KR20160084174A - Method of Screening a Patient-specific Drug by Using Variation in Nucleic Acid Sequence of Luterial - Google Patents

Abstract

Provided is a screening method of a patient-specific medicine, which can secure an excellent therapeutic effect of the patient-specific medicine. The present invention relates to a screening method of a patient-specific medicine, which comprises the following steps: (a) deriving at least one mutation codon which includes a mutation from a nucleic acid sequence of luterial derived from a patient; and (b) sorting (i) food or medicinal plant-derived luterion including a complementary codon which allows an RNAi process of at least one mutation codon derived in the step (a), or (ii) an antisense, an miRNA, a siRNA, or an aptamer which forms a complementary bond with the variation codon.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a screening method for screening a patient-specific therapeutic agent using nucleic acid sequence variation of luterian,

The present invention relates to a method for screening a patient-customized therapeutic agent, particularly a method for identifying at least one codon containing a mutation from a nucleic acid sequence of a patient-derived luterian and a lterion derived from a food or a medicinal plant capable of RNAi- Lt; RTI ID = 0.0 > miRNA, < / RTI > siRNA, or squamometer that form complementary bonds with the codon.

Various therapeutic agents for treating diseases have been developed. However, since not only different diseases are exhibited in different diseases, but disease-causing cells having various kinds of genetic characteristics are present in the same diseases, even in patients having the same disease, May be different.

Although a single therapeutic agent kills a specific disease cell to produce a therapeutic effect, it can not kill all disease-causing cells having different genetic characteristics. Therefore, a therapeutic agent suitable for a particular patient may not exhibit a therapeutic effect in another patient , But it may also cause adverse effects that intensify the disease.

Although various medicines have been developed to study various disease inducing mechanisms and to treat them, no standard method has been reported to determine whether a certain medicament should be used for a certain disease to show a desired therapeutic effect, As a result,

For example, patients with at least one mutation (at least one single nucleotide polymorphism (SNP)) may have different SNP patterns despite having the same disease. Specifically, as described in FIG. 1, even though the treatment with the therapeutic agent A 'for the treatment of the patient 1 comprising the mutation (A) of the nucleic acid sequence as compared to the normal, the therapeutic agent A' It may not exhibit an appropriate therapeutic effect.

Since patient 2 contains mutation (A) of the same nucleic acid sequence as patient 1, the therapeutic effect can be expected using therapeutic agent (A '). However, a sufficient therapeutic effect can not be expected unless the mutation (B) of the nucleic acid sequence is different from the mutation (A) of the nucleic acid sequence and the therapeutic agent for the mutation (B) of another nucleic acid sequence is not used.

Further, in the patients 3 and 4 including the mutation (B or C) of the nucleic acid sequence different from the mutation (A) of the nucleic acid sequence shown in the patient 1, sufficient therapeutic effect can not be expected even when the therapeutic agent (A ') is administered.

Even if the patient's disease is the same, it is very important to select a therapeutic agent suitable for the patient-specific mutation because the mutation that causes the disease may be different for each patient, and it is necessary to secure a method for screening the patient- It is true.

There has been an attempt to treat patients by subdividing patients based on their genetic characteristics and applying patient-tailored treatment techniques. However, there has been little development of a customized treatment selection system based on large-scale genetic information, and only in vitro / in vivo models have been developed, Scientifically, there was no case of predicting a patient-specific treatment and applying it clinically.

By predicting the susceptibility of some therapeutic agents to a metabolic disease-related cell line in a fragmentary manner and anticipating the response to the patient, it becomes possible to genetically predict the effect of the therapeutic agent, and development of a "tailor-made" therapeutic method This seems to be speeding up. However, the cell line-based genome analysis is problematic in that it does not reflect the actual clinical situation of the patient because the inherent molecular and functional characteristics of the cell line are lost during the establishment of the cell line.

The N = 1 clinical trial theory of finding the right therapeutic for each patient and providing customized chemotherapy has received great interest in recent years, but the related process has not been established.

In a situation where existing cell-based genome analysis is limited, a new approach based on genomic data analysis is required for efficient and economical personalized treatment.

Among various diseases, there are various kinds of therapeutic researches related to cancer. The types of cancer are as many as several dozen that have been found so far, and they are mainly classified according to the location of the diseased tissue. Cancer has a very rapid growth rate and invades the surrounding tissues, causing metastasis and endangering life.

Various biochemical mechanisms associated with cancer have been identified and improved prospects for diagnosis and treatment of cancer have been improved by improved detection methods for cancer, mass screening, and development of therapeutic agents and therapies for several decades .

