WO2022050476A1 - Composition for treating human immunodeficiency virus infection or infectious diseases - Google Patents

Composition for treating human immunodeficiency virus infection or infectious diseases Download PDF

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WO2022050476A1
WO2022050476A1 PCT/KR2020/012897 KR2020012897W WO2022050476A1 WO 2022050476 A1 WO2022050476 A1 WO 2022050476A1 KR 2020012897 W KR2020012897 W KR 2020012897W WO 2022050476 A1 WO2022050476 A1 WO 2022050476A1
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cell
vector
immunodeficiency virus
human immunodeficiency
present
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PCT/KR2020/012897
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French (fr)
Korean (ko)
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이형우
이상욱
브라이언윌슨
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이형우
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Priority claimed from KR1020200122316A external-priority patent/KR20220031476A/en
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Publication of WO2022050476A1 publication Critical patent/WO2022050476A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to a composition capable of treating Human Immunodeficiency Virus (HIV) infection or infectious disease.
  • HIV Human Immunodeficiency Virus
  • HIV Human immunodeficiency virus type 1
  • UAIDS Human immunodeficiency virus type 1
  • 6,800 new people are infected with the human immunodeficiency virus every day, and 5,700 die from acquired immunodeficiency syndrome (AIDS) and related diseases.
  • NRTI nucleoside reverse transcriptase inhibitor
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • proteolysis There are protease inhibitors (PI), fusion inhibitors (Fusion I), CCR5 antagonists and integrase inhibitors.
  • PI protease inhibitors
  • Fusion I fusion inhibitors
  • CCR5 antagonists CCR5 antagonists
  • integrase inhibitors Currently, there are two types of NRTIs and one type of PI or NNRTI.
  • Combination therapy with HARRT (highly active anti-retroviral therapy) is widely used as standard therapy for the initial treatment of infected people who have no treatment experience, and it significantly reduces the mortality rate from AIDS by suppressing the disease progression and prolonging the life of HIV/AIDS-infected people. has been reduced
  • One object of the present invention is to provide a pharmaceutical composition for preventing or treating Human Immunodeficiency Virus (HIV) infection or infectious disease, and a method of administering the same.
  • HIV Human Immunodeficiency Virus
  • Another object of the present invention is to provide a cell therapeutic for preventing or treating human immunodeficiency virus (HIV) infection or infectious disease.
  • HIV human immunodeficiency virus
  • Another object of the present invention is to provide a cell therapeutic agent for preventing or treating human immunodeficiency virus (HIV) infection or infectious disease, a method for administering the same, and a method for preparing the same.
  • HIV human immunodeficiency virus
  • the human immunodeficiency virus ( It relates to a pharmaceutical composition for the treatment of Human Immunodeficiency Virus (HIV) infection or infectious disease.
  • HIV Human Immunodeficiency Virus
  • the "Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin (DC-sign)" is the differentiation cluster 209 Also called (Cluster of Differentiation 209; CD209), it is a C-type lectin receptor present on the surface of macrophages and dendritic cells.
  • DC-SIGN of macrophages recognizes high-mannose type N-glycan, a type of pathogen-associated molecular pattern (PAMP) commonly found in viruses, bacteria and fungi, and has high affinity. Binds to activate phagocytosis.
  • DC-SIGN in dendritic cells mediates rolling interactions with blood endothelium, activation of CD4+ T cells, and recognition of haptens.
  • DC-SIGN in dendritic cells binds to human immunodeficiency virus (HIV) and plays a role in delivering human immunodeficiency virus (HIV) to T cells expressing CD4 and HIV-1 co-receptors [ Geijtenbeek TB, van Kooyk Y.
  • DC-SIGN a novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol. 2003;276:31-54. doi: 10.1007/978-3-662-06508-2_2. PMID: 12797442.].
  • the amino acid sequence of the DC-sign protein may be represented by SEQ ID NO: 1, and the sequence of the gene encoding it may be represented by SEQ ID NO: 2, but is not limited thereto.
  • the vector may further include a gene encoding a cluster of differentiation 4 (CD4).
  • CD4 cluster of differentiation 4
  • composition of the present invention may further include a vector comprising a gene encoding CD4.
  • CD4 Cluster of Differentiation 4
  • TCR T cell receptor
  • the amino acid sequence of the CD4 protein may be represented by SEQ ID NO: 3, and the sequence of the gene encoding it may be represented by SEQ ID NO: 4, but is not limited thereto.
  • the vector may further include a beta globin promoter, and preferably, the beta globin promoter is further added in the 5' direction of the gene encoding the DC-sign or the CD4 in the vector.
  • the beta globin promoter is further added in the 5' direction of the gene encoding the DC-sign or the CD4 in the vector.
  • Beta globin is a subunit of the hemoglobin protein located in red blood cells. Beta globin generated from the hemoglobin beta (HBB) gene and the hemoglobin alpha (HBA) gene. Alpha globin is each attached to an iron-containing molecule called heme to form hemoglobin.
  • HBB hemoglobin beta
  • HBA hemoglobin alpha
  • the "beta globin promoter” is a specific region of DNA that regulates the transcription of the hemoglobin beta (HBB) gene, and the beta globin promoter sequence may be one represented by SEQ ID NO: 5 However, any promoter sequence that controls the expression of the beta globin gene may be included without limitation.
  • the "vector” is a means capable of delivering a foreign gene to a cell and expressing it, and the vector of the present invention includes plasmids, cosmids, artificial chromosomes, and liposomes. It may be a non-viral vector such as (Liposomes), or a viral vector such as a retrovirus, an adenovirus, or an adeno-associated virus (AAV), preferably a lentivirus ( Lentivirus).
  • a non-viral vector such as (Liposomes)
  • a viral vector such as a retrovirus, an adenovirus, or an adeno-associated virus (AAV), preferably a lentivirus ( Lentivirus).
  • the "plasmids" are episomal DNA molecules separated from chromosomes and capable of independently proliferating by possessing their own origin of replication.
  • the plasmid can function as a vector by being recombined by a restriction enzyme and then transferred to a host cell.
  • the “cosmids” are plasmids using cos sites, which are the cohesive ends of pie phages, and are mainly used to make gene libraries due to the large size of insertable genes.
  • the "artificial chromosomes” are chromosomes whose structure has been artificially changed for use as a vector, such as bacterial artificial chromosomes, yeast artificial chromosomes, and human artificial chromosomes.
  • the liposomes are artificially made vesicular structures composed of one or more lipid bilayers. It is a drug carrier system that delivers The efficacy of the liposome depends on its ability to deliver and penetrate the target according to the properties of the membrane and components.
  • the "retrovirus” refers to a virus having a single-stranded positive-sense RNA as a genome that requires a DNA intermediate through reverse transcription, and a retroviral vector is a host cell. It is widely used in gene therapy because the viral vector remains stable even after being inserted into the chromosome and dividing cells.
  • the “Lentivirus” is a kind of retrovirus, and is a virus endogenous to the host (endogenous retrovirus; ERV).
  • the virion particles are slightly polymorphic, spherical, 80-100 nm in diameter, the nucleocapsids (core) are isometric, and the nucleotides are concentric rod-shaped or cone-shaped.
  • the "adenovirus (Adenovirus)" is a virus having about 36 kb DNA, and has 50 or more genes, so a vector can be generated by substituting several viral genes with genes to be expressed.
  • the "Adenovirus-associated virus (AAV)” is a satellite virus that has a very small DNA genome and requires an adenovirus. When used as a vector, it is inserted into a specific region of the human chromosome. Causes latent infection.
  • a method of gene gun, microinjection, electroporation, or lipofection may be used.
  • the "gene gun (particle bombardment gun)” is a method of introducing a biological material into living cells by accelerating a genetic material of an appropriate size at an appropriate speed.
  • the "microinjection” is referred to as a microinjection or microinjection, and is a method of injecting a microscopic amount of a genetic material, a protein, or a high molecular material such as a liposome into a cell or nucleus.
  • the "electroporation” is a method of introducing DNA into cells by suspending cells in a DNA solution and passing a pulse of DC high voltage.
  • the "Lipofection” is also called liposome transfection, a technology used to inject genetic material into cells through liposomes, and treatment of cells with a slight heat shock to increase efficiency can
  • a tag sequence may be inserted into the vector and fused.
  • the tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
  • the vector including the gene encoding the DC-sign or the vector including the gene encoding the CD4 may be transfected into a desired host cell.
  • the host cell is preferably an erythroid progenitor cell, because it cannot develop into cancer because it differentiates only with red blood cells, and the red blood cells are not destroyed and remain in the spleen within 4 months in the body.
  • the "progenitor cell” is an undifferentiated cell having self-renewal and differentiation potency, but a cell whose type of finally differentiated cell has already been determined.
  • the progenitor cells have a predetermined pathway for differentiation, but generally do not express markers of mature fully differentiated cells or do not function as mature fully differentiated cells.
  • red blood cell progenitor cells are used.
  • the "erythroid progenitor cell (erythroid precursor cell)" or “erythropoietic hematopoietic cell” are cells capable of differentiating into red blood cells, and erythrocyte differentiation (erythropoiesis) from which mature red blood cells (erythrocytes) are generated ( a) differentiating from a unipotent stem cell (hemocytoblast) into a proerythroblast; (b) differentiating from preblast cells into basophilic erythroblasts; (c) differentiating from basophilic erythroblasts into polychromatophilic erythroblasts; (d) differentiating from polybasic erythroblasts into orthochomatic erythroblasts; (e) undergoes a step of differentiation from seminal erythrocytes to reticulocytes.
  • erythrocyte differentiation erythropoiesis from which mature red blood cells (erythrocytes) are generated ( a) differentiating from a unipotent stem cell (hem
  • the red blood cell progenitor cells may be any one or more selected from the group consisting of proerythroblasts, erythroblasts, and reticulocytes, but is not limited thereto. All cells involved in the formation process.
  • the "proerythroblast (pronormoblast)" is the earliest stage cell among red blood cell progenitors, and histologically, it is difficult to distinguish it from lymphoblasts, myeloblasts, single blasts and megakaryocytes, but basophils in the cytoplasm are visible cells.
  • the "erythroblast (hematoblast)" is an immature nucleated red blood cell derived from the mesoderm, which in the future becomes red blood cells including hemoglobin. It is produced mainly in the bone marrow.
  • the erythroblasts may be any one or more selected from the group consisting of basophilic erythroblasts, polychromatic erythroblasts, and orthochromatic erythroblasts, but is not limited thereto. .
  • the "reticulocyte” is an immature red blood cell, and is a cell in which a network form of ribosomal RNA is observed when pneumethylene blue or Romanowsky stain is applied.
  • the vector when the vector is administered into the body, the vector is transfected into the red blood cell progenitor cells, and the transfected red blood cell progenitor cells can be differentiated into red blood cells.
  • the human immunodeficiency virus invading the body binds to the DC-sign and CD4 proteins expressed by the vector in the red blood cells, and the red blood cells circulate in the body and are destroyed in the spleen. Infectious diseases can be prevented, ameliorated or treated.
  • the “Human Immunodeficiency Virus (HIV)” is a kind of lentivirus that destroys the human immune system, and threatens life by destroying the human immune system so that it can lead to death due to opportunistic infection. It is a pathogen that causes Acquired Immunodeficiency Syndrome (AIDS). Human immunodeficiency virus is transmitted through body fluids such as blood transfusion, semen, vaginal fluid, cooper's fluid, and breast milk.
  • body fluids such as blood transfusion, semen, vaginal fluid, cooper's fluid, and breast milk.
  • the human immunodeficiency virus may be type 1 human immunodeficiency virus (HIV-1) or type 2 human immunodeficiency virus (HIV-2), but preferably type 1 human immunodeficiency virus ( HIV-1).
  • HIV-1 type 1 human immunodeficiency virus
  • HIV-2 type 2 human immunodeficiency virus
  • the human immunodeficiency virus may be resistant to existing anti-lentiviral agents or antiretroviral agents.
  • the anti-lentiviral agent is a nucleic acid-based reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), membrane fusion Inhibitors (fusion inhibitors; Fusion I), CCR5 antagonists (CCR5 antagonist), and may be any one or more selected from integrase inhibitors (integrase inhibitor), but is not limited thereto.
  • the human immunodeficiency virus infection may be a latent infection of the human immunodeficiency virus.
  • the "latent infection” or “latent period” refers to a disease stage in which the number of viruses in the blood decreases and clinical symptoms do not appear due to the immune action after infection with the human immunodeficiency virus.
  • the latency period of infection varies greatly from individual to individual, from 2 weeks to 20 years, and can still be transmitted to others.
  • the latent infection the number of CD4+-bearing T cells starts to decrease gradually, and the decrease is caused by viral necrosis of infected cells, increased probability of apoptosis (apoptosis) of infected T cells, or CD4+ infected CD8 cytotoxic lymphocytes. It may be based on a mechanism that recognizes and destroys T cells.
  • the human immunodeficiency virus-infected disease may be acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • AIDS syndrome or “acquired immunodeficiency syndrome” refers to a condition in which the number of CD4+ T cells has fallen below a lethal level, and cell-mediated immunity is lost and gradually exposed to opportunistic infections. is a disease
  • the progression of immunodeficiency virus infection to the acquired immunodeficiency syndrome is influenced by factors such as the virus, host, environment, and treatment. It is known that patients with AIDS usually die within one year if they do not receive a lentiviral drug.
  • prevention may include, without limitation, any action that blocks or suppresses or delays the symptoms of a disease using the pharmaceutical composition of the present invention.
  • treatment may include without limitation any action in which symptoms of a disease are improved or beneficial by using the pharmaceutical composition of the present invention.
  • the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans.
