WO2019156541A1 - 코어-쉘 구조의 마이크로입자를 유효성분으로 포함하는 혈액응고 인자 유전자 발현 증가용 조성물 - Google Patents
코어-쉘 구조의 마이크로입자를 유효성분으로 포함하는 혈액응고 인자 유전자 발현 증가용 조성물 Download PDFInfo
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- WO2019156541A1 WO2019156541A1 PCT/KR2019/001709 KR2019001709W WO2019156541A1 WO 2019156541 A1 WO2019156541 A1 WO 2019156541A1 KR 2019001709 W KR2019001709 W KR 2019001709W WO 2019156541 A1 WO2019156541 A1 WO 2019156541A1
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Definitions
- the present invention relates to a composition for increasing blood coagulation factor gene expression comprising the micro-particles of the core-shell structure as an active ingredient.
- Hemophilia is one of the serious genetic diseases.
- Hemophilia A is a gene associated with X gene that occurs in 1 in 5000 men and is a variation of the plasma glycoprotein human hemagglutinin factor 8 (also known as Factor VIII, Factor 8, FVIII, or F8), an important component of the coagulation process. Is caused by.
- the core-shell structure of the core is a halogenated hydrocarbon and / or sulfur halide
- the outer shell is composed of lipid components
- the present invention was completed by specifically confirming that the microparticles significantly increase the expression level of the coagulation factor gene when administered in vivo with the coagulation factor 8 gene.
- Patent Document 002 Republic of Korea Patent Publication No. 10-2018-0118659
- the first problem to be solved by the present invention is to provide a composition for increasing blood coagulation factor gene expression comprising the micro-particles of the core-shell structure as an active ingredient.
- the second problem to be solved by the present invention is to provide a pharmaceutical composition for the prevention or treatment of bleeding disease or bleeding comprising the composition.
- the present invention provides a composition for increasing the expression of a coagulation factor gene comprising micro-particles of the core-shell structure as an active ingredient, wherein the core is a halogenated hydrocarbon, sulfur halide or a mixture thereof as a biocompatible gas.
- the shell comprises a lipid or derivative thereof, and the coagulation factor gene is one or more genes selected from a human coagulation factor 8 gene or a variant gene thereof.
- a composition for increasing factor gene expression comprising micro-particles of the core-shell structure as an active ingredient, wherein the core is a halogenated hydrocarbon, sulfur halide or a mixture thereof as a biocompatible gas.
- the shell comprises a lipid or derivative thereof
- the coagulation factor gene is one or more genes selected from a human coagulation factor 8 gene or a variant gene thereof.
- a composition for increasing factor gene expression is provided.
- the biocompatible gas is sulfur hexafluoride, octafluoropropane, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, Chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane and mixtures thereof.
- the halogenated hydrocarbon may be a perfluorinated hydrocarbon.
- the perfluorinated hydrocarbon is perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, Perfluoropropene, perfluorobutene, perfluorobutadiene, perfluorobut-2-yne, perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclo Butane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluoromethylcyclohexane, perfluoromethylcyclohexane, perfluoromethylcyclohexane or mixtures thereof.
- the lipid may be at least one selected from the group consisting of simple lipids, phospholipids, glycero glycolipids, sphingolipids, cholesterol and cationic lipids.
- the phospholipid is a phosphatidylcholine derivative, phosphatidylethanolamine derivative, phosphatidylserine derivative, diacetylated phospholipid, L- ⁇ -dioleyl phosphatidylethanolamine, diolein, phosphatidic acid, phosphatidylglycerol, It may be selected from the group consisting of phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, polyethylene glycolated phospholipid, egg yolk lecithin, soybean lecithin and hydrogenated phospholipid.
- the glycerol glycolipid is a group consisting of sulfoxy ribosyl glycerides, diglycosyl diglycerides, digalactosyl diglycerides, galactosyl diglycerides and glycosyl diglycerides It may be selected from.
- the sphingolipid glycolipid may be galactosyl serebroside, lactosyl serebroside or ganglioside.
- the cationic lipid is 1,2-dioleoyl-3-trimethylammonio propane (DOTAP), N- (2,3-dioleyloxy propan-1-yl) -N, N, N-trimethyl ammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2- (sperminecarboxyamide) ethyl] -N, N-dimethyl-1-propaneamitrifluoro acetic acid ( DOSPA), 1,2-dimyristyloxy propyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyloxy propyl-3-diethyl hydroxyethyl ammonium bromide (DORIE) and 3 ⁇ - [N- (N'N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) may be selected from the group consisting of.
- DOTAP 1,2-dioleoyl-3-trimethyl
- the coagulation factor 8 gene is composed of an amino acid sequence represented by SEQ ID NO: 1
- the variant gene of coagulation factor 8 is composed of an amino acid sequence represented by SEQ ID NO: 3 Can be.
- the composition may increase the expression level of the coagulation factor gene by 30% or more.
- the present invention also provides a pharmaceutical composition for the prevention or treatment of bleeding diseases or bleeding comprising the composition, and human coagulation factor 8 gene or variant genes thereof.
- the bleeding disease may be hemophilia A or hemophilia B or hemophilia caused or complexed by an inhibitory antibody against coagulation factor 8 or coagulation factor 8a.
- the bleeding disease or bleeding is neonatal coagulopathy; Severe liver disease; Hypoplatelet; Congenital deficiency of factor V, VII, X or XI; And von Willebrand disease having an inhibitor against von Willebrand factor; may be one selected from the group consisting of.
- the bleeding may be due to blood loss associated with high risk surgical procedures, traumatic blood loss, blood loss due to bone marrow transplantation or blood loss associated with cerebral hemorrhage.
- composition for increasing gene expression of the present invention is administered to a living body together with a coagulation factor 8 gene or a variant gene (for example, a polynucleotide encoding a gene or a vector comprising the same), the expression of the coagulation factor gene is expressed.
- the amount can be increased by at least 30%. Accordingly, when the composition is administered together with a gene therapy agent, it is useful because a therapeutic effect can be obtained even with a small amount of genes.
- Figure 1 is a schematic diagram showing the process of designing the coagulation factor eight factor (F8M) according to an embodiment of the present invention.
- Figure 2 shows a cleavage map of the pGP vector according to an embodiment of the present invention.
