell. The ectosome fuses with the cell membrane of the target cell to deliver molecules such as various proteins, DNA, RNA, and metabolites therein, and is characterized by containing components of the derived cell. Accordingly, the present invention attempted to indirectly diagnose the condition of the target tissue, that is, the brain, using the characteristics of these ectosomes in a minimally invasive or non-invasive approach using blood.

"Brain-derived ectosome" of the present invention refers to a generic term for ectosomes secreted from various cells of brain tissue. Since the brain-derived ectosome is small in size of 0.1 to 1 μm, it is easily incorporated into the blood, and the expression level of a biomarker specifically expressed in the brain-derived ectosome can be analyzed within the brain-derived ectosome incorporated into the blood. there is.

The expression level of APP may be derived from the surface membrane protein of brain-derived ectosomes, and the expression level of Aβ may be derived from the inside of brain-derived ectosomes.

In relation to the three genes, in a specific embodiment of the present invention, the expression levels of APLP2, APP, and Aβ genes in ectosomes isolated from the blood of normal mice (C57BL/6) and dementia mice (5xFAD) It was confirmed that there was a significant difference (Fig. 1). Furthermore, multiple biomarkers, APLP2 and APP; Alternatively, as a result of confirming the difference in the expression level of the combination of APLP2 and Aβ, the expression rate was about 16 to 18 times higher in dementia subjects than in normal subjects (FIG. 2), so diagnosis can be made with higher sensitivity and specificity when multiple biomarkers are used. knew it was possible.

In another specific embodiment of the present invention, it was confirmed that the expression levels of APLP2, APP, and Aβ genes were significantly different in ectosomes isolated from the blood of a normal group in their 60s and a dementia patient group in their 60s (FIG. 3). Furthermore, multiple biomarkers, APLP2 and APP; Alternatively, as a result of confirming the difference in the expression level of the combination of APLP2 and Aβ, the expression rate was about 17 to 19 times higher in dementia subjects than normal subjects (FIG. 4), which corresponds to the experimental results in mouse ectosomes.

The term "agent for measuring the level of mRNA" of the present invention refers to an agent used in a method for measuring the level of mRNA transcribed from the gene in order to determine the expression of the three genes of the present invention included in a sample. it means. Specifically, the preparation is RT-PCR, quantitative real time PCR (quantified real time PCR), competitive RT-PCR (Competitive RT-PCR), real time RT-PCR (real time quantitative RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting), may include primers or probes capable of specifically binding to the target gene used in methods such as DNA chip analysis, but is not limited thereto.

The term "primer" refers to a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming a base pair with a complementary template, and serving as a starting point for template strand copying. refers to a short nucleic acid sequence that Primers can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

The term "probe" may be a probe capable of complementarily binding to the gene, and the nucleotide sequence of the probe is not limited as long as it can complementarily bind to each of the genes.

The term of the present invention, "agent for measuring protein level" refers to an agent used in a method for measuring the expression level of a protein encoded by the three genes of the present invention included in a sample. More specifically, the agent for measuring the level of the protein may include an antibody, peptide, or aptamer specific to the protein. Specifically, the preparation is Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, immunohistochemical staining, immunoprecipitation assay Assay), complement fixation assay, immunofluorescence, immunochromatography, FACS (fluorescence activated cell sorter analysis), and protein chip technology assay. It may include, but is not limited thereto.

The term “antibody” refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such antibodies can be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or any immunoglobulin antibody may be included as long as it has antigen-binding properties and a part thereof is also included in the antibody of the present invention.

The term "aptamer" refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may have various three-dimensional structures depending on its base sequence, and may have high affinity for a specific substance, such as an antigen-antibody reaction. An aptamer can inhibit the activity of a given target molecule by binding to the given target molecule.

The term "dementia" of the present invention is caused by a chronic or progressive disease of the brain, and degeneration, degeneration, and aging of brain tissue, central nervous system infection, cerebral infarction, brain damage, toxic metabolic disorder, and nervous system disease cause dementia. known to be the main cause. The dementia of the present invention includes all of Alzheimer's disease, vascular dementia, and other mixed dementias, and may specifically be Alzheimer's dementia, but is not limited thereto.

The term "diagnosis" of the present invention is used to determine an individual's susceptibility to a specific disease or disorder, to determine whether an individual currently has a specific disease or disorder, or to provide information on treatment efficacy. This includes monitoring the health of the object. For the purposes of the present invention, the diagnosis is to determine whether or not dementia has occurred.

Another aspect of the present invention provides a kit for diagnosing dementia comprising the composition.

The description of the terms “dementia” and “diagnosis” is as described above.

The kit of the present invention can be used to diagnose the onset of dementia by measuring the gene or protein level of APLP2, APP, or Aβ from the blood of an individual suspected of having dementia, and is not particularly limited thereto, but is not particularly limited thereto. In addition to primers, probes, antibodies, peptides or aptamers for detecting genes or proteins of, one or more other component compositions, solutions or devices suitable for the analysis method may be further included.

Specifically, the kit of the present invention may be a kit including essential elements required to perform RT-PCR. The RT-PCR kit contains, in addition to specific primer pairs for marker genes, a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- water, sterile water, and the like.