  • the pharmaceutical composition of the present invention is not limited thereto, but each can be formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, fragrances, etc., in the case of oral administration, and in the case of injections, buffers, preservatives, pain relief A topical agent, solubilizer, isotonic agent, stabilizer, etc.
  • the dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier as described above.
  • it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. there is.
  • it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.
  • the route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal, but oral or parenteral administration is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.
  • the pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated.
  • the dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.
  • the pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
  • HIV Human Immunodeficiency Virus
  • the "individual” refers to an individual in need of prevention or treatment of Human Immunodeficiency Virus (HIV) infection or infectious disease, and may include both mammals and non-mammals.
  • mammals include humans, non-human primates such as chimpanzees, other apes or monkey species; livestock animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs or cats; laboratory animals such as rodents such as rats, mice or guinea pigs, but are not limited thereto.
  • examples of the non-mammal in the present invention may include, but are not limited to, birds or fish.
  • the "administration" of the present invention means the process of introducing the active ingredient of the present invention to an individual by any suitable method, and the administration method in the treatment method of the present invention is through various routes such as oral or parenteral. may be administered.
  • a specific pharmaceutically effective amount for a target subject includes the type and extent of the reaction to be achieved, the specific composition comprising the active ingredient, including whether other agents are used in some cases, the age of the patient, Body weight, general health status, sex and diet, administration time, administration route and secretion rate of a composition containing the active ingredient, treatment period, various factors including drugs used or used together with a specific composition and well-known in the pharmaceutical field It is preferable to apply differently depending on similar factors.
  • DC-sign In the present invention, the description of DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus, its infectious disease, prevention and treatment overlaps with those described above to avoid excessive complexity of the specification. In order to avoid it, detailed description thereof will be omitted below.
  • a host cell transfected with a gene encoding DC-sign (Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin) as an active ingredient, human immunity It relates to a cell therapeutic agent for preventing or treating deficiency virus (HIV) infection or infectious disease.
  • DC-sign Densitive Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin
  • the host cell may be one further transfected with a gene encoding CD4 (Cluster of Differentiation 4).
  • the transfection may be performed using a vector including a gene encoding the DC-sign or the CD4.
  • the vector may further include a beta globin promoter, and preferably, the beta globin promoter is further added in the 5' direction of the gene encoding the DC-sign or the CD4 in the vector.
  • the expression of the DC-sign or the CD4 can be further promoted when the vector is transfected into a host cell.
  • the vector is a non-viral vector such as plasmids, cosmids, artificial chromosomes, and liposomes, or retrovirus, adenovirus, adeno- It may be a viral vector such as an associated virus (AAV), preferably a lentivirus.
  • AAV associated virus
  • a method of gene gun, microinjection, electroporation, or lipofection may be used.
  • a tag sequence may be inserted into the vector and fused.
  • the tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
  • the host cell is preferably an erythroid progenitor cell.
  • the red blood cell progenitor cells may be any one or more selected from the group consisting of proerythroblasts, erythroblasts and reticulocytes, but is not limited thereto, and red blood cells other than mature red blood cells. All cells involved in the formation process.
  • the transfected cells may be proliferated and differentiated into red blood cells, wherein the differentiation may be performed in vitro or in vivo, but preferably in vitro.
  • the human immunodeficiency virus may be type 1 human immunodeficiency virus (HIV-1) or type 2 human immunodeficiency virus (HIV-2), but preferably type 1 human immunodeficiency virus ( HIV-1).
  • HIV-1 type 1 human immunodeficiency virus
  • HIV-2 type 2 human immunodeficiency virus
  • the human immunodeficiency virus may be resistant to existing anti-lentiviral agents or antiretroviral agents.
  • the anti-lentiviral agent is a nucleic acid-based reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), membrane fusion Inhibitors (fusion inhibitors; Fusion I), CCR5 antagonists (CCR5 antagonist), and may be any one or more selected from integrase inhibitors (integrase inhibitor), but is not limited thereto.
  • the human immunodeficiency virus infection may be a latent infection of the human immunodeficiency virus.
  • the human immunodeficiency virus infection disease may be acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • the “cell therapeutic agent” refers to the proliferation or selection of living autologous, allogenic, and xenogenic cells in vitro or other methods to restore the functions of cells and tissues. It refers to medicines used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing the Cell therapy has been administered as a drug since 1993 in the United States and 2002 in Korea.
  • the cell therapeutic agent may be for treatment of human immunodeficiency virus infection or infectious disease.
  • the administration route of the cell therapeutic agent of the present invention may be administered through any general route as long as it can reach the target tissue.
  • Parenteral administration for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, may be administered intradermally, but is not limited thereto.
  • the transfected red blood cell progenitor cells can be differentiated into red blood cells.
  • the human immunodeficiency virus invading the body binds to the DC-sign and CD4 proteins expressed by the vector in the red blood cells, and the red blood cells circulate in the body and are destroyed in the spleen. Infectious diseases can be prevented, ameliorated or treated.
  • the cell therapeutic agent of the present invention may be administered by any device that allows it to migrate to a target cell.
  • the cell therapeutic agent of the present invention may be administered in an effective amount for the treatment of the human immunodeficiency virus infection or infectious disease.
  • the therapeutically effective amount refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, physician or other clinician, which an amount that induces amelioration of the symptoms of the disease or disorder being treated.
  • the cell therapeutic agent included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal content of the cell therapeutic can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, and the age, weight, general health status, sex and diet of the patient , administration time, administration route and secretion rate of the composition, treatment period, and drugs used at the same time may be adjusted according to various factors. In consideration of all of the above factors, it may include an amount capable of obtaining the maximum effect with a minimum amount without side effects.
  • DC-sign the description of the DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus and infectious diseases thereof overlaps with those described above, and in order to avoid excessive complexity of the specification, the following descriptions are made. A detailed description is omitted.
  • HIV Human Immunodeficiency Virus
  • the DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus, its infectious disease, prevention, treatment, subject and administration are overlapped with those described above. In order to avoid excessive complexity, the detailed description thereof will be omitted below.
  • transfection comprising the step of transfecting an isolated host cell with a gene encoding DC-sign (Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin) It relates to a method for preparing the infused cells.
  • DC-sign Densiclear Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin
  • the host cell is preferably an erythroid progenitor cell.
  • the red blood cell progenitor cells may be any one or more selected from the group consisting of proerythroblasts, erythroblasts and reticulocytes, but is not limited thereto, and red blood cells other than mature red blood cells. All cells involved in the formation process.
  • the step of transfecting the gene encoding CD4 (Cluster of Differentiation 4) may be further included.
  • the transfection may be performed using a vector including a gene encoding the DC-sign or the CD4.
  • the vector may further include a beta globin promoter, and preferably, the beta globin promoter is further added in the 5' direction of the gene encoding the DC-sign or the CD4 in the vector.
  • the expression of the DC-sign or the CD4 can be further promoted when the vector is transfected into a host cell.
  • the vector is a non-viral vector such as plasmids, cosmids, artificial chromosomes, and liposomes, or retrovirus, adenovirus, adeno- It may be a viral vector such as an associated virus (AAV), preferably a lentivirus.
  • AAV associated virus
  • a method of gene gun, microinjection, electroporation, or lipofection may be used.
  • a tag sequence may be inserted into the vector and fused.
  • the tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
  • the method may further include differentiating the transfected cells into red blood cells.
  • the differentiation may be performed in vitro or in vivo, but preferably in vitro.
  • the cells prepared by the above method can be used as a therapeutic agent for human immunodeficiency virus infection or infectious disease.
  • DC-sign the description of the DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus and infectious diseases thereof overlaps with those described above, and in order to avoid excessive complexity of the specification, the following descriptions are made. A detailed description is omitted.
  • the pharmaceutical composition provided in the present invention When the pharmaceutical composition provided in the present invention is used, it is possible to effectively treat human immunodeficiency virus infection, and there is an advantage in that the possibility of side effects such as cancer is low.
  • FIG. 1 shows a human beta globin promoter according to an embodiment of the present invention in Preparation Example 1 and a restriction enzyme specific to the promoter sequence.
  • FIG. 2 shows the process of constructing a beta globin promoter-based vector (pLenti6.3/beta globin) according to an embodiment of the present invention in Preparation Example 2.
  • FIG. 3 shows the confirmation of the beta globin promoter gene in the vector (pLenti6.3/beta globin) according to an embodiment of the present invention in Preparation Example 2.
  • FIG. 4 shows the process of preparing a vector (pCR8_CD4) into which the CD4 coding gene sequence according to an embodiment of the present invention is inserted in Preparation Example 3;
  • FIG. 5 shows the identification of the CD4 coding gene in the vector (pCR8_CD4) according to an embodiment of the present invention in Preparation Example 3;
  • FIG. 6 shows the process of constructing a vector (pLenti6.3_beta Pro_CD4 CD4) in which the beta globin promoter sequence and the CD4 coding gene sequence downstream of the beta globin promoter sequence according to an embodiment of the present invention were inserted in Preparation Example 3; it has been shown
  • FIG. 7 shows the process of preparing a vector (pCR8_DC-sign) into which a gene sequence encoding a DC-sign according to an embodiment of the present invention is inserted in Preparation Example 4;
  • FIG. 8 is a vector (pLenti6.3/DC-sign) in which the beta globin promoter sequence according to an embodiment of the present invention and the DC-sign coding gene sequence downstream of the beta globin promoter sequence are inserted in Preparation Example 4; It shows the manufacturing process.
  • Figure 9 confirms the gene encoding the beta globin promoter and DC-sign in the vector (pLenti6.3/DC-sign) according to an embodiment of the present invention in Preparation Example 4;
  • FIG. 10 shows CD4 expression of erythroblasts transfected according to an embodiment of the present invention in Experimental Example 1.
  • FIG. 11 shows CD4 expression of erythroblasts transfected according to an embodiment of the present invention in Experimental Example 1.
  • FIG. 12 shows CD4 expression of non-transfected control erythroblasts according to an embodiment of the present invention in Experimental Example 1.
  • FIG. 13 is a view illustrating DC-sign expression in erythroblasts transfected according to an embodiment of the present invention in Experimental Example 1 and a control group.
  • One object of the present invention is to provide a pharmaceutical composition for preventing or treating Human Immunodeficiency Virus (HIV) infection or infectious disease.
  • HIV Human Immunodeficiency Virus
  • a promoter and restriction enzymes specific for the promoter sequence were searched at the human beta-globin locus, and the results are shown in FIG. 1 .
  • the human beta globin promoter specific for erythrocyte progenitor cells and nuclear protein factors, and present on chromosome 11, was identified, and a Cla I restriction enzyme specific for the 5' direction of the promoter sequence. (ATCGAT SEQ ID NO: 6) and a Spe I restriction enzyme specific for the 3' direction (ACTAGT SEQ ID NO: 7) were identified.
  • a beta globin promoter-based vector (pLenti6.3/beta globin) by inserting the PCR-amplified beta globin promoter sequence into a lentiviral vector (pLenti6.3) using the Cla I and Spe I restriction enzymes. ) was produced. Southern blot was performed to confirm that the beta globin promoter sequence was inserted into the vector (pLenti6.3/beta globin), and the results are shown in FIG. 3 .
  • beta globin promoter gene of about 260 bp was inserted into the vector (pLenti6.3/beta globin).
  • a vector (pCR8_CD4) in which the CD4 coding gene sequence was inserted into the pCR8 vector was prepared. Southern blot was performed to confirm that the gene sequence encoding CD4 was inserted into the vector (pCR8_CD4), and the results are shown in FIG. 5 .
  • genes encoding about 2.8 kb of pCR8 vector and 1.3 kb of CD4 were identified in the vector (pCR8_CD4).
  • the vector (pCR8_CD4) into which the CD4 coding gene sequence was inserted was used as an insertion clone (Entry Clone), and the vector (pLenti6.3/beta globin) prepared in Preparation Example 2 (pLenti6.3/beta globin) was used as the destination vector (Destination Vector). As shown in 6, LR recombination reaction was performed.
  • a pLenti6.3_beta Pro_CD4 vector was constructed in which the beta globin promoter sequence and the CD4 coding gene sequence were inserted downstream of the beta globin promoter sequence.
  • a vector (pCR8_DC-sign) in which the DC-sign coding gene sequence was inserted into the pCR8 vector was prepared.
  • the vector (pCR8_DC-sign) into which the pCR8_DC-sign sequence was inserted was used as an insertion clone (Entry Clone), and the vector (pLenti6.3/beta globin) prepared in Preparation Example 2 was used as the destination vector (Destination).
  • Vector the pLenti6.3/DC-sign vector is a vector in which a DC-sign-coding gene sequence is inserted downstream of the beta globin promoter sequence as shown in FIG. 8 by performing an LR recombination reaction. was produced.
  • the gene encoding DC-sign and the human beta globin promoter were identified in the pLenti6.3/DC-sign vector.
  • the pLenti6.3_beta Pro_CD4 vector and pLenti6.3/DC-sign vector prepared in Preparation Examples 3 and 4 were transfected into a 293FT cell line and cultured in a medium. Thereafter, a recombinant lentivirus was prepared by recovering the supernatant of the medium. The recombinant lentivirus prepared as described above was infected with erythroblasts to obtain transfected erythroblasts. Flow cytometry was performed on the 3rd and 4th days after transfection to confirm the expression of CD4 and DC-sign in the transfected erythroblasts.
  • the transfected erythrocytes were reacted with a CD4 monoclonal antibody conjugated with Pacific Blue, washed with FBS, fixed, and analyzed by fluorescence-activated flow cytometry (FACS), as shown in FIGS. 10 and 11, as a control. were placed as non-recombinant lentivirus (lentivirus isotype prepared by pLenti6.3) and unstained erythroblasts. In addition, erythroblasts that were not transfected as a control were analyzed by flow cytometry and are shown in FIG. 12 .