- Figure 3 compares the expression level of F8 protein according to the administration of pGP-F8M (gene only), the pharmaceutical composition of the control group (F8M-JetPEI) and the pharmaceutical composition (F8M-MP1 and F8M-MP2) according to the embodiment It is a graph.
- Figure 4 is a graph showing the comparison of the expression level of F9 protein according to administration of the pharmaceutical composition (F9-MP1 and F9-MP2) according to the pGP-F9 (gene only) and the comparative example in the mouse.
- Figure 5 is a schematic diagram showing the domain structure constituting the eighth factor of human blood coagulation according to an embodiment of the present invention.
- the composition for increasing blood coagulation factor gene expression comprising micro-particles of the core-shell structure as an active ingredient, wherein the core is a halogenated hydrocarbon, sulfur halide or a mixture thereof as a biocompatible gas,
- the shell comprises a lipid or a derivative thereof, wherein the coagulation factor gene is expressed in one or more genes selected from a human coagulation factor 8 gene or a variant gene thereof. It provides an increasing composition.
- the "core” may be composed of halogenated hydrocarbons, sulfur halides or mixtures thereof as biocompatible gases.
- the biocompatible gas is sulfur hexafluoride, octafluoropropane, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoro Roethane, dichlorotetrafluoroethane, or mixtures thereof.
- the halogenated hydrocarbon is preferably a perfluorinated hydrocarbon.
- perfluorinated hydrocarbons include perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluoropropene, and purple Fluorobutene, perfluorobutadiene, perfluorobut-2-yne, perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane , Perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluoromethylcyclohexane, perfluoromethylcyclohexane, perfluoromethylcyclohexane, or mixtures thereof.
- biocompatible gas of the present invention sulfur hexafluoride or perfluorobutane is preferable.
- the "shell” may be composed of a component including a lipid or a derivative thereof.
- the lipid may be at least one selected from the group consisting of simple lipids, phospholipids, glycero glycolipids, sphingolipid glycolipids, cholesterol and cationic lipids, particularly phospholipids.
- phospholipids examples include phosphatidylcholine derivatives, phosphatidylethanolamine derivatives, phosphatidylserine derivatives, diacetylated phospholipids, L- ⁇ -dioleyl phosphatidylethanolamine, diolein, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, and Fingomyelin, polyethylene glycolated phospholipid, egg yolk lecithin, soybean lecithin, hydrogenated phospholipid, and the like.
- glycerol glycolipids examples include sulfoxy ribosyl glycerides, diglycosyl diglycerides, digalactosyl diglycerides, galactosyl diglycerides, glycosyl diglycerides, and the like.
- sphingoglycolipid examples include galactosyl cerebromide, lactosyl cerebromide, ganglioside and the like.
- DOTAP 1,2-dioleoyl-3- trimethylammonio propane
- DOTMA 2,3-dioleyloxy-N- [2- (sperminecarboxyamide) ethyl] -N, N-dimethyl-1-propaneamitrifluoroacetic acid
- DOSPA 1, 2-dimyristyloxy propyl-3-dimethylhydroxyethylammonium bromide
- DORIE 1,2-dioleoyloxy propyl-3-diethyl hydroxyethyl ammonium bromide
- DC-Chol 3 ⁇ - [N- (N And 'N'-dimethylaminoethyl) carbamoyl] cholesterol
- microparticles of the present invention are stabilized by a shell surrounding the gas, which is the core, to retard the diffusion of the gas into the surrounding liquid and to prevent fusion between the microparticles.
- the microparticles When the microparticles are introduced into the living body, the microparticles maintain their shape until they arrive near the target cells or tissues, and are sprayed while being destroyed near the target cells or tissues.
- the injected gas may change the cell membrane of the target cell, and may play a role of promoting the gene into the cytoplasmic environment of the target cell by the blowing force of the gas.
- the microparticles preferably have an average diameter of 1 to 10 m, preferably 2 to 8 m, more preferably 2 to 4 m.
- composition for increasing gene expression Composition for increasing gene expression
- composition for increasing gene expression according to the present invention may increase the expression level of the coagulation factor gene by at least 30% or more when administered to a living body together with the coagulation factor 8 gene or a variant gene thereof.
- the composition for increasing gene expression of the present invention was confirmed to significantly increase the amount of coagulation factor 8 gene expression when administered to mice with human coagulation factor 8 gene. More specifically, it was confirmed that when the composition for increasing gene expression of the present invention and the coagulation factor 8 gene were administered together in an animal model, the expression level of the coagulation factor 8 gene increased by 40% or more. On the other hand, in the case of the coagulation factor 9 gene, it was confirmed that the gene expression was hardly increased even when administered to the mouse with the composition for increasing gene expression of the present invention.
- composition for increasing gene expression of the present invention specifically expresses the coagulation factor gene only when administered with one or more genes selected from human coagulation factor 8 genes and variants thereof among blood coagulation factors. It is believed that there is an effect to increase the.
- the composition may further contain a pharmaceutical adjuvant such as a stabilizer, a buffer, an osmotic pressure control salt, an excipient, a preservative, or other therapeutically useful substance, and may be formulated in various oral or parenteral forms according to conventional methods. It is possible, but is preferably parenteral form. Representative of parenteral formulations are injectable formulations, preferably isotonic aqueous solutions or suspensions. Alternatively, the composition may be powdered and then suspended together with a solvent just before administration.
- the content of the microparticles is not particularly limited, but may be contained in 0.5 to 2,000 ⁇ l / ml, preferably 1 to 1,000 ⁇ l / ml. Or 5 to 2,000 ⁇ g / ml, preferably 10 to 1,000 ⁇ g / ml.
- composition is preferably administered in the form of a mixed solution mixed with the gene in terms of effectiveness.
- composition for increasing gene expression according to the present invention is administered with the following genes can further increase the expression efficiency and efficacy according to the following genes.
- blood coagulation factor 8 As used herein, the terms "blood coagulation factor 8", “factor VIII”, “FVIII”, and “F8” are used interchangeably herein.
- Mature human coagulation factor 8 is composed of 2351 amino acids (including signal peptide) arranged in a domain structure as shown in FIG. 5.
- a1, a2 and a3 are acidic regions, and acidic region a3 is known to be involved in binding of the coagulation factor 8 molecule to von Willebrand factor (vWF), which plays an important role in blood coagulation.
- vWF von Willebrand factor
- the coagulation factor eight dimer consists of a light chain (including A3, C1 and C2) and a heavy chain of variable size (including A1, A2 and B). Heavy chains are heterogeneous due to limited proteolysis in the B-domain.