Cancer can occur in lung cancer, gastric cancer (GC), breast cancer (BRC), colorectal cancer (CRC), and in virtually any tissue. Early diagnosis of cancer was based on external changes of the biotissue due to the growth of cancer cells. Recently, however, diagnosis using biomolecules such as blood, glycocyte, DNA, And detection has been attempted. The most commonly used cancer diagnosis method is a tissue sample obtained through biopsy or an image diagnosis. Among them, biopsy results in great pain for the patient, which is not only costly but also takes a long time to diagnose. In addition, if the patient is actually cancerous, there is a risk that cancer metastasis may be induced during the biopsy, and in the case where a tissue sample can not be obtained through biopsy, There is a drawback that it is not possible to diagnose the disease until the extraction is done. In the image-based diagnosis, cancer is judged based on an X-ray image or a nuclear magnetic resonance (NMR) image obtained using a contrast agent having a disease target substance, There is a possibility that the diagnosis is misdiagnosed according to the proficiency of the clinician or the reader, and there is a disadvantage that it largely depends on the precision of the device for obtaining the image. Furthermore, even the most precise apparatus can not detect tumors of a few mm or less, which is difficult to detect in the early stages of onset. In addition, in order to obtain an image, a patient or a disease-capable person is exposed to electromagnetic waves of high energy capable of causing mutation of a gene, so that it can not only cause another disease but also has a disadvantage in that the number of diagnosis through imaging is limited .

In other words, biopsy for the diagnosis of cancer involves a lot of time, cost, inconvenience, and pain. Therefore, there is a need for a method that can dramatically reduce the number of patients who need unnecessary biopsy and a method for early diagnosis of cancer .

Although prognostic criteria and molecular markers provide some guidance in predicting the fate of patients and selecting the appropriate treatment pathway, there is still a limit to accurately diagnosing the patient's prognosis. Therefore, it is urgent to study the diagnostic methods for the prediction of effective prognosis or the markers capable of diagnosing them.

Under these circumstances, the present inventors have found that the Korean patent application No. 10-2013-0082060 discloses that the diameter, area, shape or motility of mitochondrial-like micro-materials (luterian) in a patient is different from that of ruteli And can be used as an indicator to predict the diagnosis or prognosis of cancer.

In the present invention, a codon (for example, UGU, where C is mutated at the first position among the codons) containing a base having a mutation in a patient-derived lterial (for example, CGU for a normal person) is identified, SiRNA or an aptamer that forms a complementary bond with ruterion containing three bases complementary to the codon (e.g., ACA) mutated to induce RNAi expression is useful for treating the disease in the patient Based on this finding, it was confirmed that a patient-customized therapeutic agent could be screened, and the present invention was completed.

As a result of the research for the development of the therapeutic agent, 64 kinds of gene specific genes derived from lutealia derived from the patient were patterned, and a candidate therapeutic substance for inhibiting cancer-specific genes was designed for each pattern. Then, Lt; RTI ID = 0.0 > a < / RTI > patient-customized therapeutic agent.

In order to accomplish the above object, the present invention provides a method for the diagnosis of cancer, comprising the steps of: (a) deriving at least one mutation codon containing a mutation from a nucleic acid sequence of a patient-derived luterial; And (b) a food or medicinal plant-derived luterion containing (i) a complementary codon capable of RNAiing the derived at least one mutation codon, or (ii) Lt; RTI ID = 0.0 > miRNA, < / RTI > siRNA or uterus.

Based on the disease-specific gene observed in the patient-derived lterial, a codon containing at least one mutation is derived from the nucleic acid sequence of lterial, and a method for screening a patient-customized therapeutic agent suppressing the expression of the disease- It is possible to provide a new standard that can classify patients optimally and also provide the most appropriate treatment for each individual specific symptom without adverse effect while exhibiting excellent therapeutic effect.

FIG. 1 is a schematic diagram illustrating the reason for showing a difference in therapeutic effect of a therapeutic agent according to a patient.
Figure 2 is a schematic representation of the life cycle of luterian.
FIG. 3 is a photograph showing the sizes of luterian and mutant luterian.
4 is a schematic diagram showing a gene library including a gene expression pattern.
FIG. 5 is a result of a bioanalyzer analyzing whether or not RNA is contained in luterian (1: control; 2: 100-200 nm luterian; 3: 200-400 nm luterian).
6 is a result of a bioanalyzer analyzing whether or not DNA is contained in the lterial (left: control; right: lterial).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

As used herein, the terms "Luterial" and "Luterion" are living organisms present in animals and plants, respectively, ~ 500 nm / abnormal fusion stage 800 nm or more).

The luterian and lterion include DNA and RNA (Fig. 5 and Fig. 6), and are distinguished from exosomes or microvesicles in that they have motility and adhesion. Mitochondria are identified by Janus green B and fluorescent stain Rhodamine 123, Mitotracker, Acridine Orange, and DAPI. The mitochondria are identified by the Luterian temperature mitochondria It has a double-membrane structure similar to that of mitochondria and has a structure in which the internal cristae structure has not been completed. It is observed in the same laser wavelength range as that of mitochondria, Quot ;, "mitochondrial analogue ", or" proto-mitochondria ".

In animals including humans, it is present in blood, saliva, lymphatic fluid, semen and vaginal fluid, breast milk (especially colostrum), umbilical cord blood, brain cells, spinal cord and bone marrow, and is called "lterial." In addition, the substance present in plants is referred to as "lterion. &Quot; In the case of plants, they are present in large amounts especially in the stem part.