  • FACS fluorescence-activated flow cytometry
  • DC-sign PE ab conjugated with phycoerythrin
  • FACS fluorescence-activated flow cytometry
  • the present invention relates to a composition capable of treating Human Immunodeficiency Virus (HIV) infection or infectious disease.
  • HIV Human Immunodeficiency Virus

Abstract

The present invention relates to: a composition for preventing or treating human immunodeficiency virus (HIV) infection or infectious diseases, comprising as an active ingredient a vector for the transfection of erythroid progenitor cells; and a cell therapeutic agent for preventing or treating human immunodeficiency virus infection or infectious diseases, comprising as an active ingredient cells which have been transfected by means of the vector.

Description

인간 면역결핍 바이러스 감염 또는 감염 질환의 치료용 조성물Composition for the treatment of human immunodeficiency virus infection or infectious disease
본 발명은 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환을 치료할 수 있는 조성물에 관한 것이다.The present invention relates to a composition capable of treating Human Immunodeficiency Virus (HIV) infection or infectious disease.
제1형 인간 면역결핍 바이러스(Human immunodeficiency virus type; HIV)는 지금까지 약 3천 3백만명 이상이 감염된 가장 중요한 질환 중 하나인 후천면역결핍증(AIDS)의 감염인자이다. 2007년도에, 2백 10만명이 AIDS 관련 질환으로 사망하였으며, 2백 50만명의 새로운 감염자가 발생하였다. 미국 HIV/AIDS 프로그램(the Joint United Nations Programme on HIV/AIDS, UNAIDS)의 추정에 따르면, 매일 6800명이 새롭게 인간 면역결핍 바이러스에 감염되며, 5700명이 후천면역결핍증(AIDS) 및 관련 질환으로 사망한다. Human immunodeficiency virus type 1 (HIV) is an infectious agent of AIDS, one of the most important diseases that have infected more than 33 million people so far. In 2007, 2.1 million people died from AIDS-related diseases, and there were 2.5 million new infections. According to estimates by the Joint United Nations Program on HIV/AIDS (UNAIDS), 6,800 new people are infected with the human immunodeficiency virus every day, and 5,700 die from acquired immunodeficiency syndrome (AIDS) and related diseases.
1987년 최초로 HIV/AIDS 감염인 치료를 위한 항렌티바이러스제인 지도부딘(Zidovudine, AZT)이 사용된 이래, 지금까지 약 40개의 단일약제 또는 복합약제가 미국 식품의약품안전청의 허가 후 상용화되어 임상치료에 사용되고 있다. 세계적으로 사용되고 있는 항렌티바이러스제로는 6가지 계열의 약제, 즉 핵산계열 역전사효소저해제(nucleoside reverse transcriptase inhibitor; NRTI), 비핵산 계열의 역전사효소저해제(non-nucleoside reverse transcriptase inhibitor; NNRTI), 단백질분해 효소저해제(protease inhibitor; PI), 막융합저해제(fusion inhibitor; Fusion I), CCR5 길항제(CCR5 antagonist) 및 통합효소 저해제(integrase inhibitor) 등이 있으며, 현재 2 종류의 NRTIs와 1 종류의 PI 또는 NNRTI와의 병합요법이 치료경험이 없는 감염인에 대한 초기 치료에서 HARRT(highly active anti-retroviral therapy) 표준요법으로 널리 사용되어, HIV/AIDS 감염인의 질병 진전을 억제하고 삶을 연장시킴으로써 에이즈로 인한 사망률을 현저히 감소시켜왔다.Since Zidovudine (AZT), an anti-lentiviral drug, was first used for the treatment of HIV/AIDS infections in 1987, about 40 single drugs or combination drugs have been commercialized and used for clinical treatment after approval from the US Food and Drug Administration. . There are six classes of anti-lentiviral agents used worldwide, namely, nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and proteolysis. There are protease inhibitors (PI), fusion inhibitors (Fusion I), CCR5 antagonists and integrase inhibitors. Currently, there are two types of NRTIs and one type of PI or NNRTI. Combination therapy with HARRT (highly active anti-retroviral therapy) is widely used as standard therapy for the initial treatment of infected people who have no treatment experience, and it significantly reduces the mortality rate from AIDS by suppressing the disease progression and prolonging the life of HIV/AIDS-infected people. has been reduced
그러나 지도부딘 도입 6년 후인 1993년에 이 약제에 대한 내성이 확인된 이후, 항렌티바이러스제 치료경험이 있는 사람에게서 약제내성에 관한 많은 보고가 있었으며, 특히 약제경험이 전혀 없는 일차 감염인에서의 내성 출현, 교차 내성 및 여러 약제 종류에 내성을 나타냄으로써 환자 치료에 어려움을 주고 있다. However, since resistance to this drug was confirmed in 1993, six years after the introduction of zidovudine, there have been many reports of drug resistance in people who have been treated with anti-lentiviral drugs, especially resistance appearance in people with no drug experience, Cross-resistance and resistance to multiple drug types make it difficult to treat patients.
이러한 이유로, 인간 면역결핍 바이러스에 대한 약물요법이 아닌 치료제의 개발이 진행되고 있다. 미국 오레곤 건강과학대 연구팀은 거대세포바이러스(Cytomegalovirus; CMV)를 이용한 백신을 개발하였고, 템플 대학 카멜 칼릴리(Kamel Khalili) 교수는 크리스퍼(CRISPR-Cas9)를 이용한 치료제를 개발하였다. 또한, CCR5가 발현되지 않는 암환자의 골수세포를 이식받는 방법이 시도되었으나, 골수세포 기증자를 찾기 어렵고, 암이 발생할 가능성이 커서 모두 완벽한 방법은 아니다.For this reason, development of a therapeutic agent rather than a drug therapy for human immunodeficiency virus is in progress. A research team at Oregon Health Sciences University developed a vaccine using Cytomegalovirus (CMV), and Professor Kamel Khalili of Temple University developed a treatment using CRISPR-Cas9. In addition, a method of receiving bone marrow cells from a cancer patient in which CCR5 is not expressed has been attempted, but it is difficult to find a bone marrow cell donor and there is a high possibility of cancer, so not all methods are perfect.
따라서, 약제 내성 환자에 적용 가능하고, 암으로 발전 가능성이 낮은 새로운 치료 방법에 대한 요구가 증대되고 있다.Therefore, there is an increasing demand for a new treatment method applicable to drug-resistant patients and having a low possibility of developing cancer.
본 발명의 일 목적은 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료용 약학적 조성물 및 이를 투여하는 방법을 제공하고자 한다. One object of the present invention is to provide a pharmaceutical composition for preventing or treating Human Immunodeficiency Virus (HIV) infection or infectious disease, and a method of administering the same.
본 발명의 다른 목적은 인간 면역결핍 바이러스(HIV) 감염 또는 감염 질환의 예방 또는 치료용 세포 치료제를 제공하고자 한다.Another object of the present invention is to provide a cell therapeutic for preventing or treating human immunodeficiency virus (HIV) infection or infectious disease.
본 발명의 또 다른 목적은 인간 면역결핍 바이러스(HIV) 감염 또는 감염 질환의 예방 또는 치료용 세포 치료제, 이를 투여하는 방법 및 제조하는 방법을 제공하고자 한다.Another object of the present invention is to provide a cell therapeutic agent for preventing or treating human immunodeficiency virus (HIV) infection or infectious disease, a method for administering the same, and a method for preparing the same.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당 업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.
이하, 본원에 기재된 다양한 구현 예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세사항, 예컨대, 특이적 형태, 조성물 및 공정 등이 기재되어 있다. 그러나, 특정의 구현 예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구현 예" 또는 "구현 예"에 대한 본 명세서 전체를 통한 참조는 구현 예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구현 예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현된 "한 가지 구현 예에서" 또는 "구현 예"의 상황은 반드시 본 발명의 동일한 구현 예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구현 예에서 어떠한 적합한 방법으로 조합될 수 있다. 본 발명 내 특별한 정의가 없으면 본 명세서에 사용된 모든 과학적 및 기술적인 용어는 본 발명이 속하는 기술분야에서 당 업자에 의하여 통상적으로 이해되는 것과 동일한 의미를 가진다.Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, phrases of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily represent the same implementation of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable way in one or more embodiments. Unless specifically defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
본 발명의 일 구현 예에 따르면, DC-sign(Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin)을 코딩하는 유전자를 포함하는 벡터를 유효 성분으로 포함하는, 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 치료용 약학적 조성물에 관한 것이다.According to one embodiment of the present invention, the human immunodeficiency virus ( It relates to a pharmaceutical composition for the treatment of Human Immunodeficiency Virus (HIV) infection or infectious disease.
본 발명에서, 상기 “수지상 세포 특이적 세포간 접착 분자-3-G 래빙 N 온-인테그린(Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin; DC-sign)”는 분화 클러스터 209(Cluster of Differentiation 209; CD209)라고도 하며, 대식세포와 수지상세포의 표면에 존재하는 C형 렉틴 수용체이다. 대식세포의 DC-SIGN은 바이러스, 박테리아 및 진균에서 흔히 발견되는 병원체 관련 분자 패턴(PAMP)의 한 종류인 고-만노스 타입 N-글리칸(high-mannose type N-glycan)을 인식하고 높은 친화도로 결합하여 식균 작용을 활성화한다. 수지상세포의 DC-SIGN은 혈액내피와의 롤링 상호작용, CD4+ T 세포의 활성화 및 합텐(hapten)의 인식을 매개한다. 특히, 수지상 세포에서 DC-SIGN은 인간 면역결핍 바이러스(HIV)와 결합하여, CD4와 HIV-1 공동 수용체를 발현하는 T 세포에 인간 면역결핍 바이러스(HIV)를 전달하는 역할을 하는 것이 밝혀져 있다[Geijtenbeek TB, van Kooyk Y. DC-SIGN: a novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol. 2003;276:31-54. doi: 10.1007/978-3-662-06508-2_2. PMID: 12797442.].In the present invention, the "Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin (DC-sign)" is the differentiation cluster 209 Also called (Cluster of Differentiation 209; CD209), it is a C-type lectin receptor present on the surface of macrophages and dendritic cells. DC-SIGN of macrophages recognizes high-mannose type N-glycan, a type of pathogen-associated molecular pattern (PAMP) commonly found in viruses, bacteria and fungi, and has high affinity. Binds to activate phagocytosis. DC-SIGN in dendritic cells mediates rolling interactions with blood endothelium, activation of CD4+ T cells, and recognition of haptens. In particular, it has been shown that DC-SIGN in dendritic cells binds to human immunodeficiency virus (HIV) and plays a role in delivering human immunodeficiency virus (HIV) to T cells expressing CD4 and HIV-1 co-receptors [ Geijtenbeek TB, van Kooyk Y. DC-SIGN: a novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol. 2003;276:31-54. doi: 10.1007/978-3-662-06508-2_2. PMID: 12797442.].
본 발명에서, 상기 DC-sign 단백질의 아미노산 서열은 서열번호 1로 표시되는 것일 수 있으며, 이를 코딩하는 유전자의 서열은 서열번호 2로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the amino acid sequence of the DC-sign protein may be represented by SEQ ID NO: 1, and the sequence of the gene encoding it may be represented by SEQ ID NO: 2, but is not limited thereto.
본 발명에서, 상기 벡터는 CD4(Cluster of Differentiation 4)를 코딩하는 유전자를 추가로 더 포함할 수 있다. In the present invention, the vector may further include a gene encoding a cluster of differentiation 4 (CD4).
또한, 본 발명의 약학적 조성물은 CD4를 코딩하는 유전자를 포함하는 벡터를 더 포함할 수 있다.In addition, the pharmaceutical composition of the present invention may further include a vector comprising a gene encoding CD4.
본 발명에서, 상기 “분화 클러스터 4(Cluster of Differentiation 4; CD4)”는 면역 글로불린 슈퍼 패밀리의 구성원으로, T 세포 수용체(T cell receptor; TCR)의 공동 수용체이며, 항원제시세포와의 통신을 돕는 당단백질이다.In the present invention, the "Cluster of Differentiation 4 (CD4)" is a member of the immunoglobulin superfamily, is a T cell receptor (TCR) co-receptor, and helps to communicate with antigen-presenting cells. is a glycoprotein.
본 발명에서, 상기 CD4 단백질의 아미노산 서열은 서열번호 3으로 표시되는 것일 수 있으며, 이를 코딩하는 유전자의 서열은 서열번호 4로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the amino acid sequence of the CD4 protein may be represented by SEQ ID NO: 3, and the sequence of the gene encoding it may be represented by SEQ ID NO: 4, but is not limited thereto.
본 발명에서, 상기 벡터는 베타 글로빈 프로모터(beta globin promoter)를 더 포함할 수 있고, 바람직하게는 상기 벡터에서 상기 DC-sign 또는 상기 CD4를 코딩하는 유전자의 5' 방향에 상기 베타 글로빈 프로모터를 더 포함함으로써, 상기 벡터가 숙주 세포에 형질주입 시 DC-sign 또는 CD4의 발현을 보다 촉진시킬 수 있다.In the present invention, the vector may further include a beta globin promoter, and preferably, the beta globin promoter is further added in the 5' direction of the gene encoding the DC-sign or the CD4 in the vector. By including the vector, expression of DC-sign or CD4 can be further promoted when the vector is transfected into a host cell.