- the "heavy chain" includes A1 and A2, but the B-domain is partially or wholly deleted.
- the amino acid sequence of the mature wild type form of human coagulation factor 8 is shown in SEQ ID NO: 1. Reference numerals to amino acid positions of certain sequences refer to the corresponding amino acid positions within the VIII wild type protein and do not exclude the presence of mutations (eg, deletions, insertions and / or substitutions) at other positions within the mentioned sequences.
- the coding DNA sequence of SEQ ID NO: 1 corresponds to SEQ ID NO: 2.
- “Blood coagulation factor 8” includes derivatives of wild type coagulation factor 8 having coagulation promoting activity of wild type coagulation factor 8 as well as wild type coagulation factor 8.
- the derivative may have a sequence deleted, inserted and / or added as compared to the amino acid sequence of the wild type coagulation factor eight.
- Preferred derivatives are molecules of the coagulation factor 8 which have deleted all or part of the B-domain.
- the positions of amino acids indicated throughout the present specification always refer to the position of the individual amino acids in the full length mature (including signal peptide) wild type hemagglutinin factor 8.
- variable includes conservative or non-conservative substitutions, insertions, or deletions, such as amino acid sequences or nucleic acid sequences, and such changes may substantially alter the active site or active domain conferring the biological activity of each FVIII. Do not change
- the coagulation factor eight factor variant is a single-chain coagulation factor eight variant in which amino acids Asp784 to Arg1671 are deleted from the coagulation factor eight represented by SEQ ID NO: 1.
- the variant is a single-chain coagulation factor eight variant.
- the coagulation factor 8 variant is a deletion of a portion of the B-domain (residues 784-1667) and a portion of the a3 region (residues 1668-1671), wherein the variant is a coagulation factor 8, a B-domain deleted.
- the coagulation factor eight variant has an amino acid sequence represented by SEQ ID NO: 3.
- the "single coagulation factor eight" is not cleaved into two chains (e.g., heavy and light chains) by proteolysis while secreted from cells expressing the coagulation factor eight molecule, and is present as a single polypeptide chain. Refers to a blood coagulation factor eight molecule.
- the coagulation factor 8 variant may be expressed as a polynucleotide encoding the amino acid sequence.
- polynucleotide (s) refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA.
- the polynucleotides of the present invention may be single chain DNA or RNA.
- the term “polynucleotide (s)” includes DNA or RNA comprising modified bases and / or unique bases such as inosine. It is obvious that various modifications can be made to the DNA or RNA to serve a known useful purpose.
- polynucleotide (s)” includes such chemically, enzymatically or metabolically modified polynucleotides.
- coagulation Variant 8 variants can be encoded by several polynucleotides.
- the polynucleotide sequence encoding the coagulation factor eight variant available in the present invention is also interpreted to include a nucleotide sequence showing substantial identity with the amino acid sequence.
- composition for increasing gene expression according to the present invention When the composition for increasing gene expression according to the present invention is administered together with plasmids containing a single chain polynucleotide encoding the gene, it is possible to further increase the expression efficiency and efficacy thereof.
- plasmid generally refers to a circular DNA molecule formed operably linked to a vector so that a foreign gene can be expressed in a host cell.
- the plasmid can be used as a vector that is degraded by specific restriction enzymes by gene recombination to incorporate the desired gene and introduces a new gene.
- plasmids and vectors are used interchangeably herein, and those skilled in the art of genetic engineering will fully understand their meanings even if they do not distinguish their names.
- vector refers to a DNA molecule as a carrier that can stably transport a foreign gene into a host cell. To be a useful vector, it must be replicable, have a way to enter the host cell, and have a means to detect its presence.
- composition for increasing gene expression according to the present invention can further increase the expression efficiency and efficacy according to the gene when administered with expression vectors comprising a single chain polynucleotide encoding the gene.
- the term "expression” refers to the generation of the gene in a cell.
- expression vector refers to a gene construct that is capable of expressing the gene in a suitable host, and includes a gene construct comprising essential regulatory elements operably linked to express the gene insert.
- operably linked refers to the functional linkage of a nucleic acid expression control sequence and a polynucleotide encoding the gene to perform a general function.
- a promoter and a polynucleotide encoding the gene can be operably linked to affect the expression of the polynucleotide.
- Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.
- Expression vectors of the present invention are prepared using a plasmid, a vector or a viral vector, but is not limited thereto.
- Suitable expression vectors can include expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and the like, and can be prepared in a variety of ways, and the promoters of the vectors may be constitutive or inducible. Can be. Since plasmids are the most commonly used form of current vectors, "plasmid” and “vector” are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors.
- Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include several to hundreds of plasmid vectors per host cell, and (b) restrictions on which foreign DNA fragments can be inserted. It has a structure including an enzyme cleavage site. Although no suitable restriction enzyme cleavage site is present, synthetic oligonucleotide adapters or linkers according to conventional methods can be used to facilitate ligation of the vector and foreign DNA.
- Skeletal vectors that can be used in the present invention are not particularly limited, but the group consisting of pCDNA3.1, pGP, pEF, pVAX, pUDK, pCK, pQE40, pT7, pET / Rb, pET28a, pET-22b (+) and pGEX
- Various vectors selected from can be used, and it is preferable in terms of effect to be prepared using one vector selected from the group consisting of pGP, pEF, pCK, pUDK and pVAX.
- the expression vector of the present invention may be an expression vector comprising a pGP vector having a cleavage map of FIG.
- the present invention also provides a pharmaceutical composition for preventing or treating bleeding disease or bleeding comprising the composition for increasing gene expression, and the coagulation factor 8 gene or a variant gene thereof.
- the bleeding disease may be hemophilia A, hemophilia 8 or hemophilia caused or complexed with inhibitory antibodies against hemagglutination factor 8 or hemagglutination factor a.
- the bleeding disease or bleeding may include neonatal coagulopathy; Severe liver disease; Hypoplatelet; Congenital deficiency of factor V, VII, X or XI; And von Willebrand disease having an inhibitor against von Willebrand factor; may be one selected from the group consisting of.
- the bleeding may be due to blood loss during a high risk surgical procedure, traumatic blood loss, blood loss due to bone marrow transplantation or blood loss associated with cerebral hemorrhage.