Normal lterial and lterion sizes range from 50 to 200 nm, and mutant lterials and mutant lterion fuse to form mutants and have a size of several tens of micrometers. Ruterian and lterion may be referred to as immature mitochondrial steps involving mRNA and miRNA (seldom, including DNA). Luteal and ruterion are characterized by the fact that they are not dissolved in the digestive juices and are introduced into the blood and are expected to be involved in signaling, cell differentiation, cell death, as well as regulation of cell cycle and cell growth.

The inventors of the present invention have found that blood-derived ruterium is closely related to the diagnosis of cancer (Korean Patent Application No. 10-2013-0082060). Specifically, the normal Tertiary and Lterion are expected to play a role in blocking the growth of cancer cells and returning the cells to a healthy immune system. Its role is in RNA interference (RNA interference), which has the potential to normalize genes . ≪ / RTI >

The term "RNAi (RNA interference)" means the introduction of double-stranded RNA (dsRNA) consisting of a strand having a sequence homologous to the mRNA of the target gene and a strand having a sequence complementary thereto, Thereby inhibiting the expression of the target gene.

Luterian, which has a size of less than 200 nm, for example, 50-150 nm, in normal ruteri and ruterion is expected to prevent the growth of cancer cells and return the cells to a healthy immune system, RNA interference (RNA interference), which has the potential to induce cell death. When the information system in the normal luteal RNA is instructed to deviate from the normal orbit and produce a protein causing an abnormal disease, it acts to suppress the occurrence of diseases such as cancer by artificially interfering with it, and the size is 200-500 nm or more When mature, it is also involved in energy metabolism and energy metabolism. When specific wavelengths are irradiated, it reacts like a chloroplast, amplifying light energy by reaction expression. Thus, failure to perform a normal role in these ruteriacs can lead to critical disorders in homeostasis and ATP production, leading to diseases both in respiration and in energy metabolism.

Conversely, ruteri- als that do not play a normal role vary in size and morphology, and exhibit ecological and behavioral differences from normal ruteriacs. Specifically, in the case of normal luterian, a double-spore does not grow after the formation of a double-spore. However, in the case of a mutant luterian found in a body fluid discharged from a patient suffering from a cancer patient or a chronic disease, , And has a size of more than 600-800 nm and a size of 200 μm (200,000 nm) or more. Similar to the virus, the virus infects red blood cells, white blood cells, platelets, and the like, or exhibits a characteristic of coagulating with other lterials (FIGS. 2 and 3). To distinguish these ruterians from normal ruterians, they are called "mutant lterials", "mutant lterials" and "mutant luterials".

On the other hand, in the case of ruteri- al derived from the blood of a human or an animal, it is difficult to observe it because it is rapidly dissolvable in vivo and disappears or its morphology changes. When it is placed under abnormal environment, normal ruteri- al mutation It is difficult to use it in the treatment of diseases due to mutation of ruteriol.

However, unlike plant derived ruterian, plant-derived ruterian does not dissolve or disappear quickly at room temperature and has a characteristic that it is not mutated by fusion even after storage for a long period of time. In addition, it is also possible to inhibit the growth of the patient's blood-derived mutant ruteriac so as not to cause further fussion, inhibit the maturation of the ruterian in the blood-derived maturation stage of the patient, It can be inhibited from growing by fusion.

The characteristics of the plant-derived lterion are expected to be due to the RNAi function of lteriolin. Such RNAi function can be used to inhibit or prevent the mutagenization or maturation of lterials derived from a patient having a specific disease, To treat or prevent certain diseases, or to screen such substances.

As a result of separating and observing these lterials, it was confirmed that the separated lterials had the following characteristics:

(a) in the case of a normal person, it is circular or elliptical with a size smaller than red blood cells (50 to 200 nm);

(b) nucleic acid content;

(c) Immunochemical fluorescence staining shows a similar response to mitochondria;

(d) indicate fusion and / or fission ecological form;

(e) mature to a size of up to 300 nm in the absence of fusion, mature with a DNA-containing mitochondria, mimicking a mitochondria-like structure on a SEM or TEM electron micrograph;

(f) increases in size up to several thousand nm when fusion occurs;

(g) In the case of a patient-derived, a mutant ruteri- al having a larger size (longer than 500 nm in diameter) than that of a normal human and having a nonuniform shape is generated; And

(h) Represents a light reaction different from that of liquid aerosol.

In addition, it may be characterized as having one or more of the following characteristics:

(i) indicates autofluorescence;

(j) producing ATP at a size of 200-400 nm;

(k) adherence;

(l) bilayer structure;

(m) In certain conditions, the mutant lterian bursts and has a stemness after bursting; And

(n) p53 gene and telomere control function.

In this regard, lutealia may be isolated, for example, from body fluids discharged from a patient, and the term " body fluids discharged from a patient "as used herein refers to blood, saliva, lymphatic fluid, semen, , Umbilical cord blood, brain cells, spinal cord or bone marrow, but is not limited thereto.

In one embodiment, the luterian can be isolated from the blood, which is readily obtainable from the patient, and the luterian contained in the blood can be isolated, for example, by the following steps.

A method for separating lutealia from blood includes a first separation step of separating blood-derived material having a size of platelets and platelets or more from the blood; A second separation step of centrifugally separating the blood separated from the platelets and the blood-derived substance having the size of the platelets or more; A third separation step of separating luterian from the supernatant obtained by the centrifugation; And washing the separated lterials.