본 발명에서 상기 “베타 글로빈(beta globin)"은 적혈구 안에 위치한 헤모글로빈 단백질의 서브유닛이다. 헤모글로빈 베타(Hemoglobin Beta; HBB) 유전자로부터 생성된 베타 글로빈과 헤모글로빈 알파(Hemoglobin Alpha; HBA) 유전자로부터 생성된 알파 글로빈은 각각 헴(heme)이라 불리는 철-함유 분자에 부착되어 헤모글로빈을 형성한다.In the present invention, the "beta globin" is a subunit of the hemoglobin protein located in red blood cells. Beta globin generated from the hemoglobin beta (HBB) gene and the hemoglobin alpha (HBA) gene. Alpha globin is each attached to an iron-containing molecule called heme to form hemoglobin.
본 발명에서 상기 “베타 글로빈 프로모터(beta globin promoter)"는 상기 헤모글로빈 베타(Hemoglobin Beta; HBB) 유전자의 전사를 조절하는 DNA의 특정 부위이며, 상기 베타 글로빈 프로모터 서열은 서열번호 5로 표시되는 것일 수 있으나, 상기 베타 글로빈 유전자의 발현을 조절하는 프로모터 서열이라면 제한없이 포함될 수 있다.In the present invention, the "beta globin promoter" is a specific region of DNA that regulates the transcription of the hemoglobin beta (HBB) gene, and the beta globin promoter sequence may be one represented by SEQ ID NO: 5 However, any promoter sequence that controls the expression of the beta globin gene may be included without limitation.
본 발명에서 상기 "벡터(vector)"는 외래 유전자를 세포로 전달하여 발현할 수 있는 수단으로, 본 발명의 상기 벡터는 플라스미드(plasmids), 코스미드(cosmids), 인공 염색체(artificial chromosomes), 리포솜(Liposomes)과 같은 비바이러스성 벡터이거나, 레트로바이러스(retrovirus), 아데노바이러스(Adenovirus), 아데노-관련 바이러스(Adenovirus-associated virus; AAV)와 같은 바이러스성 벡터일 수 있으며, 바람직하게는 렌티바이러스(Lentivirus)일 수 있다.In the present invention, the "vector" is a means capable of delivering a foreign gene to a cell and expressing it, and the vector of the present invention includes plasmids, cosmids, artificial chromosomes, and liposomes. It may be a non-viral vector such as (Liposomes), or a viral vector such as a retrovirus, an adenovirus, or an adeno-associated virus (AAV), preferably a lentivirus ( Lentivirus).
본 발명에서 상기 “플라스미드(plasmids)”는 염색체와 분리되어 있고 자신의 복제기점을 소유하여 독립적으로 증식 가능한 에피솜 DNA 분자이다. 상기 플라스미드는 제한효소에 의해 재조합된 후 숙주 세포에 전달되어 벡터의 기능을 할 수 있다.In the present invention, the "plasmids" are episomal DNA molecules separated from chromosomes and capable of independently proliferating by possessing their own origin of replication. The plasmid can function as a vector by being recombined by a restriction enzyme and then transferred to a host cell.
본 발명에서 상기 “코스미드(cosmids)”는 파이 파지의 점착성 말단(cohesive ends)인 cos 부위를 이용한 플라스미드로, 삽입할 수 있는 유전자의 크기가 커서 유전자 라이브러리를 만드는데 주로 사용된다.In the present invention, the “cosmids” are plasmids using cos sites, which are the cohesive ends of pie phages, and are mainly used to make gene libraries due to the large size of insertable genes.
본 발명에서 상기 “인공 염색체(artificial chromosomes)”는 벡터로 사용하기 위하여 인위적으로 구조를 변화시킨 염색체로, 박테리아 인공염색체, 효모 인공염색체, 인간 인공염색체 등이 있다.In the present invention, the "artificial chromosomes" are chromosomes whose structure has been artificially changed for use as a vector, such as bacterial artificial chromosomes, yeast artificial chromosomes, and human artificial chromosomes.
본 발명에서 상기 “리포솜(Liposomes)”은 인위적으로 만든 1개 이상의 지질 2중층으로 되어 있는 소낭 구조물로, 세포막의 형태와 유사하고 다양한 물질을 혼입하는 능력 때문에 핵산뿐만 아니라 펩티드, 항체, 앱타머 등을 전달하는 약물 운반체 시스템이다. 상기 리포솜의 효능은 막과 성분의 특성에 따른 표적 전달 및 침투 능력에 달려 있다.In the present invention, the "liposomes" are artificially made vesicular structures composed of one or more lipid bilayers. It is a drug carrier system that delivers The efficacy of the liposome depends on its ability to deliver and penetrate the target according to the properties of the membrane and components.
본 발명에서 상기 “레트로바이러스(retrovirus)”는 역전사(reverse transcription)를 통한 DNA 중간체를 필수로 하는 단일가닥의 양성 센스(positive-sense) RNA를 게놈으로 가지는 바이러스로, 레트로바이러스 벡터는 숙주 세포의 염색체에 삽입되어 세포분열을 하여도 바이러스 벡터가 안정적으로 유지되므로 유전자 치료에 많이 사용된다.In the present invention, the "retrovirus" refers to a virus having a single-stranded positive-sense RNA as a genome that requires a DNA intermediate through reverse transcription, and a retroviral vector is a host cell. It is widely used in gene therapy because the viral vector remains stable even after being inserted into the chromosome and dividing cells.
본 발명에서, 상기 “렌티바이러스(Lentivirus)”는 레트로바이러스의 일종으로, 숙주에 내인성인 바이러스(endogenous retrovirus; ERV)이다. 비리온 입자는 약간 다형성의 직경 80 내지 100nm의 구형이고, 뉴클레오캡시드(코어)는 등척성이며, 뉴클레오티드는 동심 막대 형상 또는 원추 형상이다. In the present invention, the “Lentivirus” is a kind of retrovirus, and is a virus endogenous to the host (endogenous retrovirus; ERV). The virion particles are slightly polymorphic, spherical, 80-100 nm in diameter, the nucleocapsids (core) are isometric, and the nucleotides are concentric rod-shaped or cone-shaped.
본 발명에서 상기 “아데노바이러스(Adenovirus)”는 약 36kb DNA를 가지는 바이러스로, 50가지 이상의 유전자를 가지고 있어 몇 가지의 바이러스 유전자를 발현하려는 유전자로 치환하여 벡터를 생성할 수 있다.In the present invention, the "adenovirus (Adenovirus)" is a virus having about 36 kb DNA, and has 50 or more genes, so a vector can be generated by substituting several viral genes with genes to be expressed.
본 발명에서 상기 “아데노바이러스-관련 바이러스(Adenovirus-associated virus; AAV)”는 DNA 유전체가 매우 작고, 아데노바이러스가 필요한 인공위성바이러스(Satellite virus)로, 벡터로 사용할 경우 사람 염색체의 특정한 부위에 삽입되어 잠복감염(latent infection)을 일으킨다. In the present invention, the "Adenovirus-associated virus (AAV)" is a satellite virus that has a very small DNA genome and requires an adenovirus. When used as a vector, it is inserted into a specific region of the human chromosome. Causes latent infection.
본 발명에서, 상기 벡터를 세포로 주입하기 위하여 유전자총(gene gun), 미세주입법(microinjection), 전기천공법(electroporation) 또는 리포펙션(lipofection)의 방법에 의할 수 있다.In the present invention, in order to inject the vector into the cells, a method of gene gun, microinjection, electroporation, or lipofection may be used.
본 발명에서 상기 “유전자총(gene gun, particle bombardment gun)”은 적당한 크기의 유전물질을 적정 속도로 가속시켜 살아 있는 세포 내로 생물학적인 물질을 도입하는 방법이다.In the present invention, the "gene gun (particle bombardment gun)" is a method of introducing a biological material into living cells by accelerating a genetic material of an appropriate size at an appropriate speed.
본 발명에서 상기 “미세주입법(microinjection)”은 현미주입법 혹은 미량주입법이라고 하며 세포 또는 핵 등에 극미량의 유전물질, 단백질 또는 리포솜 등의 고분자 물질을 주입하는 방법이다.In the present invention, the "microinjection" is referred to as a microinjection or microinjection, and is a method of injecting a microscopic amount of a genetic material, a protein, or a high molecular material such as a liposome into a cell or nucleus.
본 발명에서 상기 “전기천공법(electroporation)”은 세포를 DNA용액에 현탁한 후 직류고전압의 펄스를 통과시켜 세포 내에 DNA를 도입시키는 방법이다.In the present invention, the "electroporation" is a method of introducing DNA into cells by suspending cells in a DNA solution and passing a pulse of DC high voltage.
본 발명에서 상기 “리포펙션(Lipofection)”은 리포솜 형질주입(liposome transfection)이라고도 하며, 리포솜을 통해 세포에 유전자 물질을 주입하는 데 사용되는 기술로, 약간의 열 충격으로 세포를 처리하여 효율을 높일 수 있다.In the present invention, the "Lipofection" is also called liposome transfection, a technology used to inject genetic material into cells through liposomes, and treatment of cells with a slight heat shock to increase efficiency can
또한, 본 발명에서는 상기 벡터에 삽입된 유전자의 정제를 용이하게 하기 위하여 태그 서열을 상기 벡터 상에 삽입하여 융합시킬 수 있다. 상기 태그로는 헥사-히스티딘 태그, 헤마글루티닌 태그, myc 태그 또는 flag 태그를 포함하나 이에 한정되는 것은 아니며 당업자에게 알려진 정제를 용이하게 하는 태그는 모두 본 발명에서 이용 가능하다. In addition, in the present invention, in order to facilitate purification of the gene inserted into the vector, a tag sequence may be inserted into the vector and fused. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
본 발명의 약학적 조성물에서 상기 DC-sign을 코딩하는 유전자를 포함하는 벡터 또는 상기 CD4를 코딩하는 유전자를 포함하는 벡터는 목적하는 숙주 세포에 형질주입될 수 있다. In the pharmaceutical composition of the present invention, the vector including the gene encoding the DC-sign or the vector including the gene encoding the CD4 may be transfected into a desired host cell.
본 발명에서 상기 숙주 세포는 적혈구 전구 세포(erythroid progenitor cell)인 것이 적혈구로만 분화되어 암으로 발전할 수 없고, 상기 적혈구는 체내에서 4개월 내에 비장에서 파괴되어 잔류되지 않으므로, 바람직하다.In the present invention, the host cell is preferably an erythroid progenitor cell, because it cannot develop into cancer because it differentiates only with red blood cells, and the red blood cells are not destroyed and remain in the spleen within 4 months in the body.
본 발명에서 상기 “전구 세포(progenitor cell)”는 자기 복제능 및 분화능(differentiation potency)을 가진 미분화 세포이지만, 최종적으로 분화하는 세포의 종류가 이미 결정되어 있는 세포이다. 상기 전구 세포는 분화 경로가 예정되어 있으나, 일반적으로 성숙한 완전히 분화된 세포의 마커를 발현하지는 않거나, 성숙한 완전히 분화된 세포로서는 기능하지 않는다. 본 발명에서는 적혈구 전구 세포를 사용한다.In the present invention, the "progenitor cell" is an undifferentiated cell having self-renewal and differentiation potency, but a cell whose type of finally differentiated cell has already been determined. The progenitor cells have a predetermined pathway for differentiation, but generally do not express markers of mature fully differentiated cells or do not function as mature fully differentiated cells. In the present invention, red blood cell progenitor cells are used.
본 발명에서 상기 ”적혈구 전구 세포(erythroid progenitor cell, erythroid precursor cell)” 내지 “적혈구계 조혈 세포”는 적혈구로 분화될 수 있는 세포로, 성숙한 적혈구(erythrocyte)가 생성되는 적혈구 분화(erythropoiesis)는 (a) 단일분화성 줄기세포(unipotent stem cell, hemocytoblast)에서 전적아세포(proerythroblast)로 분화하는 단계; (b) 전적아세포에서 호염기성 적아세포(basophilic erythroblast)로 분화하는 단계; (c) 호염기성 적아세포에서 다염기성 적아세포(polychromatophilic erythroblast)로 분화하는 단계; (d) 다염기성 적아세포에서 정염성 적아세포(orthochomatic erythroblast)로 분화하는 단계; (e) 정염성 적아세포에서 망상적혈구(reticulocyte)로 분화하는 단계를 거친다.In the present invention, the "erythroid progenitor cell (erythroid precursor cell)" or "erythropoietic hematopoietic cell" are cells capable of differentiating into red blood cells, and erythrocyte differentiation (erythropoiesis) from which mature red blood cells (erythrocytes) are generated ( a) differentiating from a unipotent stem cell (hemocytoblast) into a proerythroblast; (b) differentiating from preblast cells into basophilic erythroblasts; (c) differentiating from basophilic erythroblasts into polychromatophilic erythroblasts; (d) differentiating from polybasic erythroblasts into orthochomatic erythroblasts; (e) undergoes a step of differentiation from seminal erythrocytes to reticulocytes.
본 발명에서, 상기 적혈구 전구 세포는 전적아세포(proerythroblast), 적아세포(erythroblast) 및 망상적혈구(reticulocyte)로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되지 않고, 성숙이 끝난 적혈구를 제외한 적혈구 형성 과정에 포함된 모든 세포를 의미한다.In the present invention, the red blood cell progenitor cells may be any one or more selected from the group consisting of proerythroblasts, erythroblasts, and reticulocytes, but is not limited thereto. All cells involved in the formation process.
본 발명에서 상기 “전적아세포(proerythroblast, pronormoblast)”는 적혈구 전구 세포 중 가장 초기 단계 세포로, 조직학적으로는 림프 모세포, 골수 모세포, 단일 모세포 및 거핵 모세포들과 구별이 어려우나, 세포질에서 호염기성을 보이는 세포이다.In the present invention, the "proerythroblast (pronormoblast)" is the earliest stage cell among red blood cell progenitors, and histologically, it is difficult to distinguish it from lymphoblasts, myeloblasts, single blasts and megakaryocytes, but basophils in the cytoplasm are visible cells.