- the pharmaceutical composition for preventing or treating the bleeding disease or bleeding has a good therapeutic effect due to the increased expression of the gene in the body even with a small amount of the coagulation factor 8 gene or a variant gene thereof. It can be usefully used for the prevention or treatment of.
- the composition for increasing gene expression the coagulation factor 8 or a variant gene thereof may be included in a ratio of 1: 0.5 to 30 by volume (w / v).
- the pharmaceutical composition of the present invention may be for gene therapy.
- compositions described herein can be formulated into therapeutic pharmaceutical preparations.
- compositions of the present invention may be formulated in lyophilized or stable liquid form.
- the composition of the present invention can be lyophilized through various procedures known in the art. Lyophilized formulations are reconstituted prior to use with the addition of one or more pharmaceutically acceptable diluents, such as sterile water for injection or sterile saline.
- the formulation of the composition is delivered to the subject by any pharmaceutically suitable means of administration.
- Various delivery systems are known and can be used to administer the composition by any convenient route.
- the composition of the present invention is administered systemically.
- the insert proteins of the invention may be delivered parenterally (eg intravenously, subcutaneously, intramuscularly, intraperitoneally, intracranially, intrapulmonally, intranasally or transdermally) or intestine (eg oral, vaginal) according to conventional methods. Or rectal) for delivery.
- the most preferred route of administration is intravenous and intramuscular administration.
- These formulations may be administered continuously by infusion or bolus injection. Some formulations include sustained release systems.
- compositions of the present invention are administered to a patient in a therapeutically effective amount meaning a dose sufficient to produce the desired effect while preventing or reducing the severity or spread of the condition or indication to be treated without reaching a dose that causes unacceptable side effects.
- the exact dose depends on various factors such as signs, dosage form, mode of administration, etc. and should be determined preclinical and clinically for each indication.
- composition of the present invention may be administered alone or in combination with other therapeutic agents. Such agents may be included as part of the same agent.
- the present invention is a subject suffering from a bleeding disease, such as hemophilia A, hemophilia B or acquired hemophilia; Or an individual suffering from chronic or liver disease.
- the method of treatment may comprise administering to a subject an effective amount of a pharmaceutical composition of the present invention.
- genes such as the coagulation factor 8 gene or variants thereof of the present invention may be administered at a dosage of 10 ng to 100 mg. Dosages may be the same or different each time when the administration of the coagulation factor 8 or variant thereof, or polynucleotide encoding the same is repeated more than once.
- the gene of the full-length coagulation factor 8 (F8) represented by SEQ ID NO: 1 was produced by requesting from Genscript Corporation (USA).
- a full length factor VIII gene represented by SEQ ID NO: 1 was used as a template, and fragment 1 represented by SEQ ID NO: 4 was prepared by polymerase chain reaction (PCR) using primers 1 and 2. It was. PCR was made by mixing 2 ⁇ l of template DNA, 1 ⁇ l of 10 pmol / ⁇ l primer, 2.5 ⁇ l of 2.5mM dNTP, 1 ⁇ l of pfu enzyme mix (Enzynomics, Korea), 2.5 ⁇ l of 10x buffer, and 50 ⁇ l of sterilized tertiary distilled water. Thereafter, the solution was reacted for 30 seconds at 95 ° C., 30 seconds at 60 ° C., and 30 seconds at 72 ° C.
- PCR polymerase chain reaction
- FIG. 1 is a schematic diagram showing the process of designing the coagulation factor eight factor (F8M) according to an embodiment of the present invention.
- the primers are summarized in Table 1 below.
- F8 ATGCAAATAGAGCTCTCCACCTGCTTCTTT 2
- F8M-1 R
- F8M-2 F8M-2 (F) CCACAATTCCAGAAAATACTACTCTTCAGTCAGATCAAGAGGAAATTGAC 4
- F8 final F8 final (F) GCTAGCATGCAAATAGAGCTCTCCACCTGC 6
- F8 final GCGGCCGCTCAGTAGAGGTCCTGTGCCTCGCA
- composition for increasing gene expression Composition for increasing gene expression
- compositions F8M-MP1 and F8M-MP2 were prepared by mixing 15 ⁇ l of the respective gene expression increasing compositions (MP1 and MP2) with 70 ⁇ g / 35 ⁇ l of the pGP-F8M prepared above.
- composition for increasing gene expression Composition for increasing gene expression
- compositions F9-MP1 and F9-MP2 were prepared by mixing 15 ⁇ l of the respective gene expression increasing compositions (MP1 and MP2) with 70 ⁇ g / 35 ⁇ l of the pGP-F9 prepared above.
- composition for increasing gene expression Composition for increasing gene expression
- a suspension (corresponding to a composition for increasing gene expression) was prepared by mixing 128 ⁇ l of JetPEI with 2 ml 5% glucose according to the in vivo JetPEI (Polyplus, USA) manufacturer's manual.
- the pharmaceutical composition F8M-JetPEI was prepared by mixing 15 ⁇ l of the composition and 70 ⁇ g / 35 ⁇ l of the prepared pGP-F8M.
- mice were slaughtered and muscles at the injection site were cut out. Each of the cut muscles was pulverized using liquid nitrogen and a protein extraction kit (Cellbiolabs, USA), and total protein was isolated. The isolated total protein mass was measured using a DC protein assay kit (Bio-Rad laboratories, USA).
- the administration of the pharmaceutical composition (example) according to the present invention shows a statistically significantly higher expression level compared to the case of administration of the gene alone or the composition of the control group.
- the group administered with F8M-MP1 showed 44% higher expression than the group administered with gene only (pGP-F8M), and the group administered with F8M-MP2 compared with the group administered with gene only. 116% high expression level.
- the administration of the composition (example) according to the present invention shows a significantly higher expression compared to the case of administration of the gene alone, or the administration of the composition of the control or comparative example. Able to know.
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Abstract
Description
본 발명은 코어-쉘 구조의 마이크로 입자를 유효성분으로 포함하는 혈액응고 인자 유전자 발현 증가용 조성물에 관한 것이다.
혈우병(Hemophilia)은 심각한 유전 질환 중 하나이다. A형 혈우병은 X 유전자 관련 질환으로 남성 5000명 중 1명에서 발병하고, 혈액응고 과정의 중요한 성분인 혈장 당단백질 인간 혈액응고 제8인자(Factor VIII, Factor 8, FVIII 또는 F8이라고도 함)의 변이에 의해 발생된다.