In addition, the kit of the present invention may be a kit including essential elements required to perform a microarray chip. The microarray chip kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. It can be easily prepared by a manufacturing method commonly used in the art. In order to fabricate a microarray chip, a micropipetting method using a piezoelectric method or a pin type is used to immobilize the searched marker as a probe DNA molecule on a substrate of a DNA chip. It is preferable to use a method using a spotter of, but is not limited thereto. The substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto. . In addition, the substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane, but is not limited thereto.

In addition, the kit of the present invention may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody.

Another aspect of the present invention is (a) in a biological sample isolated from a subject suspected of having dementia, APLP2 (amyloid beta precursor like protein 2), APP (amyloid precursor protein), and Aβ (amyloid beta) Measuring the level of mRNA or protein expressed therefrom of one or more genes selected from the group consisting of; and (b) comparing the measured level with the level measured in a sample isolated from a normal individual.

The terms "APLP2", "APP", "Aβ", "dementia", and "diagnosis" are explained as described above.

The term "subject" of the present invention means a subject for whom dementia is to be diagnosed. At this time, the subject may be included without limitation as long as it is an animal that can develop dementia, such as a dog, horse, cow, rat, goat, rabbit, chicken, duck, goose, as well as a human.

The term "sample" as used herein refers to cell culture supernatant, whole blood, serum, plasma, ascites fluid, cerebrospinal fluid, bone marrow aspirate, bronchoalveolar lavage, urine, semen, vaginal fluid, mucus, It may be obtained from saliva, sputum, or other sources known in the art, including purified lysates from biological tissue samples, or other tissues, including, for example, brain tissue.

The biological sample of the present invention may specifically include blood, and more specifically may include ectosomes of blood. Since the ectosomes pass through the blood brain barrier and are secreted into the blood, it is obvious that a large amount of brain-derived ectosomes secreted from the brain are mixed in human blood. According to previous studies, it is known that brain-derived ectosomes have a surface antibody called CD171, and only brain-derived ectosomes can be isolated by isolating ectosomes having CD171 surface antibody from blood.

The term "normal subject" of the present invention refers to an individual who has not been diagnosed with dementia, and in a sample isolated from a normal individual and a sample isolated from an individual whose diagnosis of dementia is to be predicted, the mRNA of the gene or the protein expressed therefrom By measuring and comparing expression levels, it is possible to predict whether a subject suspected of having dementia develops dementia.

The method for providing information for diagnosis of dementia of the present invention measures the level of mRNA of the gene or protein expressed therefrom measured in a biological sample isolated from an individual suspected of having dementia, and isolated from a normal individual. If it is significantly higher than the level measured in the tested sample, it can be determined that the risk of developing dementia is high.

Another aspect of the present invention is a biomarker for diagnosing dementia, including at least one selected from the group consisting of APLP2 (amyloid beta precursor like protein 2), APP (amyloid precursor protein), and Aβ (amyloid beta) composition is provided.

The term "biomarker" of the present invention is a substance capable of diagnosing dementia, that is, a substance capable of distinguishing and diagnosing the occurrence of dementia in a biological sample, and a polypeptide or nucleic acid showing a significant difference between a normal individual and an individual with dementia. , organic biomolecules such as lipids, glycolipids, glycoproteins, sugars, and the like. For the purposes of the present invention, the biomarker supports the onset of dementia when the level of increased expression of one or more genes selected from the group consisting of APLP2, APP, and Aβ.

Another aspect of the present invention is the mRNA of one or more genes selected from the group consisting of amyloid beta precursor like protein 2 (APLP2), amyloid precursor protein (APP), and amyloid beta (Aβ), or a protein expressed therefrom. Provided is a composition for diagnosing the onset of dementia, including an agent for measuring the level.

In the present invention, by using a biomarker specifically expressed in brain-derived ectosomes, it is possible to diagnose a disease state of the brain without sampling brain tissue or imaging. Specifically, it is possible to diagnose dementia non-invasively by isolating brain tissue-specific and brain region-specific expression ectosomes from blood, using specific discovery biomarkers, and comparing their profiles.

1 is a diagram showing the result of confirming the expression of a specific gene in ectosomes extracted from the blood of mice of a normal group and a dementia group.
2 is a diagram showing the results of confirming multi-biomarker gene expression in ectosomes extracted from the blood of normal and dementia group mice.
3 is a diagram showing the results of confirmation of specific gene expression in ectosomes extracted from blood samples of patients in their 60's normal group and dementia group.
4 is a diagram showing the results of confirming multi-biomarker gene expression in ectosomes extracted from blood samples of patients in their 60s, normal group and dementia group.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Experimental Example 1. Verification of biomarkers in mouse ectosomes

Blood was drawn from five 6-month-old C57BL/6 mice and five 6-month-old 5xFAD mice, ectosomes were isolated, and the expression of three genes expressed in the isolated ectosomes was analyzed by quantitative real-time genetic analysis.