본 발명에서 상기 “적아세포(erythroblast, hematoblast)”는 중배엽에서 유래한 미성숙의 유핵적혈구로서 장차 헤모글로빈을 포함하여 적혈구 세포가 되며, 배일 때는 간, 비장(지라), 림프절 등에서 만들어지나, 성체가 되면 골수 내에서 주로 만들어진다. In the present invention, the "erythroblast (hematoblast)" is an immature nucleated red blood cell derived from the mesoderm, which in the future becomes red blood cells including hemoglobin. It is produced mainly in the bone marrow.
본 발명에서, 상기 적아세포는 호염기성 적아세포(basophilic erythroblast), 다염성 적아세포(polychromatic erythroblast) 및 정염성 적아세포(orthochromatic erythroblast)로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되지 않는다.In the present invention, the erythroblasts may be any one or more selected from the group consisting of basophilic erythroblasts, polychromatic erythroblasts, and orthochromatic erythroblasts, but is not limited thereto. .
본 발명에서 상기 “망상적혈구(reticulocyte)”는 미성숙한 적혈구로, 뉴메틸렌 블루나 로마노스키 염색(Romanowsky stain)을 할 경우 리보솜 RNA의 그물 형태가 관찰되는 세포이다.In the present invention, the "reticulocyte" is an immature red blood cell, and is a cell in which a network form of ribosomal RNA is observed when pneumethylene blue or Romanowsky stain is applied.
본 발명의 약학적 조성물에서, 상기 벡터를 체내로 투여하면, 상기 벡터는 상기 적혈구 전구 세포에 형질주입되고, 상기 형질주입된 적혈구 전구 세포는 적혈구로 분화될 수 있다. 이때 체내로 침입한 인간 면역결핍 바이러스가 상기 적혈구 세포 내 벡터에 의해 발현된 DC-sign 및 CD4 단백질에 결합하게 되는데, 상기 적혈구 세포는 체내를 순환하다 비장에서 파괴되므로, 인간 면역결핍 바이러스의 감염 또는 감염 질환이 예방, 개선 또는 치료될 수 있다. In the pharmaceutical composition of the present invention, when the vector is administered into the body, the vector is transfected into the red blood cell progenitor cells, and the transfected red blood cell progenitor cells can be differentiated into red blood cells. At this time, the human immunodeficiency virus invading the body binds to the DC-sign and CD4 proteins expressed by the vector in the red blood cells, and the red blood cells circulate in the body and are destroyed in the spleen. Infectious diseases can be prevented, ameliorated or treated.
본 발명에서 상기 “인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV)”는 인간의 면역체계를 파괴하는 렌티바이러스의 일종으로, 기회감염에 의해 사망에 이를 수 있도록 인간의 면역체계를 무너뜨려 생명을 위협하는 후천면역결핍증후군(Acquired immunodeficiency syndrome; AIDS)을 일으키는 병원체이다. 인간 면역결핍 바이러스는 수혈, 정액, 질액, 쿠퍼액, 모유 등의 체액으로 전염되고, 공기 중에서 자유롭게 떠다니는 바이러스 상태이나 체액속에서는 감염된 백혈구의 형태로 존재한다. In the present invention, the “Human Immunodeficiency Virus (HIV)” is a kind of lentivirus that destroys the human immune system, and threatens life by destroying the human immune system so that it can lead to death due to opportunistic infection. It is a pathogen that causes Acquired Immunodeficiency Syndrome (AIDS). Human immunodeficiency virus is transmitted through body fluids such as blood transfusion, semen, vaginal fluid, cooper's fluid, and breast milk.
본 발명에서, 상기 인간 면역 결핍 바이러스는 제1형 인간 면역 결핍 바이러스(HIV-1) 또는 제2형 인간 면역 결핍 바이러스(HIV-2)일 수 있으나, 바람직하게는 제1형 인간 면역 결핍 바이러스(HIV-1)일 수 있다.In the present invention, the human immunodeficiency virus may be type 1 human immunodeficiency virus (HIV-1) or type 2 human immunodeficiency virus (HIV-2), but preferably type 1 human immunodeficiency virus ( HIV-1).
본 발명에서, 상기 인간 면역결핍 바이러스는 기존 항렌티바이러스제 내지 항레트로바이러스제에 내성을 가진 것일 수 있다. 상기 항렌티바이러스제는 핵산계열 역전사효소저해제(nucleoside reverse transcriptase inhibitor; NRTI), 비핵산 계열의 역전사효소저해제(non-nucleoside reverse transcriptase inhibitor; NNRTI), 단백질분해 효소저해제(protease inhibitor; PI), 막융합저해제(fusion inhibitor; Fusion I), CCR5 길항제(CCR5 antagonist) 및 통합효소 저해제(integrase inhibitor)에서 선택된 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the human immunodeficiency virus may be resistant to existing anti-lentiviral agents or antiretroviral agents. The anti-lentiviral agent is a nucleic acid-based reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), membrane fusion Inhibitors (fusion inhibitors; Fusion I), CCR5 antagonists (CCR5 antagonist), and may be any one or more selected from integrase inhibitors (integrase inhibitor), but is not limited thereto.
본 발명에서, 상기 인간 면역결핍 바이러스 감염은 상기 인간 면역결핍 바이러스의 잠복 감염일 수 있다.In the present invention, the human immunodeficiency virus infection may be a latent infection of the human immunodeficiency virus.
본 발명에서 상기 “잠복 감염” 내지 “잠복기”는 인간 면역결핍 바이러스 감염 후 면역 작용으로 인해 혈중 바이러스 수는 줄어들고 임상 증상이 나타나지 않는 질환 단계이다. 상기 잠복 감염 기간은 2주에서 20년으로 개인별로 매우 다르고 여전히 다른 사람을 전염시킬 수 있다. 상기 잠복 감염에서 CD4+를 가진 T 세포의 개체 수가 점점 감소하기 시작하는데, 감소되는 원인은 감염된 세포의 바이러스성 괴사, 감염된 T세포의 아포토시스(세포자살, apoptosis) 확률 증가 또는 CD8 세포독성 림프구가 감염된 CD4+ T 세포를 인지하여 파괴하는 기작에 의할 수 있다.In the present invention, the "latent infection" or "latent period" refers to a disease stage in which the number of viruses in the blood decreases and clinical symptoms do not appear due to the immune action after infection with the human immunodeficiency virus. The latency period of infection varies greatly from individual to individual, from 2 weeks to 20 years, and can still be transmitted to others. In the latent infection, the number of CD4+-bearing T cells starts to decrease gradually, and the decrease is caused by viral necrosis of infected cells, increased probability of apoptosis (apoptosis) of infected T cells, or CD4+ infected CD8 cytotoxic lymphocytes. It may be based on a mechanism that recognizes and destroys T cells.
본 발명에서 상기 인간 면역결핍 바이러스 감염 질환은 후천면역결핍증후군(Acquired immunodeficiency syndrome; AIDS)일 수 있다. 상기 “후천면역결핍증후군(에이즈, Acquired immunodeficiency syndrome; AIDS)” 내지 “후천성면역결핍증후군”은 CD4+ T 세포의 수가 치명적인 수준 이하로 내려간 결과, 세포매개성 면역이 상실되어 점차 기회감염에 쉽게 노출되는 질환이다. 면역결핍 바이러스 감염이 상기 후천면역결핍증후군으로 진행은 바이러스, 숙주, 환경 및 치료 여부의 요인에 의해 영향을 받는다. 후천면역결핍증후군 환자가 렌티바이러스제를 처방받지 않으면 보통 1년 이내에 사망하는 것으로 알려져 있다.In the present invention, the human immunodeficiency virus-infected disease may be acquired immunodeficiency syndrome (AIDS). The above “acquired immunodeficiency syndrome (AIDS)” or “acquired immunodeficiency syndrome” refers to a condition in which the number of CD4+ T cells has fallen below a lethal level, and cell-mediated immunity is lost and gradually exposed to opportunistic infections. is a disease The progression of immunodeficiency virus infection to the acquired immunodeficiency syndrome is influenced by factors such as the virus, host, environment, and treatment. It is known that patients with AIDS usually die within one year if they do not receive a lentiviral drug.
한편, 본 발명에서, "예방"은 본 발명의 약학적 조성물을 이용하여 질환의 증상을 차단하거나, 그 증상을 억제 또는 지연시키는 모든 행위라면 제한없이 포함할 수 있다. Meanwhile, in the present invention, "prevention" may include, without limitation, any action that blocks or suppresses or delays the symptoms of a disease using the pharmaceutical composition of the present invention.
또한, 본 발명에서, "치료"는 본 발명의 약학적 조성물을 이용하여 질환의 증상이 호전되거나 이롭게 되는 모든 행위라면 제한없이 포함할 수 있다.In addition, in the present invention, "treatment" may include without limitation any action in which symptoms of a disease are improved or beneficial by using the pharmaceutical composition of the present invention.
본 발명에서, 상기 약학적 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학적 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. In the present invention, the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans.
본 발명의 약학적 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 약학적 조성물은 약제적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용되는 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 약학적 조성물의 제형은 상술한 바와 같은 약학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형할 수 있다.The pharmaceutical composition of the present invention is not limited thereto, but each can be formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. can The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, fragrances, etc., in the case of oral administration, and in the case of injections, buffers, preservatives, pain relief A topical agent, solubilizer, isotonic agent, stabilizer, etc. can be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. can be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.
본 발명에 따른 약학적 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥 내, 근육 내, 동맥 내, 골수 내, 경막 내, 심장 내, 경피, 피하, 복강 내, 비강 내, 장관, 국소, 설하 또는 직장이 포함되나, 경구 또는 비경구 투하가 바람직하다. The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal, but oral or parenteral administration is preferred.
본 발명에서, "비경구"는 피하, 피내, 정맥 내, 근육 내, 관절 내, 활액낭 내, 흉골 내, 경막 내, 병소 내 및 두개골 내 주사 또는 주입기술을 포함한다. 본 발명의 약학적 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.As used herein, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.
본 발명의 약학적 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여 시간, 투여 경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학적 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물 형태, 투여 경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50mg/kg 또는 0.001 내지 50mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형될 수 있다.The pharmaceutical composition of the present invention depends on several factors including the activity of the specific compound used, age, weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
본 발명의 다른 구현 예에서는, 본 발명에 따른 상기 조성물을 유효량으로 개체에 투여하는 단계를 포함하는, 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료 방법에 관한 것이다.In another embodiment of the present invention, it relates to a method for preventing or treating Human Immunodeficiency Virus (HIV) infection or infectious disease, comprising administering to a subject an effective amount of the composition according to the present invention.
본 발명에서 상기 “개체”란, 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료가 필요한 개체로서, 포유동물 및 비-포유동물을 모두 포함할 수 있다. 여기서, 상기 포유동물의 예로는 인간, 비-인간 영장류, 예컨대 침팬지, 다른 유인원 또는 원숭이 종; 축산 동물, 예컨대 소, 말, 양, 염소, 돼지; 사육 동물, 예컨대 토끼, 개 또는 고양이; 실험 동물, 예를 들어 설치류, 예컨대 래트, 마우스 또는 기니아 피그 등을 포함할 수 있으나, 이에 제한되는 것은 아니다. 또한, 본 발명에서 상기 비-포유동물의 예로는 조류 또는 어류 등을 포함할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the "individual" refers to an individual in need of prevention or treatment of Human Immunodeficiency Virus (HIV) infection or infectious disease, and may include both mammals and non-mammals. Here, examples of such mammals include humans, non-human primates such as chimpanzees, other apes or monkey species; livestock animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs or cats; laboratory animals such as rodents such as rats, mice or guinea pigs, but are not limited thereto. In addition, examples of the non-mammal in the present invention may include, but are not limited to, birds or fish.
본 발명의 상기 “투여”란, 임의의 적절한 방법으로 개체에게 본 발명의 유효성분을 도입하는 과정을 의미하는 것으로서, 본 발명의 상기 치료 방법에서 투여 방법은 경구 또는 비경구 등의 다양한 경로를 통해 투여될 수 있다.The "administration" of the present invention means the process of introducing the active ingredient of the present invention to an individual by any suitable method, and the administration method in the treatment method of the present invention is through various routes such as oral or parenteral. may be administered.
본 발명의 목적상, 목적하는 개체에 대한 구체적인 약학적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 상기 유효성분을 포함하는 조성물, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 상기 유효성분을 포함하는 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.For the purposes of the present invention, a specific pharmaceutically effective amount for a target subject includes the type and extent of the reaction to be achieved, the specific composition comprising the active ingredient, including whether other agents are used in some cases, the age of the patient, Body weight, general health status, sex and diet, administration time, administration route and secretion rate of a composition containing the active ingredient, treatment period, various factors including drugs used or used together with a specific composition and well-known in the pharmaceutical field It is preferable to apply differently depending on similar factors.
본 발명에서, 상기 DC-sign, CD4, 벡터, 베타 글로빈 프로모터, 적혈구 전구 세포, 형질주입, 인간 면역결핍 바이러스, 이의 감염 질환, 예방 및 치료에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다.In the present invention, the description of DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus, its infectious disease, prevention and treatment overlaps with those described above to avoid excessive complexity of the specification. In order to avoid it, detailed description thereof will be omitted below.