More specifically, the first separating step may comprise taking blood from the patient and passing through a filter having a diameter of 0.8-1.2 [mu] m or more in diameter to separate the unfiltered material. The second separation step may include repeatedly centrifuging at 1200 to 5000 rpm for 5 to 10 minutes to remove a common microbeptide such as an exosome to obtain a supernatant. The third separation step may include a step of irradiating the supernatant obtained through centrifugal separation with infrared rays to separate the lutealia particles having mobility and collected by using a pipette. Since luterian has characteristics of autofluorescence and motility, it is possible to identify luterian particles from the supernatant when irradiating infrared rays as described above. At this time, separation can be carried out using a pipette while observing lterial particles moving with a dark-field microscope or a confocal microscope. The lterial separated in the third step may be passed through a filter having a cavity of 50 nm diameter, and only the unfiltered portion may be washed with PBS to obtain lterial. Since the lutealia has a diameter of 50 nm or more in length, the blood-derived fine material other than luterian can be removed through the above process. By this process, luterian having a long diameter of 50-800 nm can be obtained, which can be observed through a dark-field microscope or a confocal microscope. The obtained luterian was irradiated with a laser beam having a wavelength of 50-200 nm (generator) / 200-400 nm (mature stage) / 400-600 nm (splitter) / 600-800 nm And hyperopia).

In some cases, the luterian isolated from the body fluid discharged from the patient sometimes dissolves rapidly in vivo, and disappears or changes its morphology. Therefore, it is difficult to observe it. Therefore, it is cultured so as not to exceed a specific size (500 nm) .

Specifically, water may be added to the lterial and incubated at 18 to 30 ° C (preferably 20 to 25 ° C) under IR light irradiation.

The water added during the culture may be, but is not limited to, saline solution or PBS solution. The blood-derived luteriol before culture can be obtained according to the above separation method, and the size of the luteal can be 50 to 200 nm. The size of the cultured blood derived lterial after culturing may be 300 to 500 nm. At this time, it is possible to keep the size of the lterial not to exceed 500 nm while observing with a microscope. When the culture is finished, it can be classified according to size, cooled to minus 80 ° C or stored with nitrogen or stored. can do.

The lutealia cultured as described above can be stored without changing its shape or activity without changing its characteristics for a certain period of time, so that it can be effectively used for diagnosing diseases and predicting prognosis using lterial.

"Without change in lterial characteristics" means that the morphology or size of the lterial maintains its shape in the medium composition almost similar to that before the culture. Further, the activity such as the mobility of ruteriac such as the nano-tracking speed is maintained to a value similar to that before culturing.

Luterian can be classified into four types of 50-200nm / 200-400nm / 400-600nm / 600-800nm, and four species can be classified into generator-mature-shear-shear-hyperdelivery respectively. For the study of RNAi characteristics, a generator size of 50-200 nm or a maturation period of 200-400 nm can be used. Generation of ruterium can be used when the test period is long. For mitochondrial characterization studies, 400-600 nm. For disease prediction studies and prognosis, 600-800 nm And mature lterial.

(A) is a step of deriving at least one codon containing a mutation from the nucleic acid sequence of the patient-derived lterial from the nucleic acid sequence of the patient-derived lterial. "Codon" according to the present invention may mean three bases representing a gene expression unit designating an amino acid. Specifically, the codon is provided in the form of single stranded RNA or DNA. In the case of DNA, U is represented by T in the nucleotide sequence, and the sequence is included in the scope of the present invention. This is something that is obvious to those who have knowledge.

"A codon containing a mutation" according to the present invention means a nucleotide which is different from a normal one in any one or two or more positions of three bases, and the mutation can be specifically detected in a patient suffering from a normal non- For example, a SNP contained in one of three nucleotides constituting a codon.

The mutation can be determined by comparing the nucleic acid sequence of the patient-derived luterian with the nucleic acid sequence of the normal luterian to identify at least one sequence difference. Nucleotide sequence comparisons may be performed using methods within the purview of those skilled in the art.

In one embodiment, the mutation may comprise a single or multiple SNPs derived by comparing the nucleic acid sequence of a patient-derived lterial to the nucleic acid sequence of a normal luteal origin.

A SNP is a single nucleotide polymorphism that occurs in one of about 1000 nucleotides on the human genome and takes the form of a single nucleotide mutation between individuals of the same species. The significance of such a single nucleotide polymorphism in genetics varies greatly depending on its location. For example, when a single nucleotide polymorphism is present at a position encoding a protein, it can affect the structure of the protein, thereby changing the function of the protein, and can also be associated with disease.

In another example, when a single nucleotide polymorphism is present on another gene that does not encode a protein, that is, when it is present in a promoter or an intron, there is a difference in the expression level of the protein with respect to each, And the protein may be abnormally expressed through a metamorphic alternative splicing process.

For example, a SNP-containing codon containing a lterali-derived disease-specific mutation can be identified from a patient. When CGUs encode arginine and are converted to T (U) GU (C -> T (U) SNP), tumors, especially leukemia, can occur. In addition, GGT (U) can predict the frequency of tumor disease when expressed as GT (U) T (U) (G -> T (U) SNP) in coding glycine.