본 발명의 발명자들은 적은 양의 유전자로도 치료 효과를 얻을 수 있는 유전자 치료제를 개발하기 위해 연구하던 중, 코어가 할로겐화 탄화수소 및/또는 할로겐화 황이고, 외곽 쉘이 지질 성분으로 구성된 코어-쉘 구조의 마이크로 입자가 상기 혈액응고 제8인자 유전자와 함께 생체 내에 투여되는 경우 상기 혈액응고 인자 유전자의 발현량을 현저히 증가시킴을 구체적으로 확인하여 본 발명을 완성하였다.
(특허문헌 001) 대한민국등록특허 제10-1542752호
(특허문헌 002) 대한민국공개특허 제10-2018-0118659호
본 발명이 해결하고자 하는 첫 번째 과제는 코어-쉘 구조의 마이크로 입자를 유효성분으로 포함하는 혈액응고 인자 유전자 발현 증가용 조성물을 제공하는 것이다.
상기한 목적을 달성하기 위하여 본 발명은 코어-쉘 구조의 마이크로 입자를 유효성분으로 포함하는 혈액응고 인자 유전자의 발현 증가용 조성물로서, 상기 코어는 생적합성 기체로서 할로겐화 탄화수소, 할로겐화 황 또는 이들의 혼합물이고, 상기 쉘은 지질 또는 이의 유도체를 포함하여 구성되며, 상기 혈액응고 인자 유전자는 인간 혈액응고 제8인자(Factor VIII) 유전자 또는 이의 변이체 유전자 중에서 선택되는 1종 이상의 유전자인 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물을 제공한다.
본 발명의 일 실시예에 있어서, 상기 할로겐화 탄화수소는 퍼플루오르화 탄화수소일 수 있다.
본 발명의 일 실시예에 있어서, 상기 퍼플루오르화 탄화수소는 퍼플루오로메탄, 퍼플루오로에탄, 퍼플루오로프로판, 퍼플루오로부탄, 퍼플루오로펜탄, 퍼플루오로헥산, 퍼플루오로헵탄, 퍼플루오로프로펜, 퍼플루오로부텐, 퍼플루오로부타디엔, 퍼플루오로부트-2-인, 퍼플루오로시클로부탄, 퍼플루오로메틸시클로부탄, 퍼플루오로디메틸시클로부탄, 퍼플루오로트리메틸시클로부탄, 퍼플루오로시클로펜탄, 퍼플루오로메틸시클로펜탄, 퍼플루오로디메틸시클로펜탄, 퍼플루오로메틸시클로헥산, 퍼플루오로메틸시클로헥산, 퍼플루오로메틸시클로헥산 또는 그것의 혼합물일 수 있다.
본 발명의 일 실시예에 있어서, 상기 지질은 단순지질, 인지질, 글리세로 당지질, 스핑고 당지질, 콜레스테롤 및 양이온 지질로 이루어진 군에서 선택된 1종 이상일 수 있다.
본 발명의 일 실시예에 있어서, 상기 혈액응고 제8인자 유전자는 서열번호 1로 표시되는 아미노산 서열로 이루어진 것이고, 상기 혈액응고 제8인자의 변이체 유전자는 서열번호 3으로 표시되는 아미노산 서열로 이루어진 것일 수 있다.
본 발명의 일 실시예에 있어서, 상기 조성물은 상기 혈액응고 인자 유전자의 발현량을 30% 이상 증가시킬 수 있다.
또한, 본 발명은 상기 조성물, 및 인간 혈액응고 제8인자 유전자 또는 이의 변이체 유전자를 포함하는 출혈 질환 또는 출혈의 예방 또는 치료용 약학적 조성물을 제공한다.
본 발명의 일 실시예에 있어서, 상기 출혈 질환 또는 출혈은 신생 응고병증; 중증 간질환; 저혈소판증; 인자 V, VII, X 또는 XI의 선천성 결핍; 및 폰 빌레브란트 인자에 대한 저해제를 갖는 폰 빌레브란트병;으로 이루어진 군으로부터 선택된 하나일 수 있다.
본 발명의 일 실시예에 있어서, 상기 출혈은 고위험 외과 수술 과정의 혈액 손실, 외상성 혈액 손실, 골수 이식에 의한 혈액 손실 또는 뇌출혈과 연관된 혈액손실로 인한 것일 수 있다.
본 발명의 유전자 발현 증가용 조성물은 혈액응고 제8인자 유전자 또는 이의 변이체 유전자(예를 들어, 유전자를 암호화하는 폴리뉴클레오타이드 또는 이를 포함하는 벡터)와 함께 생체에 투여되는 경우 상기 혈액응고 인자 유전자의 발현량을 적어도 30% 이상 증가시킬 수 있다. 이에 따라, 상기 조성물은 유전자 치료제와 함께 투여하는 경우, 적은 양의 유전자로도 치료 효과를 얻을 수 있어 유용하다.
도 3은 마우스에서 pGP-F8M(유전자만), 대조군의 약학 조성물(F8M-JetPEI) 및 실시예에 따른 약학 조성물(F8M-MP1 및 F8M-MP2)의 투여에 따른 F8 단백질의 발현량을 비교하여 나타내는 그래프이다.
도 4는 마우스에서 pGP-F9(유전자만) 및 비교예에 따른 약학 조성물(F9-MP1 및 F9-MP2)의 투여에 따른 F9 단백질 발현량을 비교하여 나타내는 그래프이다.
본 발명의 일 측면에 따르면, 코어-쉘 구조의 마이크로 입자를 유효성분으로 포함하는 혈액응고 인자 유전자 발현 증가용 조성물로서, 상기 코어는 생적합성 기체로서 할로겐화 탄화수소, 할로겐화 황 또는 이들의 혼합물이고, 상기 쉘은 지질 또는 이의 유도체를 포함하여 구성되며, 상기 혈액응고 인자 유전자는 인간 혈액응고 제8인자(Factor VIII) 유전자 또는 이의 변이체 유전자 중에서 선택되는 1종 이상의 유전자인 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물을 제공한다.
상기 할로겐화 탄화수소로는 퍼플루오르화 탄화수소인 것이 바람직하다.