본 발명의 또 다른 구현 예에 따르면, DC-sign(Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin)을 코딩하는 유전자가 형질주입된 숙주 세포를 유효성분으로 포함하는, 인간 면역결핍 바이러스(HIV) 감염 또는 감염 질환의 예방 또는 치료용 세포 치료제에 관한 것이다.According to another embodiment of the present invention, comprising a host cell transfected with a gene encoding DC-sign (Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin) as an active ingredient, human immunity It relates to a cell therapeutic agent for preventing or treating deficiency virus (HIV) infection or infectious disease.
본 발명에서, 상기 숙주 세포는 CD4(Cluster of Differentiation 4)를 코딩하는 유전자가 추가로 형질주입된 것일 수 있다.In the present invention, the host cell may be one further transfected with a gene encoding CD4 (Cluster of Differentiation 4).
본 발명에서 상기 형질주입은 상기 DC-sign 또는 상기 CD4를 코딩하는 유전자를 포함하는 벡터를 사용하여 수행될 수 있다. In the present invention, the transfection may be performed using a vector including a gene encoding the DC-sign or the CD4.
본 발명에서, 상기 벡터는 베타 글로빈 프로모터(beta globin promoter)를 더 포함할 수 있고, 바람직하게는 상기 벡터에서 상기 DC-sign 또는 상기 CD4를 코딩하는 유전자의 5' 방향에 상기 베타 글로빈 프로모터를 더 포함함으로써, 상기 벡터가 숙주 세포에 형질주입 시 상기 DC-sign 또는 상기 CD4의 발현을 보다 촉진시킬 수 있다.In the present invention, the vector may further include a beta globin promoter, and preferably, the beta globin promoter is further added in the 5' direction of the gene encoding the DC-sign or the CD4 in the vector. By including, the expression of the DC-sign or the CD4 can be further promoted when the vector is transfected into a host cell.
본 발명에서 상기 벡터는 플라스미드(plasmids), 코스미드(cosmids), 인공 염색체(artificial chromosomes), 리포솜(Liposomes)과 같은 비바이러스성 벡터이거나, 레트로바이러스(retrovirus), 아데노바이러스(Adenovirus), 아데노-관련 바이러스(AAV)와 같은 바이러스성 벡터일 수 있으며, 바람직하게는 렌티바이러스(Lentivirus)일 수 있다.In the present invention, the vector is a non-viral vector such as plasmids, cosmids, artificial chromosomes, and liposomes, or retrovirus, adenovirus, adeno- It may be a viral vector such as an associated virus (AAV), preferably a lentivirus.
본 발명에서, 상기 벡터를 세포로 주입하기 위하여 유전자총(gene gun), 미세주입법(microinjection), 전기천공법(electroporation) 또는 리포펙션(lipofection)의 방법에 의할 수 있다.In the present invention, in order to inject the vector into the cells, a method of gene gun, microinjection, electroporation, or lipofection may be used.
또한, 본 발명에서는 상기 벡터에 삽입된 유전자의 정제를 용이하게 하기 위하여 태그 서열을 상기 벡터 상에 삽입하여 융합시킬 수 있다. 상기 태그로는 헥사-히스티딘 태그, 헤마글루티닌 태그, myc 태그 또는 flag 태그를 포함하나 이에 한정되는 것은 아니며 당업자에게 알려진 정제를 용이하게 하는 태그는 모두 본 발명에서 이용 가능하다. In addition, in the present invention, in order to facilitate purification of the gene inserted into the vector, a tag sequence may be inserted into the vector and fused. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
본 발명에서 상기 숙주 세포는 적혈구 전구 세포(erythroid progenitor cell)인 것이 바람직하다. In the present invention, the host cell is preferably an erythroid progenitor cell.
본 발명에서, 상기 적혈구 전구 세포는 전적아세포(proerythroblast), 적아세포(erythroblast) 및 망상적혈구(reticulocyte)로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되지 않고, 성숙이 끝난 적혈구를 제외한 적혈구 형성 과정에 포함된 모든 세포를 의미한다.In the present invention, the red blood cell progenitor cells may be any one or more selected from the group consisting of proerythroblasts, erythroblasts and reticulocytes, but is not limited thereto, and red blood cells other than mature red blood cells. All cells involved in the formation process.
본 발명에서, 상기 형질주입된 세포는 증식되어 적혈구로 분화될 수 있고, 이때 상기 분화는 체외(in vitro) 또는 체내(in vivo)에서 이루어질 수 있으나, 바람직하게는 체외에서 이루어질 수 있다.In the present invention, the transfected cells may be proliferated and differentiated into red blood cells, wherein the differentiation may be performed in vitro or in vivo, but preferably in vitro.
본 발명에서, 상기 인간 면역 결핍 바이러스는 제1형 인간 면역 결핍 바이러스(HIV-1) 또는 제2형 인간 면역 결핍 바이러스(HIV-2)일 수 있으나, 바람직하게는 제1형 인간 면역 결핍 바이러스(HIV-1)일 수 있다.In the present invention, the human immunodeficiency virus may be type 1 human immunodeficiency virus (HIV-1) or type 2 human immunodeficiency virus (HIV-2), but preferably type 1 human immunodeficiency virus ( HIV-1).
본 발명에서, 상기 인간 면역결핍 바이러스는 기존 항렌티바이러스제 내지 항레트로바이러스제에 내성을 가진 것일 수 있다. 상기 항렌티바이러스제는 핵산계열 역전사효소저해제(nucleoside reverse transcriptase inhibitor; NRTI), 비핵산 계열의 역전사효소저해제(non-nucleoside reverse transcriptase inhibitor; NNRTI), 단백질분해 효소저해제(protease inhibitor; PI), 막융합저해제(fusion inhibitor; Fusion I), CCR5 길항제(CCR5 antagonist) 및 통합효소 저해제(integrase inhibitor)에서 선택된 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the human immunodeficiency virus may be resistant to existing anti-lentiviral agents or antiretroviral agents. The anti-lentiviral agent is a nucleic acid-based reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), membrane fusion Inhibitors (fusion inhibitors; Fusion I), CCR5 antagonists (CCR5 antagonist), and may be any one or more selected from integrase inhibitors (integrase inhibitor), but is not limited thereto.
본 발명에서, 상기 인간 면역결핍 바이러스 감염은 상기 인간 면역결핍 바이러스의 잠복 감염일 수 있다.In the present invention, the human immunodeficiency virus infection may be a latent infection of the human immunodeficiency virus.
본 발명에서, 상기 인간 면역결핍 바이러스 감염 질환은 후천면역결핍증후군(AIDS)일 수 있다.In the present invention, the human immunodeficiency virus infection disease may be acquired immunodeficiency syndrome (AIDS).
본 발명에서, 상기 “세포 치료제”란 세포와 조직의 기능을 복원시키기 위하여 살아있는 자가(autologous), 동종(allogenic), 이종(xenogenic) 세포를 체외에서 증식 또는 선별하거나 여타한 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 말한다. 미국은 1993년부터, 우리나라는 2002년부터 세포 치료제를 의약품으로 관리하고 있다. 상기 세포 치료제는 인간 면역결핍 바이러스 감염 또는 감염 질환의 치료를 위한 것일 수 있다.In the present invention, the “cell therapeutic agent” refers to the proliferation or selection of living autologous, allogenic, and xenogenic cells in vitro or other methods to restore the functions of cells and tissues. It refers to medicines used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing the Cell therapy has been administered as a drug since 1993 in the United States and 2002 in Korea. The cell therapeutic agent may be for treatment of human immunodeficiency virus infection or infectious disease.
본 발명의 세포 치료제의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 비경구 투여, 예를 들어, 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여될 수 있으나, 이에 제한되지는 않는다.The administration route of the cell therapeutic agent of the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, may be administered intradermally, but is not limited thereto.
본 발명의 세포 치료제에서, 상기와 같이 형질주입된 적혈구 전구 세포는 적혈구로 분화될 수 있다. 이때 체내로 침입한 인간 면역결핍 바이러스가 상기 적혈구 세포 내 벡터에 의해 발현된 DC-sign 및 CD4 단백질에 결합하게 되는데, 상기 적혈구 세포는 체내를 순환하다 비장에서 파괴되므로, 인간 면역결핍 바이러스의 감염 또는 감염 질환이 예방, 개선 또는 치료될 수 있다. In the cell therapeutic agent of the present invention, the transfected red blood cell progenitor cells can be differentiated into red blood cells. At this time, the human immunodeficiency virus invading the body binds to the DC-sign and CD4 proteins expressed by the vector in the red blood cells, and the red blood cells circulate in the body and are destroyed in the spleen. Infectious diseases can be prevented, ameliorated or treated.
본 발명의 상기 세포 치료제는 표적 세포로 이동할 수 있도록 하는 임의의 장치에 의해 투여될 수도 있다.The cell therapeutic agent of the present invention may be administered by any device that allows it to migrate to a target cell.
본 발명의 세포 치료제는 상기 인간 면역결핍 바이러스 감염 또는 감염 질환의 치료를 위한 유효량으로 투여될 수 있다. 상기 치료를 위한 유효량(therapeutically effective amount)은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다. The cell therapeutic agent of the present invention may be administered in an effective amount for the treatment of the human immunodeficiency virus infection or infectious disease. The therapeutically effective amount refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, physician or other clinician, which an amount that induces amelioration of the symptoms of the disease or disorder being treated.
본 발명의 조성물에 포함되는 세포 치료제는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로 최적의 세포 치료제 함량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 포함할 수 있다.It is apparent to those skilled in the art that the cell therapeutic agent included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal content of the cell therapeutic can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, and the age, weight, general health status, sex and diet of the patient , administration time, administration route and secretion rate of the composition, treatment period, and drugs used at the same time may be adjusted according to various factors. In consideration of all of the above factors, it may include an amount capable of obtaining the maximum effect with a minimum amount without side effects.
본 발명에서, 상기 DC-sign, CD4, 벡터, 베타 글로빈 프로모터, 적혈구 전구 세포, 형질주입, 인간 면역결핍 바이러스 및 이의 감염 질환에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다.In the present invention, the description of the DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus and infectious diseases thereof overlaps with those described above, and in order to avoid excessive complexity of the specification, the following descriptions are made. A detailed description is omitted.
본 발명의 또 다른 구현 예에서는, 본 발명에 따른 상기 세포 치료제를 유효량으로 개체에 투여하는 단계를 포함하는, 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료 방법에 관한 것이다.In another embodiment of the present invention, it relates to a method for preventing or treating Human Immunodeficiency Virus (HIV) infection or infectious disease, comprising administering to an individual an effective amount of the cell therapeutic agent according to the present invention. will be.
본 발명에서, 상기 DC-sign, CD4, 벡터, 베타 글로빈 프로모터, 적혈구 전구 세포, 형질주입, 인간 면역결핍 바이러스, 이의 감염 질환, 예방, 치료, 개체 및 투여에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다.In the present invention, the DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus, its infectious disease, prevention, treatment, subject and administration are overlapped with those described above. In order to avoid excessive complexity, the detailed description thereof will be omitted below.
본 발명의 또 다른 구현 예에 따르면, 분리된 숙주 세포에 DC-sign(Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin)을 코딩하는 유전자를 형질주입 시키는 단계를 포함하는, 형질주입된 세포를 제조하는 방법에 관한 것이다.According to another embodiment of the present invention, transfection comprising the step of transfecting an isolated host cell with a gene encoding DC-sign (Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin) It relates to a method for preparing the infused cells.
본 발명에서 상기 숙주 세포는 적혈구 전구 세포(erythroid progenitor cell)인 것이 바람직하다. In the present invention, the host cell is preferably an erythroid progenitor cell.
본 발명에서, 상기 적혈구 전구 세포는 전적아세포(proerythroblast), 적아세포(erythroblast) 및 망상적혈구(reticulocyte)로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되지 않고, 성숙이 끝난 적혈구를 제외한 적혈구 형성 과정에 포함된 모든 세포를 의미한다.In the present invention, the red blood cell progenitor cells may be any one or more selected from the group consisting of proerythroblasts, erythroblasts and reticulocytes, but is not limited thereto, and red blood cells other than mature red blood cells. All cells involved in the formation process.
본 발명에서, CD4(Cluster of Differentiation 4)를 코딩하는 유전자를 형질주입 시키는 단계를 더 포함할 수 있다.In the present invention, the step of transfecting the gene encoding CD4 (Cluster of Differentiation 4) may be further included.
본 발명에서 상기 형질주입은 상기 DC-sign 또는 상기 CD4를 코딩하는 유전자를 포함하는 벡터를 사용하여 수행될 수 있다. In the present invention, the transfection may be performed using a vector including a gene encoding the DC-sign or the CD4.
본 발명에서, 상기 벡터는 베타 글로빈 프로모터(beta globin promoter)를 더 포함할 수 있고, 바람직하게는 상기 벡터에서 상기 DC-sign 또는 상기 CD4를 코딩하는 유전자의 5' 방향에 상기 베타 글로빈 프로모터를 더 포함함으로써, 상기 벡터가 숙주 세포에 형질주입 시 상기 DC-sign 또는 상기 CD4의 발현을 보다 촉진시킬 수 있다.In the present invention, the vector may further include a beta globin promoter, and preferably, the beta globin promoter is further added in the 5' direction of the gene encoding the DC-sign or the CD4 in the vector. By including, the expression of the DC-sign or the CD4 can be further promoted when the vector is transfected into a host cell.
본 발명에서 상기 벡터는 플라스미드(plasmids), 코스미드(cosmids), 인공 염색체(artificial chromosomes), 리포솜(Liposomes)과 같은 비바이러스성 벡터이거나, 레트로바이러스(retrovirus), 아데노바이러스(Adenovirus), 아데노-관련 바이러스(AAV)와 같은 바이러스성 벡터일 수 있으며, 바람직하게는 렌티바이러스(Lentivirus)일 수 있다.In the present invention, the vector is a non-viral vector such as plasmids, cosmids, artificial chromosomes, and liposomes, or retrovirus, adenovirus, adeno- It may be a viral vector such as an associated virus (AAV), preferably a lentivirus.