Specifically, RNA was extracted from the isolated ectosome, 2 μg of the extracted RNA was dissolved in DEPC purified water, 1 μL of oligo(dt) 12-18 and 1 μL of 10 mM dNTP were added, reacted at 70 ° C for 5 minutes, Immediately cooled on ice, 4 μL of 5x reaction (first strand) buffer, 2 μL of 100 mM DTT, and 40 units of RNase inhibitor were added and reacted at 42 ° C for 50 minutes and then at 65 ° C for 10 minutes to synthesize cDNA. 5 μL of the synthesized cDNA was added with 1 μL of each of the three types of dementia-related genes and brain-specific gene primer pairs, 12.5 μL of SYBR, and the rest of distilled water to make 25 μL, followed by reaction at 94°C for 5 minutes, followed by 95°C for 1 minute. , The process of 55 ℃ 30 seconds was repeated for 40 cycles, and the reaction was recorded in real time for each cycle. In this experiment, the period recorded as causing a certain amount of reaction was calculated in proportion to the normal group and used as the result value.

As a result, as shown in FIG. 1, gene expression rates of APP, Aβ, and APLP2 were about 2 to 3 times higher in dementia blood ectosomes than in normal blood ectosomes.

In addition, in order to confirm the difference between the dementia group and the normal group by multiple biomarkers in each ectosome, only ectosomes expressing APLP2 were isolated from animal samples and human samples, and RNA was extracted from the isolated ectosomes. After dissolving 2 μg of RNA in DEPC purified water, add 1 μL of oligo(dt) 12-18 and 1 μL of 10 mM dNTP, react at 70 ° C for 5 minutes, cool on ice immediately, and add 5x reaction (first strand) buffer 4 μL, 2 μL of 100 mM DTT, and 40 units of RNase inhibitor were added, reacted at 42°C for 50 minutes, and then reacted at 65°C for 10 minutes to synthesize cDNA. 5 μL of the synthesized cDNA was added with 1 μL of each of the three types of dementia-related gene primer pairs, 12.5 μL of SYBR, and the remaining distilled water to make 25 μL, followed by reaction at 94°C for 5 minutes, 95°C for 1 minute, 55°C 30 The process of seconds was repeated for 40 cycles, and the reaction was checked and recorded in real time for each cycle. After the repeat cycle, the reaction was terminated by reacting at 72 ° C. for 10 minutes. In this experiment, the period recorded as causing a certain amount of reaction was calculated in proportion to the normal group and used as the result value. As shown in FIG. 2, the dementia group showed about 16 to 18 times higher expression rate than the normal group It was found that it can be used with higher sensitivity and specificity values when diagnosing dementia by using multiple biomarkers.

Experimental Example 2. Verification of biomarkers in ectosomes of dementia patients

Blood was drawn from 5 normal people in their 60s and 5 people diagnosed with dementia in their 60s, ectosomes were isolated, and the expression of three genes expressed in the isolated ectosomes was analyzed by quantitative real-time genetic analysis.

Specifically, RNA was extracted from the isolated ectosome, 2 μg of the extracted RNA was dissolved in DEPC purified water, 1 μL of oligo(dt) 12-18 and 1 μL of 10 mM dNTP were added, reacted at 70 ° C for 5 minutes, Immediately cooled on ice, 4 μL of 5x reaction (first strand) buffer, 2 μL of 100 mM DTT, and 40 units of RNase inhibitor were added and reacted at 42 ° C for 50 minutes and then at 65 ° C for 10 minutes to synthesize cDNA. 5 μL of the synthesized cDNA was added with 1 μL of each of the three types of dementia-related genes and brain-specific gene primer pairs, 12.5 μL of SYBR, and the rest of distilled water to make 25 μL, followed by reaction at 94°C for 5 minutes, followed by 95°C for 1 minute. , The process of 55 ℃ 30 seconds was repeated for 40 cycles, and the reaction was recorded in real time for each cycle. In this experiment, the period recorded as causing a certain amount of reaction was calculated in proportion to the normal group and used as the result value.

As a result, as shown in FIG. 3, gene expression rates of APP, Aβ, and APLP2 were about 2 to 3 times higher in dementia blood ectosomes than in normal blood ectosomes.

In addition, as a result of confirming the difference between the dementia group and the normal group by multiple biomarkers in each ectosome, as shown in FIG. 4, it was confirmed that the dementia group showed an expression rate about 17 to 19 times higher than that of the normal group. This corresponds to the experimental results in the mouse ectosome of Experimental Example 1, and it was found that it can be used with higher sensitivity and specificity values when diagnosing dementia using multiple biomarkers.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (1)

APLP2 (amyloid beta precursor like protein 2) and APP (amyloid precursor protein) specifically expressed in brain-derived ectosomes; or APLP2 and Aβ (amyloid beta); including an agent for measuring the level of mRNA or protein expressed therefrom;
The agent for measuring the mRNA level of the gene includes a primer or probe that specifically binds to the gene,
In the dementia diagnosis kit, wherein the agent for measuring the level of the protein includes an antibody, peptide, or aptamer specific to the protein,
The kit for diagnosing dementia further comprises necessary elements for immunological detection of antibodies, RT-PCR, and microarray chip.

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