The present invention is based on the number of theoretical variations that can occur in patients with codons containing three nucleotides. Specifically, considering that the number of SNPs in which any one of three nucleotides of the codon is different from that of a normal one and the number of SNPs in which the disease can be induced is based on any one of four base types and three nucleotide positions, Is based on a maximum of 64 (= 4 ³ ).

Based on this, it is possible to classify 64 mutation patterns that can be found in patients. FIG. 4 shows a pattern table in which the nucleic acid sequence of the patient-derived lterali was classified into 64 patterns according to the mutation.

Referring to FIG. 4, in relation to the 64 codon patterns in which the lutealia-derived disease-specific gene is classified, amino acids are encoded from the species A, T, G and C or RNA cases A, U, G and C in the case of DNA Were combined and classified into 64 expression patterns.

Quot; complementary codon "according to the present invention is a nucleic acid molecule containing three bases capable of inhibiting the expression of RNAi by making a mutation-containing codon RNAi or a complementary binding with a mutation-containing codon, and directly binding to a mutation-containing codon May refer to three nucleotide-containing sequences that reduce their activity.

(B) is a step of (i) selecting a luterion derived from a food or a medicinal plant containing a complementary codon capable of RNAi the derived at least one mutation codon.

Means that the food or medicinal plant-derived lterion has been isolated in a manner similar to lterials isolated from blood.

Unlike blood derived ruteriac, the ruterion derived from the food or the medicinal plant does not dissolve or disappear in a short time at room temperature, and is not mutated by fusion even when stored for a long period of time. In addition, it inhibits the growth of the patient so as not to cause further fission by reacting with the mutated lterial from the blood of the patient, and inhibits the maturation of the luteal in the blood-derived maturation stage of the patient, Can be inhibited from being mutated or fused to grow.

The characteristics of the food or medicinal plant-derived lterion are expected to be due to the RNAi function of lterion. The RNAi function can be used to inhibit or prevent the mutagenization or maturation of lutealia derived from a patient having a specific disease, Can be used as a substance to treat or prevent a specific disease.

The lterion derived from the food or medicinal plant has a density of 1 or less and a density higher than that of the lipid and the lipid and a density lower than that of the protein, and thus can be separated from the plant by the steam distillation method, but is not limited thereto.

More specifically, the plant may use the medicinal plants selected from Tables 1 to 4. Lterion is present in all plants and thus is not limited to the medicinal plants described in Tables 1 to 4.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

In addition, it is expected that ruterion is distributed in a large amount on the stem side of a plant, and it is preferable to separate ruterion from the stem portion.

The food or medicinal plant-derived lterion may be separated by the following steps.

(a) adding a solvent to a food or a medicinal plant and shaking it while bubbling intermittently with air or oxygen at 50 to 90 占 폚; (b) collecting vapor or gas vaporized by said shaking and then cooling to obtain a condensate; (c) filtering the obtained condensate by using a filter having voids of 0.8 to 1.2 mu m; (d) centrifuging the filtered condensate; And (e) separating the food or medicinal plant-derived lterion from the centrifuged supernatant.

The step (a) may be performed by shaking the plant at 50 to 90 ° C for about 8 to 10 hours, and bubbling with oxygen for 20 to 30 minutes every 2 to 3 hours to prevent the lterion of the plant from entangling . The condensate of step (c) may be irradiated with an IR light for 20 to 60 minutes (preferably 30 to 40 minutes) before being passed through the filter to prevent deformation of the lterion. The centrifugation in step (d) may be repeatedly performed at 1200 to 5000 rpm for 5 to 10 minutes. When the supernatant obtained through centrifugal separation in the step (e) is irradiated with an IR light, the lterionic particles having mobility in response to the IR light can be separated using a pipette. The lterion separated by the step (e) is passed through a filter having a cavity of 50 nm diameter, and only the unfiltered portion is washed with PBS, and the lterion can be obtained by collecting the portion caught by the filter. Through the above process, fine materials other than ruterion can be removed, and ruterion having a size of 50 nm or more can be obtained.

The lterion derived from the food or medicinal plant obtained by the above process can be observed through a dark-field microscope or a confocal microscope. The lterion can be observed in succession using filters of 200 nm, 400 nm, 600 nm and 800 nm, respectively, Generator) / 200-400 nm (mature stage) / 400-600 nm (splitter) / 600-800 nm (hyperfractionation).

The food or medicinal plant derived lterion separated as described above has the following characteristics:

(a) a circular or elliptical shape having a diameter of 50 to 800 nm;

(b) nucleic acid content;

(c) Immunochemical fluorescence staining shows a similar response to mitochondria;

(d) indicate fusion and / or fission ecological form;

(e) mature to a size of up to 500 nm in the absence of fusion, mature with a DNA-containing mitochondria, mimic mitochondria on a SEM or TEM electron micrograph;

(f) exhibits a different light response than exosomes; And

(g) Fission occurs during IR irradiation.

In some cases, the lterion may be further characterized as having one or more of the following properties:

(i) indicates autofluorescence;

(j) producing ATP at a size of 200-400 nm;

(k) adherence;

(l) bilayer structure; And

(m) Reaction with blood-derived ruteri- al inhibits the fusion of blood-derived ruteri- al or promotes fission.