본 발명에서, 상기 벡터를 세포로 주입하기 위하여 유전자총(gene gun), 미세주입법(microinjection), 전기천공법(electroporation) 또는 리포펙션(lipofection)의 방법에 의할 수 있다.In the present invention, in order to inject the vector into the cells, a method of gene gun, microinjection, electroporation, or lipofection may be used.
또한, 본 발명에서는 상기 벡터에 삽입된 유전자의 정제를 용이하게 하기 위하여 태그 서열을 상기 벡터 상에 삽입하여 융합시킬 수 있다. 상기 태그로는 헥사-히스티딘 태그, 헤마글루티닌 태그, myc 태그 또는 flag 태그를 포함하나 이에 한정되는 것은 아니며 당업자에게 알려진 정제를 용이하게 하는 태그는 모두 본 발명에서 이용 가능하다. In addition, in the present invention, in order to facilitate purification of the gene inserted into the vector, a tag sequence may be inserted into the vector and fused. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
본 발명에서, 상기 형질주입된 세포를 적혈구로 분화시키는 단계를 더 포함할 수 있다. 이때, 이때 상기 분화는 체외(in vitro) 또는 체내(in vivo)에서 이루어질 수 있으나, 바람직하게는 체외에서 이루어질 수 있다.In the present invention, the method may further include differentiating the transfected cells into red blood cells. In this case, the differentiation may be performed in vitro or in vivo, but preferably in vitro.
본 발명에서 상기 방법으로 제조된 세포는 인간 면역결핍 바이러스 감염 또는 감염 질환에 대한 치료제로 사용될 수 있다.In the present invention, the cells prepared by the above method can be used as a therapeutic agent for human immunodeficiency virus infection or infectious disease.
본 발명에서, 상기 DC-sign, CD4, 벡터, 베타 글로빈 프로모터, 적혈구 전구 세포, 형질주입, 인간 면역결핍 바이러스 및 이의 감염 질환에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다.In the present invention, the description of the DC-sign, CD4, vector, beta globin promoter, erythroid progenitor cell, transfection, human immunodeficiency virus and infectious diseases thereof overlaps with those described above, and in order to avoid excessive complexity of the specification, the following descriptions are made. A detailed description is omitted.
본 발명에서 제공하는 약학적 조성물을 사용하는 경우 인간 면역 결핍 바이러스 감염증을 효과적으로 치료할 수 있을 뿐만 아니라, 암과 같은 부작용의 발생 가능성이 낮은 장점이 있다. When the pharmaceutical composition provided in the present invention is used, it is possible to effectively treat human immunodeficiency virus infection, and there is an advantage in that the possibility of side effects such as cancer is low.
도 1은 준비예 1에서 본 발명의 일 실시예에 따른 인간 베타 글로빈 프로모터(human beta globin promoter) 및 상기 프로모터 서열에 특이적이 제한효소를 나타낸 것이다.1 shows a human beta globin promoter according to an embodiment of the present invention in Preparation Example 1 and a restriction enzyme specific to the promoter sequence.
도 2는 준비예 2에서 본 발명의 일 실시예에 따른 베타 글로빈 프로모터 기반 벡터(pLenti6.3/beta globin)를 제작하는 과정을 나타낸 것이다.2 shows the process of constructing a beta globin promoter-based vector (pLenti6.3/beta globin) according to an embodiment of the present invention in Preparation Example 2.
도 3은 준비예 2에서 본 발명의 일 실시예에 따른 벡터(pLenti6.3/beta globin)에서 베타 글로빈 프로모터 유전자를 확인한 것이다.3 shows the confirmation of the beta globin promoter gene in the vector (pLenti6.3/beta globin) according to an embodiment of the present invention in Preparation Example 2.
도 4는 준비예 3에서 본 발명의 일 실시예에 따른 CD4를 코딩하는 유전자 서열을 삽입한 벡터(pCR8_CD4)를 제작하는 과정을 나타낸 것이다.4 shows the process of preparing a vector (pCR8_CD4) into which the CD4 coding gene sequence according to an embodiment of the present invention is inserted in Preparation Example 3;
도 5는 준비예 3에서 본 발명의 일 실시예에 따른 벡터(pCR8_CD4)에서 CD4를 코딩하는 유전자를 확인한 것이다.FIG. 5 shows the identification of the CD4 coding gene in the vector (pCR8_CD4) according to an embodiment of the present invention in Preparation Example 3;
도 6은 준비예 3에서 본 발명의 일 실시예에 따른 베타 글로빈 프로모터 서열 및 상기 베타 글로빈 프로모터 서열의 하류에 CD4를 코딩하는 유전자 서열이 삽입된 벡터(pLenti6.3_beta Pro_CD4 CD4)를 제작하는 과정을 나타낸 것이다.6 shows the process of constructing a vector (pLenti6.3_beta Pro_CD4 CD4) in which the beta globin promoter sequence and the CD4 coding gene sequence downstream of the beta globin promoter sequence according to an embodiment of the present invention were inserted in Preparation Example 3; it has been shown
도 7은 준비예 4에서 본 발명의 일 실시예에 따른 DC-sign을 코딩하는 유전자 서열을 삽입한 벡터(pCR8_DC-sign)를 제작하는 과정을 나타낸 것이다.7 shows the process of preparing a vector (pCR8_DC-sign) into which a gene sequence encoding a DC-sign according to an embodiment of the present invention is inserted in Preparation Example 4;
도 8은 준비예 4에서 본 발명의 일 실시예에 따른 베타 글로빈 프로모터 서열 및 상기 베타 글로빈 프로모터 서열의 하류에 DC-sign을 코딩하는 유전자 서열이 삽입된 벡터(pLenti6.3/DC-sign)를 제작하는 과정을 나타낸 것이다.8 is a vector (pLenti6.3/DC-sign) in which the beta globin promoter sequence according to an embodiment of the present invention and the DC-sign coding gene sequence downstream of the beta globin promoter sequence are inserted in Preparation Example 4; It shows the manufacturing process.
도 9는 준비예 4에서 본 발명의 일 실시예에 따른 벡터(pLenti6.3/DC-sign)에서 베타 글로빈 프로모터 및 DC-sign을 코딩하는 유전자를 확인한 것이다.Figure 9 confirms the gene encoding the beta globin promoter and DC-sign in the vector (pLenti6.3/DC-sign) according to an embodiment of the present invention in Preparation Example 4;
도 10은 실험예 1에서 본 발명의 일 실시예에 따른 형질주입된 적아세포의 CD4 발현을 확인한 것이다.10 shows CD4 expression of erythroblasts transfected according to an embodiment of the present invention in Experimental Example 1. FIG.
도 11은 실험예 1에서 본 발명의 일 실시예에 따른 형질주입된 적아세포의 CD4 발현을 확인한 것이다.11 shows CD4 expression of erythroblasts transfected according to an embodiment of the present invention in Experimental Example 1.
도 12는 실험예 1에서 본 발명의 일 실시예에 따른 형질주입되지 않은 대조군 적아세포의 CD4 발현을 확인한 것이다.12 shows CD4 expression of non-transfected control erythroblasts according to an embodiment of the present invention in Experimental Example 1. FIG.
도 13은 실험예 1에서 본 발명의 일 실시예에 따른 형질주입된 적아세포와 대조군에서의 DC-sign 발현을 확인한 것이다.13 is a view illustrating DC-sign expression in erythroblasts transfected according to an embodiment of the present invention in Experimental Example 1 and a control group.
본 발명의 일 목적은 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료용 약학적 조성물을 제공하고자 한다. One object of the present invention is to provide a pharmaceutical composition for preventing or treating Human Immunodeficiency Virus (HIV) infection or infectious disease.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
[준비예 1] 적혈구 전구 세포 형질주입을 위한 베타 글로빈 프로모터 확인[Preparation Example 1] Confirmation of beta globin promoter for red blood cell progenitor cell transfection
적혈구 전구 세포 및 핵 단백질 인자에 특이적인 형질주입을 위하여 베타 글로빈 유전자좌(Human beta-globin locus)에서 프로모터 및 상기 프로모터의 서열에 특이적인 제한효소를 탐색하여 도 1과 같이 나타내었다.For specific transfection of erythrocyte progenitor cells and nuclear protein factors, a promoter and restriction enzymes specific for the promoter sequence were searched at the human beta-globin locus, and the results are shown in FIG. 1 .
도 1에서처럼, 적혈구 전구 세포 및 핵 단백질 인자에 특이적이고, 염색체 11번에 존재하는, 인간 베타 글로빈 프로모터(human beta globin promoter)를 확인하였고, 상기 프로모터 서열의 5' 방향에 특이적인 ClaI 제한효소(ATCGAT 서열번호 6) 및 3' 방향에 특이적인 SpeI 제한효소(ACTAGT 서열번호 7)를 확인하였다.As shown in FIG. 1 , the human beta globin promoter, specific for erythrocyte progenitor cells and nuclear protein factors, and present on chromosome 11, was identified, and a Cla I restriction enzyme specific for the 5' direction of the promoter sequence. (ATCGAT SEQ ID NO: 6) and a Spe I restriction enzyme specific for the 3' direction (ACTAGT SEQ ID NO: 7) were identified.
[준비예 2] 베타 글로빈 프로모터를 포함하는 벡터 제작[Preparation Example 2] Construction of a vector containing a beta globin promoter
도 2와 같이, 상기 ClaI 제한효소 및 SpeI 제한효소를 이용하여 렌티바이러스 벡터(pLenti6.3)에 PCR로 증폭된 베타 글로빈 프로모터 서열을 삽입하여 베타 글로빈 프로모터 기반 벡터(pLenti6.3/beta globin)를 제작하였다. 상기 벡터(pLenti6.3/beta globin)에서 베타 글로빈 프로모터 서열이 삽입된 것을 확인하기 위하여 서던 블롯을 실시하였고, 그 결과를 도 3과 같이 나타내었다.As shown in FIG. 2, a beta globin promoter-based vector (pLenti6.3/beta globin) by inserting the PCR-amplified beta globin promoter sequence into a lentiviral vector (pLenti6.3) using the Cla I and Spe I restriction enzymes. ) was produced. Southern blot was performed to confirm that the beta globin promoter sequence was inserted into the vector (pLenti6.3/beta globin), and the results are shown in FIG. 3 .
도 3과 같이, 상기 벡터(pLenti6.3/beta globin)에서 약 260bp의 베타 글로빈 프로모터 유전자가 삽입된 것을 확인할 수 있었다.As shown in FIG. 3 , it was confirmed that the beta globin promoter gene of about 260 bp was inserted into the vector (pLenti6.3/beta globin).
[준비예 3] CD4 발현을 위한 벡터 제작[Preparation Example 3] Preparation of vector for CD4 expression
도 4와 같이 pCR8 벡터에 CD4를 코딩하는 유전자 서열을 삽입한 벡터(pCR8_CD4)를 제작하였다. 상기 벡터(pCR8_CD4)에서 CD4를 코딩하는 유전자 서열이 삽입된 것을 확인하기 위하여 서던 블롯을 실시하였고, 그 결과를 도 5와 같이 나타내었다.As shown in FIG. 4, a vector (pCR8_CD4) in which the CD4 coding gene sequence was inserted into the pCR8 vector was prepared. Southern blot was performed to confirm that the gene sequence encoding CD4 was inserted into the vector (pCR8_CD4), and the results are shown in FIG. 5 .
도 5와 같이, 상기 벡터(pCR8_CD4)에서 약 2.8kb의 pCR8 벡터 및 1.3kb의 CD4를 코딩하는 유전자를 확인할 수 있었다.As shown in FIG. 5 , genes encoding about 2.8 kb of pCR8 vector and 1.3 kb of CD4 were identified in the vector (pCR8_CD4).
상기 CD4를 코딩하는 유전자 서열을 삽입한 벡터(pCR8_CD4)를 삽입 클론(Entry Clone)으로 하고, 준비예 2에서 제작된 벡터(pLenti6.3/beta globin)를 목적 벡터(Destination Vector)로 하여, 도 6과 같이 LR 재조합 반응(LR recombination reaction)을 실시하였다.The vector (pCR8_CD4) into which the CD4 coding gene sequence was inserted was used as an insertion clone (Entry Clone), and the vector (pLenti6.3/beta globin) prepared in Preparation Example 2 (pLenti6.3/beta globin) was used as the destination vector (Destination Vector). As shown in 6, LR recombination reaction was performed.
그 결과 도 6와 같이 베타 글로빈 프로모터 서열 및 상기 베타 글로빈 프로모터 서열의 하류에 CD4를 코딩하는 유전자 서열이 삽입된 벡터인 pLenti6.3_beta Pro_CD4 벡터를 제작하였다.As a result, as shown in FIG. 6 , a pLenti6.3_beta Pro_CD4 vector was constructed in which the beta globin promoter sequence and the CD4 coding gene sequence were inserted downstream of the beta globin promoter sequence.