상기 퍼플루오르화 탄화수소로는, 퍼플루오로메탄, 퍼플루오로에탄, 퍼플루오로프로판, 퍼플루오로부탄, 퍼플루오로펜탄, 퍼플루오로헥산, 퍼플루오로헵탄, 퍼플루오로프로펜, 퍼플루오로부텐, 퍼플루오로부타디엔, 퍼플루오로부트-2-인, 퍼플루오로시클로부탄, 퍼플루오로메틸시클로부탄, 퍼플루오로디메틸시클로부탄, 퍼플루오로트리메틸시클로부탄, 퍼플루오로시클로펜탄, 퍼플루오로메틸시클로펜탄, 퍼플루오로디메틸시클로펜탄, 퍼플루오로메틸시클로헥산, 퍼플루오로메틸시클로헥산, 퍼플루오로메틸시클로헥산 또는 그것의 혼합물을 들 수 있다.
상기 지질은 단순지질, 인지질, 글리세로 당지질, 스핑고 당지질, 콜레스테롤 및 양이온 지질로 이루어진 군에서 선택된 1종 이상일 수 있는데, 특히 인지질인 것이 바람직하다.
상기 스핑고 당지질로는, 갈락토실 세레브로시드, 락토실 세레브로시드, 강글리오사이드 등을 들 수 있다.
마이크로 입자
본 발명의 마이크로 입자는 코어인 기체를 둘러싸는 쉘에 의해 안정화되어, 기체의 주위 액체로의 확산을 지연시키고 마이크로 입자 사이의 융합을 방지한다.
상기 마이크로 입자는 생체 내에 투입되는 경우 표적 세포 또는 조직 부근에 도착하기까지는 모양을 유지하다가 표적 세포 또는 조직 근처에서 파괴되면서 기체를 분사하게 된다. 이때 분사되는 기체는 표적 세포의 세포막에 변화를 일으키고, 기체의 분사력에 의해 유전자가 표적 세포의 세포질 환경 중으로 진입될 수 있도록 촉진하는 역할을 할 수 있다.
상기 마이크로 입자는 평균 직경이 1 내지 10 ㎛, 바람직하게는 2 내지 8 ㎛, 더욱 바람직하게는 2 내지 4 ㎛인 것이 바람직하다.
최근, 기체를 코어로 하는 코어-쉘 마이크로 입자와 관련하여 다양한 연구가 이루어져 왔다. 기존 연구들에서는 마이크로 입자와 함께 초음파를 조사하지 않으면 유전자 발현 증가 효과가 나타나지 않았다.
구체적으로, Sang-Chol Lee et al., Korean Circulation J 2006;36:32-38; "저주파 초음파를 이용한 미세기포 파괴를 통한 근육조직으로의 유전자 전달 증강에 대한 연구"에는, 초음파 조사 없이 luciferase 유전자-마이크로 입자 혼합물만 주입하는 경우에는 유전자 발현 증가 효과가 나타나지 않는다는 내용이 개시되어 있다.
ZP Shen et al., Gene Therapy(2008) 15, 1147-1155; "Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery"에는 초음파 조사 없이 luciferase 유전자-마이크로 입자 혼합물만 주입하는 경우에는 유전자 발현 증가 효과가 나타나지 않는다는 내용이 개시되어 있다.
또한, Xingsheng Li et al., J Ultrasound Med 2008; 27:453-460; "Experimental Research on Therapeutic Angiogenesis Induced by Hepatocyte Growth Factor Directed by Ultrasound-Targeted Microbubble Destruction in Rats"에는 초음파 조사 없이 HGF 유전자-리포좀 마이크로 입자 혼합물을 주입하는 경우에는 유전자 발현 증가 효과가 나타나지 않는다는 내용이 개시되어 있다.
그러나, 본 발명자들은 상기 마이크로 입자의 용도에 대하여 연구하던 중 본 발명에 따른 마이크로 입자를 혈액응고 인자 중에서도 혈액응고 제8인자 유전자 또는 이의 변이체와 함께 주입하는 경우에는 특이하게도 초음파의 조사 없이도 상기 유전자의 발현량을 현저히 증가시키는 것을 확인하여 본 발명을 완성하게 되었다. 한편, 하기 실험예에서 구체적으로 나타낸 바와 같이, 상기 혈액응고 제8인자 유전자 또는 이의 변이체 유전자 이외의 혈액응고 인자 유전자(예를 들어, 혈액응고 제9인자(Factor IX, Factor 9, FIX 또는 F9라고도 함))에서는 상기 마이크로 입자로 인한 유전자 발현량의 증가 효과가 나타나지 않았다.
본 발명에 따른 유전자 발현 증가용 조성물은 혈액응고 제8인자 유전자 또는 이의 변이체 유전자와 함께 생체에 투여되는 경우 상기 혈액응고 인자 유전자의 발현량을 적어도 30% 이상 증가시킬 수 있다.
즉, 본 발명의 유전자 발현 증가용 조성물은 혈액응고 인자 중에서도 인간 혈액응고 제8인자 유전자 및 이의 변이체 유전자 중에서 선택되는 1종 이상의 유전자와 함께 투여되는 경우에만 특이적으로 상기 혈액응고 인자 유전자의 발현량을 증가시키는 효과가 있는 것으로 판단된다.
본 발명의 유전자 발현 증가용 조성물에서, 상기 마이크로 입자의 함량은 특별히 제한되지 않으나, 0.5 내지 2,000 ㎕/㎖, 바람직하게는 1 내지 1,000 ㎕/㎖로 함유될 수 있다. 또는, 5 내지 2,000 ㎍/㎖, 바람직하게는 10 내지 1,000 ㎍/㎖로 함유될 수 있다.
상기 마이크로 입자의 함량이 상기 범위를 벗어나는 경우에는 기대하는 효과를 얻을 수 없게 된다.
본 발명에 따른 유전자 발현 증가용 조성물은 하기 유전자들과 함께 투여되는 경우 하기 유전자의 발현 효율 및 이에 따른 효능을 더욱 증대시킬 수 있다.
본 발명의 실시예에 따른 혈액응고 제8인자 변이체는 단일쇄의 혈액응고 제8인자 변이체로서 서열번호 1로 표시되는 혈액응고 제8인자로부터 Asp784 내지 Arg1671의 아미노산이 결실된 것이다. 상기 변이체는 단일쇄의 혈액응고 제8인자 변이체이다. 상기 혈액응고 제8인자 변이체는 B-도메인 일부(잔기들 784-1667) 및 a3 영역의 일부(잔기들 1668-1671)가 결실된 것으로, 상기 변이체는 혈액응고 제8인자, B-도메인 결실된 혈액응고 제8인자에 비해 단백질 발현량이 증대되고 혈액 응고 활성 및 안정성이 향상된다. 상기 혈액응고 제8인자 변이체는 서열번호 3으로 표시되는 아미노산 서열을 갖는다.