The above steps can be used to isolate motile 50-800 nm lterion reacting to an IR light source and observe mobility through a dark-field microscope or confocal microscope.

The food or medicinal plant-derived lterion can be classified into four types of 50-200 nm / 200-400 nm / 400-600 nm / 600-800 nm. It should be noted that it is easy to dissolve or change its morphology after separation of ruterion, and it is desirable to keep it freezing below minus 80 ℃ within a short time in long term storage. In short-term storage (live state), the image is preferably about 4 ° C, preferably stored in a saline solution of 0.5%, and irradiated at a low temperature of about 30 cm. For short-term storage, it can be stored in PBS solution, or it can be packed with nitrogen by sealed storage. Optionally, the food or medicinal plant lterion may contain one or more preservatives selected from the group consisting of flavonoids picetin, butaine and sulforetin.

In some cases, water is added to ruterion and cultured at 18 to 30 ° C under irradiation with IR light to cultivate plant lterion, or water is added to 400 to 800 nm plant-derived ruterion, and irradiated at 18 to 30 ° C Preferably 20 to 25 < 0 > C) (thereby inducing fission of the plant lterion). The water added during the culture may be, but is not limited to, saline solution or PBS solution. Plant-derived ruterion before cultivation can be obtained according to the above-described separation method, and the size of the ruterion having a size of 50 to 200 nm can be used. The size of the plant-derived lterion cultured according to the culture method of the present invention after culturing may be 300 to 500 nm. At this time, it is possible to keep the size of the lterion not to exceed 500 nm while observing with a microscope. When the culture is completed, it can be classified according to size, cooled to minus 80 ° C or stored with nitrogen, Can be added.

In one embodiment, the food or medicinal plant-derived lterion may be a lterione separated from nexia (an allergen-removed russilla extract).

The complementary codon can be derived from sequence analysis of food or medicinal plant-derived lterion separated from the above, and sequence analysis of food or medicinal plant-derived lterion can be performed by a method within the range usually used by those skilled in the art For example, using PCR through primer production.

For the therapeutic effect of the disease, a sufficient number of complementary codons capable of RNAi-encoding the mutation-containing codons can be expected as the number of the codons contained in the food or medicinal plant-derived lterion is sufficient. For this purpose, 2-20 mutations A food or medicinal plant-derived lterion containing a complementary codon capable of RNAi-containing codons can be screened with a desirable therapeutic agent.

The food or medicinal plant-derived lterion containing the complementary codon capable of RNAi can have a size of, for example, 200 nm or less, and preferably has a size of 50-150 nm to exhibit a desired therapeutic effect.

For example, in step (a) above, a codon containing two or more mutations from the nucleic acid sequence can be derived. Wherein the patient-specific therapeutic agent comprises a food or medicinal plant-derived lterion containing two or more complementary codons capable of RNAiing two or more codons in step (b), or a lterion that forms a complementary bond with said two or more mutation codons Screening may include screening for antisense miRNA, siRNA, or squamous cell.

Furthermore, it may be a codon containing three or more mutations, wherein the patient-specific therapeutic agent is a food containing three or more complementary codons capable of RNAi three or more codons in step (b) or A medicinal plant-derived lterion, or an antisense miRNA, siRNA or uterum that forms a complementary bond with the three or more mutation codons.

(B) further comprises the step of: (ii) selecting an antisense miRNA, siRNA or uterum that forms a complementary bond with the mutation codon. In the present invention, antisense miRNAs, siRNAs, or aptamers that form a complementary bond with a mutation codon can be used as a patient-specific therapeutic agent by directly inhibiting the expression of mutant codons.

The miRNA is a non-coding single-stranded RNA molecule of 21 to 25 nucleotides that controls the expression of eukaryotic genes. It binds to the mRNA 3 'UTR (untranslated region) of a specific gene, It is known to inhibit. In fact, miRNAs studied in animals reduce protein expression without affecting mRNA levels of specific genes. miRNA binds complementarily to specific mRNAs linked to RNA-induced silencing complex (RISC), but the central portion of the miRNA remains a mismatch, and unlike conventional siRNAs, miRNAs do not degrade mRNA. However, unlike these animal miRNAs, food or medicinal plant miRNAs are completely in agreement with the target mRNA and induce RNA interference by inducing mRNA degradation.

The siRNA plays a predetermined role in RNA interference (RNAi) pathway, in particular RNA silencing, a sequence-specific RNA degradation process promoted by double-stranded RNA (dsRNA). siRNAs are double stranded with small 3'-overhangs and are derived from longer dsRNA precursors that induce silencing. This serves as a guide to the destruction of the target RNA.

The aptamer is a nucleic acid molecule that exhibits a high degree of specific binding affinity to a molecule through interactions other than classic Watson-click base pairing. The aptamers are those capable of specifically binding to a selected target and modulating the activity of the target, for example, by blocking the ability of the target to function via typing. Aptamers are 10-15 kDa in size (30-45 nucleotides), bind to their target with an affinity of less than nanomolar, and exhibit differentiation for closely related targets.