[준비예 4] DC-sign 발현을 위한 벡터 제작[Preparation Example 4] Vector construction for DC-sign expression
도 7과 같이 pCR8 벡터에 DC-sign을 코딩하는 유전자 서열을 삽입한 벡터(pCR8_DC-sign)를 제작하였다. 또한, 도 8과 같이 상기 pCR8_DC-sign 서열을 삽입한 벡터(pCR8_DC-sign)를 삽입 클론(Entry Clone)으로 하고, 준비예 2에서 제작된 벡터(pLenti6.3/beta globin)를 목적 벡터(Destination Vector)로 하여, LR 재조합 반응(LR recombination reaction)을 실시하여, 도 8과 같이 상기 베타 글로빈 프로모터 서열의 하류에 DC-sign을 코딩하는 유전자 서열이 삽입된 벡터인 pLenti6.3/DC-sign 벡터를 제작하였다.As shown in FIG. 7, a vector (pCR8_DC-sign) in which the DC-sign coding gene sequence was inserted into the pCR8 vector was prepared. In addition, as shown in FIG. 8, the vector (pCR8_DC-sign) into which the pCR8_DC-sign sequence was inserted was used as an insertion clone (Entry Clone), and the vector (pLenti6.3/beta globin) prepared in Preparation Example 2 was used as the destination vector (Destination). Vector), the pLenti6.3/DC-sign vector is a vector in which a DC-sign-coding gene sequence is inserted downstream of the beta globin promoter sequence as shown in FIG. 8 by performing an LR recombination reaction. was produced.
상기 pLenti6.3/DC-sign 벡터의 DC-sign을 코딩하는 유전자 및 베타 글로빈 프로모터의 재조합 반응을 확인하기 위하여, 서던 블롯을 실시하였고, 그 결과를 도 9와 같이 나타내었다.In order to confirm the recombination reaction of the gene encoding the DC-sign of the pLenti6.3/DC-sign vector and the beta globin promoter, Southern blot was performed, and the results are shown in FIG. 9 .
도 9과 같이, 상기 pLenti6.3/DC-sign 벡터에서 DC-sign을 코딩하는 유전자 및 인간 베타 글로빈 프로모터를 확인할 수 있었다.As shown in FIG. 9 , the gene encoding DC-sign and the human beta globin promoter were identified in the pLenti6.3/DC-sign vector.
[실험예 1] 적아세포 형질주입 및 유전자 발현 확인[Experimental Example 1] Erythroblast transfection and gene expression confirmation
준비예 3 및 4에서 제작된 pLenti6.3_beta Pro_CD4 벡터 및 pLenti6.3/DC-sign 벡터를 293FT 세포주에 형질주입(transfection)시켜 배지에 배양하였다. 이후 상기 배지의 상층액을 회수하는 방법으로 재조합 렌티바이러스를 제조하였다. 상기와 같이 제조된 재조합 렌티바이러스를 적아세포(erythroblast)에 감염시켜 형질주입된 적아세포를 얻었다. 상기 형질주입된 적아세포의 CD4 및 DC-sign 발현 여부를 확인하기 위하여 형질주입된 후 3일 및 4일차에 유세포분석을 실시하였다.The pLenti6.3_beta Pro_CD4 vector and pLenti6.3/DC-sign vector prepared in Preparation Examples 3 and 4 were transfected into a 293FT cell line and cultured in a medium. Thereafter, a recombinant lentivirus was prepared by recovering the supernatant of the medium. The recombinant lentivirus prepared as described above was infected with erythroblasts to obtain transfected erythroblasts. Flow cytometry was performed on the 3rd and 4th days after transfection to confirm the expression of CD4 and DC-sign in the transfected erythroblasts.
상기 형질주입된 적아세포에 Pacific Blue로 컨쥬게이트된 CD4 단일클론항체를 넣고 반응시킨 후, FBS로 세척한 다음 고정하여 형광 활성 유세포측정기(FACS)로 분석하여 도 10 및 11에 나타내었고, 대조군으로는 재조합되지 않은 렌티바이러스(pLenti6.3에 의해 제조된 렌티바이러스 isotype) 및 염색되지 않은 적아세포로 두었다. 또한, 대조군으로 형질주입되지 않은 적아세포를 유세포측정기로 분석하여 도 12에 나타내었다.The transfected erythrocytes were reacted with a CD4 monoclonal antibody conjugated with Pacific Blue, washed with FBS, fixed, and analyzed by fluorescence-activated flow cytometry (FACS), as shown in FIGS. 10 and 11, as a control. were placed as non-recombinant lentivirus (lentivirus isotype prepared by pLenti6.3) and unstained erythroblasts. In addition, erythroblasts that were not transfected as a control were analyzed by flow cytometry and are shown in FIG. 12 .
도 10에서 보는 바와 같이, 형질주입된 적아세포는 형질주입된지 3일 후에는 CD4를 발현하는 것을 확인할 수 있었고, 도 11에서 보는 바와 같이 형질주입된 적아세포는 형질주입된지 4일 후에도 CD4를 발현하는 것을 확인할 수 있었다. 이는 도 12에서 나타난 대조군에서의 CD4 발현량과 차이가 있었다.As shown in FIG. 10 , it was confirmed that the transfected erythroblasts expressed CD4 3 days after transfection, and as shown in FIG. 11 , the transfected erythroblasts expressed CD4 even 4 days after transfection. could confirm that This was different from the CD4 expression level in the control group shown in FIG. 12 .
또한, 상기 형질주입된 적아세포에 피코에리드린(phycoerythrin)으로 컨쥬게이트된 DC-sign 항체(DC-sign PE ab)를 넣고 반응시킨 후, FBS로 세척한 다음 고정하여 형광 활성 유세포측정기(FACS)로 분석하였고, 대조군으로는 형질주입되지 않은 적아세포 및 염색되지 않은 적아세포로 두어 도 13에 나타내었다. In addition, a DC-sign antibody (DC-sign PE ab) conjugated with phycoerythrin was added to the transfected erythroblasts and reacted, washed with FBS and fixed, followed by fluorescence-activated flow cytometry (FACS) was analyzed, and as controls, untransfected erythroblasts and unstained erythroblasts were shown in FIG. 13 .
도 13에서 보는 바와 같이, 형질주입된 적아세포는 DC-sign을 발현하는 것을 확인할 수 있었다. As shown in FIG. 13 , it was confirmed that the transfected erythroblasts expressed DC-sign.
상기 결과를 이상에서 본 발명에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.Although the above results have been described in detail with respect to the present invention above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.
본 발명은 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환을 치료할 수 있는 조성물에 관한 것이다.The present invention relates to a composition capable of treating Human Immunodeficiency Virus (HIV) infection or infectious disease.

Claims (23)

  1. DC-sign(Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin)을 코딩하는 유전자를 포함하는 벡터를 유효성분으로 포함하는, 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료용 약학적 조성물.Human Immunodeficiency Virus (HIV) infection or infection comprising a vector containing a gene encoding DC-sign (Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin) as an active ingredient A pharmaceutical composition for preventing or treating a disease.
  2. 제 1항에 있어서,The method of claim 1,
    상기 벡터는 CD4(Cluster of Differentiation 4)를 코딩하는 유전자를 추가로 더 포함하는 것인, 약학적 조성물.The vector further comprises a gene encoding a CD4 (Cluster of Differentiation 4), the pharmaceutical composition.
  3. 제 1항에 있어서,The method of claim 1,
    CD4를 코딩하는 유전자를 포함하는 벡터를 더 포함하는, 약학적 조성물.A pharmaceutical composition, further comprising a vector comprising a gene encoding CD4.
  4. 제 1항에 있어서,The method of claim 1,
    상기 벡터는 베타 글로빈 프로모터(beta globin promoter)를 더 포함하는 것인, 약학적 조성물.The vector further comprises a beta globin promoter (beta globin promoter), the pharmaceutical composition.
  5. 제 1항에 있어서,The method of claim 1,
    상기 벡터는 플라스미드(plasmids), 코스미드(cosmids), 인공 염색체(artificial chromosomes), 리포솜(Liposomes), 레트로바이러스(retrovirus), 아데노바이러스(Adenovirus) 또는 아데노-관련 바이러스(Adenovirus-associated virus; AAV)인, 약학적 조성물.The vector includes plasmids, cosmids, artificial chromosomes, liposomes, retroviruses, adenoviruses, or adenovirus-associated virus (AAV). Phosphorus, pharmaceutical composition.
  6. 제 1항에 있어서,The method of claim 1,
    상기 벡터는 목적하는 숙주 세포에 형질주입되는 것인, 약학적 조성물.The vector is to be transfected into a host cell of interest, the pharmaceutical composition.
  7. 제 6항에 있어서,7. The method of claim 6,
    상기 숙주 세포는 적혈구 전구 세포(erythroid progenitor cell)인, 약학적 조성물. The host cell is an erythroid progenitor cell, the pharmaceutical composition.
  8. 제 7항에 있어서,8. The method of claim 7,
    상기 적혈구 전구 세포는 전적아세포(proerythroblast), 적아세포(erythroblast) 및 망상적혈구(reticulocyte)로 구성된 군으로부터 선택되는 어느 하나 이상인, 약학적 조성물.The erythrocyte progenitor cell is any one or more selected from the group consisting of proerythroblast, erythroblast and reticulocyte, a pharmaceutical composition.
  9. 제 7항에 있어서,8. The method of claim 7,
    상기 적혈구 전구 세포는 적혈구로 분화되는 것인, 약학적 조성물.The erythrocyte progenitor cells will be differentiated into erythrocytes, a pharmaceutical composition.
  10. 제 1항에 있어서,The method of claim 1,
    상기 인간 면역결핍 바이러스 감염은 상기 인간 면역결핍 바이러스의 잠복 감염인, 약학적 조성물.wherein the human immunodeficiency virus infection is a latent infection of the human immunodeficiency virus.
  11. 제 1항에 있어서,The method of claim 1,
    상기 인간 면역결핍 바이러스 감염 질환은 후천면역결핍증후군(Acquired immunodeficiency syndrome; AIDS)인, 약학적 조성물.The human immunodeficiency virus infection disease is acquired immunodeficiency syndrome (AIDS), a pharmaceutical composition.
  12. DC-sign(Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin)을 코딩하는 유전자가 형질주입된 숙주 세포를 유효성분으로 포함하는, 인간 면역결핍 바이러스(HIV) 감염 또는 감염 질환의 치료용 세포 치료제.Human immunodeficiency virus (HIV) infection or infectious disease comprising a host cell transfected with a gene encoding DC-sign (Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin) as an active ingredient Therapeutic cell therapy.
  13. 제 12항에 있어서,13. The method of claim 12,
    상기 숙주 세포는 CD4(Cluster of Differentiation 4)를 코딩하는 유전자가 추가로 형질주입된 것인, 세포 치료제.The host cell is a gene encoding CD4 (Cluster of Differentiation 4) is additionally transfected, the cell therapeutic agent.
  14. 제 12항에 있어서,13. The method of claim 12,
    상기 형질주입은 벡터를 사용하여 수행되는, 세포 치료제.The transfection is performed using a vector, a cell therapeutic agent.
  15. 제 14항에 있어서,15. The method of claim 14,
    상기 벡터는 베타 글로빈 프로모터(beta globin promoter)를 더 포함하는 것인, 세포 치료제.The vector further comprises a beta globin promoter (beta globin promoter), the cell therapeutic agent.
  16. 제 14항에 있어서,15. The method of claim 14,
    상기 벡터는 플라스미드(plasmids), 코스미드(cosmids), 인공 염색체(artificial chromosomes), 리포솜(Liposomes), 레트로바이러스(retrovirus), 아데노바이러스(Adenovirus) 또는 아데노-관련 바이러스(AAV)인, 세포 치료제.The vector is plasmids, cosmids, artificial chromosomes, liposomes, retroviruses, adenoviruses, or adeno-associated viruses (AAV), cell therapeutics.
  17. 제 12항에 있어서, 13. The method of claim 12,
    상기 인간 면역결핍 바이러스 감염은 상기 인간 면역결핍 바이러스의 잠복 감염인, 세포 치료제.wherein the human immunodeficiency virus infection is a latent infection of the human immunodeficiency virus.
  18. 제 12항에 있어서, 13. The method of claim 12,
    상기 인간 면역결핍 바이러스 감염 또는 감염 질환은 후천면역결핍증후군(AIDS)인, 세포 치료제.The human immunodeficiency virus infection or infectious disease is acquired immunodeficiency syndrome (AIDS), a cell therapeutic agent.
  19. 분리된 숙주 세포에 DC-sign(Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin)을 코딩하는 유전자를 형질주입 시키는 단계를 포함하는, 형질주입된 세포를 제조하는 방법.A method for producing a transfected cell, comprising transfecting an isolated host cell with a gene encoding DC-sign (Dendritic Cell-Specific intercellular adhesion molecule-3-G rabbing N on-integrin).
  20. 제 19항에 있어서,20. The method of claim 19,
    상기 숙주 세포는 적혈구 전구 세포(erythroid progenitor cell)인, 형질주입된 세포를 제조하는 방법.The method for producing a transfected cell, wherein the host cell is an erythroid progenitor cell.
  21. 제 19항에 있어서,20. The method of claim 19,
    CD4(Cluster of Differentiation 4)를 코딩하는 유전자를 형질주입 시키는 단계를 더 포함하는, 형질주입된 세포를 제조하는 방법.A method for producing a transfected cell, further comprising the step of transfecting a gene encoding CD4 (Cluster of Differentiation 4).
  22. 제 1항 내지 제 11항 중 어느 하나의 조성물을 유효량으로 개체에 투여하는 단계를 포함하는, 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료 방법.A method for preventing or treating Human Immunodeficiency Virus (HIV) infection or an infectious disease, comprising administering to an individual an effective amount of the composition of any one of claims 1 to 11.
  23. 제 12항 내지 제 18항 중 어느 하나의 세포 치료제를 유효량으로 개체에 투여하는 단계를 포함하는, 인간 면역결핍 바이러스(Human Immunodeficiency Virus; HIV) 감염 또는 감염 질환의 예방 또는 치료 방법.A method for preventing or treating Human Immunodeficiency Virus (HIV) infection or an infectious disease, comprising administering to an individual an effective amount of the cell therapeutic agent according to any one of claims 12 to 18.
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