상기 "단일쇄의 혈액응고 제8인자"는 상기 혈액응고 제8인자 분자를 발현하는 세포로부터 분비되는 동안 단백질 분해에 의해 두 쇄(예: 중쇄와 경쇄)로 절단되지 않고, 단일 폴리펩타이드 쇄로 존재하는 혈액응고 제8인자 분자를 의미한다.
본 명세서에서, "벡터"라는 용어는 외래 유전자를 숙주 세포 내로 안정적으로 운반할 수 있는 운반체로서의 DNA 분자를 말한다. 유용한 벡터가 되기 위해서는 복제될 수 있어야 하며, 숙주 세포 내로 유입할 수 있는 방안을 갖추어야 하고, 자신의 존재를 검출할 수 있는 수단을 구비하여야 한다.
본 발명의 발현벡터는 플라스미드, 벡터 또는 바이러스 벡터를 이용하여 제작되나, 이에 한정되는 것은 아니다. 적합한 발현벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 등을 포함할 수 있으며, 목적에 따라 다양하게 제조될 수 있고, 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 플라스미드가 현재 벡터의 가장 통상적으로 사용되는 형태이므로, 본 발명의 명세서에서 "플라스미드(plasmid)" 및 "벡터(vector)"는 때로 상호 교환적으로 사용된다. 본 발명의 목적상, 플라스미드 벡터를 이용하는 게 바람직하다. 이러한 목적에 사용될 수 있는 전형적인 플라스미드 벡터는 (a) 숙주세포당 수 개에서 수백 개의 플라스미드 벡터를 포함하도록 복제가 효율적으로 이루어지도록 하는 복제 개시점, 및 (b) 외래 DNA 절편이 삽입될 수 있는 제한효소 절단부위를 포함하는 구조를 지니고 있다. 적절한 제한효소 절단 부위가 존재하지 않을지라도, 통상의 방법에 따른 합성 올리고 뉴클레오타이드 어댑터(oligonucleotide adaptor) 또는 링커(linker)를 사용하면 벡터와 외래 DNA를 용이하게 라이게이션(ligation)할 수 있다.
또한, 본 발명은 상기의 유전자 발현 증가용 조성물, 및 상기의 혈액응고 제8인자 유전자 또는 이의 변이체 유전자를 포함하는 것을 특징으로 하는 출혈 질환 또는 출혈의 예방 또는 치료용 약학적 조성물을 제공한다.
상기 출혈 질환은 A형 혈우병, 혈액응고 제8인자 또는 혈액응고 제8인자a에 대한 억제성 항체에 의하여 야기되거나 복합화된 혈우병 또는 B형 혈우병일 수 있다. 또한, 상기 출혈 질환 또는 출혈은 신생 응고병증; 중증 간질환; 저혈소판증; 인자 V, VII, X 또는 XI의 선천성 결핍; 및 폰 빌레브란트 인자에 대한 저해제를 갖는 폰 빌레브란트병;으로 이루어진 군으로부터 선택된 하나일 수 있다.
그리고, 상기 출혈은 고위험 외과 수술 과정의 혈액 손실, 외상성 혈액 손실, 골수 이식에 의한 혈액 손실 또는 뇌출혈과 연관된 혈액손실로 인한 것일 수 있다.
상기 출혈 질환 또는 출혈의 예방 또는 치료용 약학적 조성물은 적은 양의 혈액응고 제8인자 유전자 또는 이의 변이체 유전자로도 체내에서 상기 유전자의 발현이 증가됨으로 인해 우수한 치료 효과를 나타내므로 상기 출혈 질환 또는 출혈의 예방 또는 치료에 유용하게 이용될 수 있다.
본 발명의 약학 조성물에서, 상기 유전자 발현 증가용 조성물 : 상기 혈액응고 제8인자 또는 이의 변이체 유전자는 1 : 0.5 내지 30 부피비(w/v)로 포함될 수 있다.
이러한 약제학적 담체 및 부형제 뿐만 아니라 적당한 약제학적 제형은 당업계에 공지되어 있다(예컨대, "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor & Francis(2000) 또는 "Handbook of Pharmaceutical Excipients", 3rd edition, Kibbe et al., Pharmaceutical Press(2000)). 특히, 본 발명의 약학적 조성물은 동결건조 형태 또는 안정한 액체 형태로 제형화될 수 있다. 본 발명의 조성물은 당업계에 공지된 다양한 절차를 통해 동결건조될 수 있다. 동결건조된 제형은 주사용 멸균수 또는 멸균 생리식염수와 같은 하나 이상의 약제학적으로 허용되는 희석제를 첨가하여 사용하기 전에 재구성시킨다.
또한, 본 발명은 A형 혈우병, B형 혈우병 또는 후천적 혈우병과 같은 출혈 질환을 앓고 있는 개체; 또는 만성 질환 또는 간 질환을 앓고 있는 개체;를 치료하는 방법에 관한 것이다. 상기 치료 방법은 본 발명의 약학적 조성물을 유효량으로 개체에게 투여하는 단계를 포함할 수 있다.
본 발명의 일 구현예에 따르면, 본 발명의 혈액응고 제8인자 유전자 또는 이의 변이체 등의 유전자는 10 ng 내지 100 mg의 투여량으로 투여될 수 있다. 상기 혈액응고 제8인자 또는 이의 변이체, 또는 이를 암호화하는 폴리뉴클레오타이드의 투여가 일회를 초과하여 반복될 때 투여량은 매회 동일하거나 다를 수 있다.