An antisense miRNA, siRNA, or aptamer comprising the complementary codon may be in a form comprising additional sequences capable of hybridizing with a sequence around a codon for complementary binding to a codon comprising a mutation.

In one embodiment, the total length of the antisense miRNA, siRNA, or squamoma, including additional sequences that can hybridize with the sequences around the complementary codon and codon, can be, for example, 9 to 200 nucleotides (nts) , Preferably 9 to 100 nts, and more preferably 9 to 50 nts. More preferably, it is characterized by being 9 nts or more, or less than 85 nts. When the length of the total nucleic acid sequence is within the above range, the in vivo stability is high and the toxicity is low, and a codon suitable for the treatment of the desired disease is targeted, and the complementary codon showing the corresponding RNAi effect can be set, A therapeutic agent showing a therapeutic effect can be screened.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: Isolation of patient-derived lterials

The normal and mutant lterials from various patients were isolated. At the end of the non-small cell lung cancer, 50 cc of blood was collected and the unfiltered material was separated by passing through a filter having a diameter of 0.8 μm or more. The filtered blood was centrifuged repeatedly at 1200 ~ 5000 rpm for 5-10 minutes to remove normal microbejets such as exosome to obtain supernatant. The supernatant was irradiated with visible light, and the luteal particles having mobility and gathering were separated using a pipette. Since luterian has characteristics of autofluorescence and motility, it is possible to identify luterian particles upon irradiation of visible light as described above.

At this time, separation was performed using a pipette while observing lterial particles moving with a dark-field microscope or a confocal microscope. The separated lterials were passed through a filter having pores with a diameter of 50 nm, and only unfiltered portions were washed with PBS to obtain lterials. By this process, luterian having a long diameter of 50-800 nm can be obtained, which can be observed through a dark-field microscope or a confocal microscope. The obtained lterials were classified into 50-200 nm (generators) / 200-400 nm (mature stage) / 400-600 nm (splitter) / 600-800 nm (hyperfractionation) depending on their sizes.

Example 2: Isolation of nucleic acid (RNA / DNA) derived from ruteri

(AllPrep DNA / RNA micro kit: Cat 80284) to isolate total RNA and DNA from 200-400 nm lterials isolated in Example 1. Experion RNA (DNA) StdSens (Bio- Rad) chip.

After centrifugation (8000 g, 1 hour and 30 minutes), ruterium was recovered and added to 50 μl of RLT plus (detergents) of degradation buffer in the kit, 3.5 μl of beta-mercaptoethanol was added, and a 20 gauge needle- And the solution was passed 5-10 times to dissolve the lterial. The sample digestion buffer was transferred to an AllPrep DNA spin column and centrifuged (≥8000 g for 15 seconds) to separate the DNA from the column and RNA from the column.

First, the same volume of 350 μl of 70% ethanol was added to the buffer, and 700 μl of the mixture was transferred to an RNease MinElute spin column (≥8000 g for 15 seconds) and the buffer passed through the column was removed. Column washing was performed stepwise using 700 [mu] l of each of RW1, 500 [mu] l of RPE buffer and 80% of ethanol. All centrifugation (≥8000 g 15 sec) used above proceeded under the same conditions. To obtain the RNA, 14 μl of RNeasy-free solution was added to the column, followed by centrifugation (≥8000 g for 60 seconds) to isolate the rutile RNA.

Example 3 Sequence Analysis of Luteli

Primer was prepared and the sequence of the lterial nucleic acid isolated in Example 3 was analyzed by PCR. The nucleic acid derived from luterian isolated in Example 2 was transferred to a test tube and 400 μl of extraction buffer (200 mM Tris-HCl, pH 8.0; 200 mM NaCl; 25 mM EDTA; 0.5% SDS) and Proteinase K (20 mg / , Mixed well in a buffer solution with a glass rod, and incubated at 37 DEG C for 1 hour.

The same amount of 2X CTAB buffer solution was added to the mixed solution, and the mixture was allowed to stand at 65 ° C for 15 minutes, mixed with chloroform: isoamyl alcohol (24: 1), thoroughly mixed and then centrifuged at 12,000 rpm. The supernatant was transferred to a new tube, 0.7 volumes of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes and centrifuged at 12,000 rpm for 5 minutes to precipitate DNA. The DNA precipitate was washed with 70% ethanol, vacuum-dried and dissolved in 100 μl of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). 2 μl of 10 mg / ml RNase was added and the RNA was removed by treatment at 37 ° C for 30 minutes. The separated DNA was quantified by electrophoresis on agarose gel (1.0%) and comparing with quantified DNA. 50 ng of DNA isolated from the sample, 4 kinds of dNTPs (each 2.5 mM), Taq polymerase (2.5 units, manufactured by Promega), 20 pmol of primer and buffer (10 mM Tris- HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin) and analyzed by GeneAmp PCR system 9700 (Applied Biosystem).

DNA denaturation, annealing with a primer, and DNA polymerisation were repeated 35 times in total. After the reaction, the PCR amplified product was electrophoresed on 2% agarose gel, stained with ethidium bromide, and detected by irradiating various DNA bands with UV. Subsequently, mutant codons frequently present in patients with certain diseases were identified.

Example 4: Isolation of Plant-derived Lterion

100 g of medicinal plant lacquer (Rhus verniciflua stokes) is cut to 20 to 30 times the size of a 2-3 liter container size and placed in a container. 500 to 800 g (preferably 6 to 600 g ) Was put into a container, and then bathing was conducted at 80 캜 or lower. Bubbling with oxygen for 20 to 30 minutes was performed every 3 hours for about 8 hours, so that the plant luteolin was not entangled. The vaporized steam, which is steamed at the time of extraction after the bubbling, is collected in a flask. The condensed water obtained by cooling the condensed water was irradiated with IR light (wavelength: 3 to 1000 탆 or more, preferably 200 to 600 탆) for 1-2 hours to prevent mutation of ruterion. Thereafter, the condensate was filtered using a filter having a diameter of 0.8 μm or more, and only the condensate having passed through the filter was centrifuged repeatedly at 1200 to 5000 rpm for 5 to 10 minutes. The supernatant obtained through centrifugation is irradiated with an IR light to separate mature luteolin particles using a pipette, and the separated luteolin is passed through a filter having pores with a diameter of 50 nm Only the unfiltered portion was washed with PBS to obtain ruterion derived from lacquer tree.

By this process, luterion having a long diameter of 50-800 nm can be obtained, which can be observed through a dark-field microscope or a confocal microscope. The obtained lterion was classified into 50-200 nm (generator) / 200-400 nm (mature stage) / 400-600 nm (splitter) / 600-800 nm (hyperfractionation) depending on the size.

In the same manner, lterion was obtained from the medicinal plants described in Tables 1 to 4.

Example 5: Culture of lterion

(1) PBS was added to ruterion having a size of about 50 to 200 nm in the ruterion obtained in Example 4, irradiated with IR light, and cultured at 18 to 30 ° C for about 3 hours. Immediately after irradiation of the IR light, the size of the lterion was observed with a microscope at intervals of about 1 hour. After about 1 to 6 hours, it was confirmed that the lterione having a size of about 200 nm before cultivation was grown to about 500 nm. Thus, it can be seen that when the plant-derived rterion is added with water and cultured under irradiation with IR light at 18 to 30 ° C, its size can be grown up to about 500 nm.

(2) PBS was added to ruterion of about 400 to 800 nm in size obtained in Example 4, and the mixture was irradiated with IR light, followed by incubation at 18 to 30 ° C for about 3 hours. The size and condition of ruterion were confirmed by a microscope at about 1 hour from immediately after irradiation of the IR light. After about 1 to 6 hours, it was confirmed that the ruterion having a size of about 400 to 800 nm before cultivation was not grown but fissioned.

Example 6: Isolation of nucleic acid (RNA / DNA) derived from ruterion

Using the same method as in Example 2, RNA and DNA were isolated from the lterion isolated in Example 5.

Example 7 Sequence Analysis of Lterion

A primer was prepared in the same manner as in Example 3, and sequenced using PCR. DNA denaturation, annealing with a primer, and DNA polymerization were repeated. The PCR amplified product was electrophoresed and then stained with thiodium bromide to detect various DNA bands Respectively. Thereafter, a lterion containing a complementary codon for the mutated codon of luterian analyzed in Example 3 was selected through a hybridization reaction.

Example 8: Efficacy of therapeutic agents

Based on the sequence of the various medicinal plant-derived lteriones analyzed in Example 7, the medicinal plant lterion having a codon complementary to the various patient-derived lterials analyzed in Example 3 was selected as the customized treatment for the patient.

The medicinal plant lterion selected in Example 7 was treated with the patient-derived mutant lterial of Example 3 and observed under a microscope. As a result, it was confirmed that the mutant lterial was not observed and the lterial was changed into a size of 20 to 200 nm (long diameter 200 nm) while maintaining the circular / elliptical shape, and recovered to a normal state. In addition, it was confirmed that the mobility of ruterian in cancer patients recovered to normal (nanotracking speed of 12 nm / sec or more).

It was thus found that the medicinal plant lterion of Example 7 can inhibit the expression of the mutation codon derived from the patient-derived mutant lterial of Example 3.

Having thus described the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the invention will be defined by the appended claims and their equivalents.

Claims (5)

(a) deriving at least one mutation codon containing a mutation from a nucleic acid sequence of a patient-derived luterial; And
(b) a food or medicinal plant-derived luterion containing (i) a complementary codon capable of RNAiing the derived at least one mutation codon, or (ii) an antisense < / RTI > selecting a miRNA, siRNA or platemer.
The method according to claim 1,
Wherein the mutation comprises single or multiple SNPs derived from a nucleic acid sequence of a patient-derived lterial compared to a nucleic acid sequence of a normal luteal origin.
The method according to claim 1,
Wherein said step (b) comprises the step of selecting a food or medicinal plant-derived lterion containing a complementary codon capable of RNAi the 2-20 mutation-containing codons.
The method according to claim 1,
Wherein the complementary codon is derived from sequence analysis of food or medicinal plant-derived lterion.
The method according to claim 1,
Wherein said antisense miRNA, siRNA or aptamer has a sequence length of 9-200 nts.

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