서열번호 1로 표시되는 전장 혈액응고 제8인자(full length factor VIII) 유전자를 주형으로 하고, 프라이머 1과 2를 이용하여 중합효소연쇄반응 (PCR)을 통해 서열번호 4로 표시되는 절편 1을 제조하였다. PCR은 주형 DNA 2㎕, 10pmol/㎕ 프라이머 각 1㎕, 2.5mM dNTP 2.5㎕, pfu enzyme mix(Enzynomics, Korea) 1㎕와 10x buffer 2.5㎕와 멸균한 3차 증류수를 50㎕까지 채워서 혼합액을 만든 후, 이 용액을 95 ℃에서 30초, 60 ℃에서 30초, 72 ℃에서 30초 동안 반응시키는 과정을 40회 반복하여 수행하였다. 프라이머 3과 4를 이용하여 위의 방법과 같은 PCR을 통해 서열번호 5로 표시되는 절편 2를 제조하였다. 이후, 상기 절편 1과 2를 프라이머 5 및 6을 이용하여 overlapping PCR을 수행함으로써, 단일쇄 혈액응고 제8인자 변이체를 제조하였다. overlapping PCR은 DNA절편을 각각 2㎕ 넣고 다른 효소와 완충제, 프라이머 등과 반응시간 및 방법은 PCR과 동일하게 사용하였다. 도 1은 본 발명의 일 실시예에 따른 혈액응고 제8인자 변이체(F8M)를 디자인하는 과정을 나타내는 모식도이다.
프라이머번호 | 프라이머명 | 염기서열 |
1 | F8(F) | ATGCAAATAGAGCTCTCCACCTGCTTCTTT |
2 | F8M-1(R) | TCTGACTGAAGAGTAGTATTTTCTGGAATTGTGGTGGCATTAAAT |
3 | F8M-2(F) | CCACAATTCCAGAAAATACTACTCTTCAGTCAGATCAAGAGGAAATTGAC |
4 | F8(R) | TCAGTAGAGGTCCTGTGCCTCGCAGCCCAG |
5 | F8 final(F) | GCTAGCATGCAAATAGAGCTCTCCACCTGC |
6 | F8 final(R) | GCGGCCGCTCAGTAGAGGTCCTGTGCCTCGCA |
서열번호 6으로 표시되는 전장 혈액응고 제9인자(GenBank 염기서열 FR846239.1참고)의 유전자를 바이오닉스사(대한민국)에 의뢰하여 제작하였다.
이 등(Lee Y, et al. Improved expression of vascular endothelial growth factor by naked DNA in mouse skeletal muscles: implication for gene therapy of ischemic diseases. Biochem. Biophys. Res. Commun. 2000;272(1):230-235)의 논문을 참고하여 pCK 플라스미드를 합성한 후 하기 표 2의 프라이머 7 및 8을 이용하여 위에서 기술한 방법과 동일한 방법으로 PCR을 하여 절편을 얻고 EcoRI 효소로 37 ℃에서 1시간 동안 반응한 후 Expin Gel SV (GeneAll, Korea) kit를 이용하여 DNA를 정제하였다. 이후, T4 ligase를 이용하여 30분간 ligation한 후 E.coli에 overnight 배양하였다. 다음날 colony를 배양한 후 mini-prep하여 DNA를 분리하여 서열번호 7로 표시되는 pGP 플라스미드를 제작하였다. 도 2는 본 발명의 일 실시예에 따른 pGP 벡터의 개열지도를 나타낸 것이다.
상기에서 준비한 유전자들 각각과 pGP 플라스미드를 각각 NheI과 NotI 효소로 1시간 동안 절단하고 아가로즈젤에 전기영동하여 절편을 분리하였다. 분리된 절편을 T4 ligase를 이용하여 30분간 ligation하고 E. Coli에 overnight 배양하였다. 다음날 colony를 배양한 후 mini-prep하여 DNA를 분리하여 NheI과 NotI으로 확인하였다. 클로닝이 완료된 DNA는 제한효소로 확인된 E. Coli supernatant를 4 L 플라스크 두 병에 kanamycin과 함께 넣고 overnight 배양한 후 Endofree Giga prep. kit (Qiagen, USA)를 이용하여 플라스미드 DNA를 생산하고 동물실험에 사용하였다. 상기 제조된 각각의 플라스미드 DNA를 하기 표 3에 정리하여 나타내었다.
유전자 | 플라스미드 DNA |
혈액응고 제8인자(F8) | pGP-F8 |
혈액응고 제8인자 변이체(F8M) | pGP-F8M |
혈액응고 제9인자(F9) | pGP-F9 |
실시예: 유전자 발현 증가용 조성물 및 F8M을 포함하는 약학 조성물의 제조(F8M-MP1 및 F8M-MP2)
평균 직경이 약 2.5 ㎛이고 코어는 헥사플루오르화황, 쉘은 지질을 포함하여 구성되었으며 표준코드 621400210으로 표시되는 코어-쉘 구조의 마이크로 입자를 브라코이미징코리아로부터 구입하였다(MP1). 또한, 평균 직경이 약 2.4 내지 3.6 ㎛이고, 코어는 퍼플루오로부탄, 쉘은 지질 및 계면활성제를 포함하여 구성되었으며 표준코드 646300210으로 표시되는 코어-쉘 구조의 마이크로 입자를 지이헬스케어 에이에스 한국지점으로부터 구입하였다(MP2).
상기 각각의 유전자 발현 증가용 조성물(MP1 및 MP2) 15 ㎕와 상기 제조된 pGP-F8M 70 ㎍/35 ㎕을 혼합하여 각각의 약학 조성물 F8M-MP1 및 F8M-MP2를 제조하였다.
비교예: 유전자 발현 증가용 조성물 및 F9를 포함하는 약학 조성물의 제조(F9-MP1 및 F9-MP2)
상기 실시예와 동일한 방법으로 유전자 발현 증가용 조성물을 제조하였다.
상기 각각의 유전자 발현 증가용 조성물(MP1 및 MP2) 15 ㎕와 상기 제조된 pGP-F9 70㎍/35㎕을 혼합하여 각각의 약학 조성물 F9-MP1 및 F9-MP2를 제조하였다.
in vivo JetPEI(Polyplus, USA)의 제조사의 매뉴얼에 따라 JetPEI 128㎕를 2ml 5% glucose와 혼합하여 현탁액(유전자 발현 증가용 조성물에 대응)을 제조하였다.
상기 조성물 15 ㎕와 상기 제조된 pGP-F8M 70 ㎍/35 ㎕을 혼합하여 약학 조성물 F8M-JetPEI를 제조하였다.
실험예 : 마우스에서의 단백질 발현량
투여 후 7일째에 마우스를 도살하고 주사한 부위의 근육을 잘라내었다. 그리고, 상기 잘라낸 각각의 근육들을 액체 질소 및 단백질 추출킷트(Cellbiolabs, USA)를 이용하여 분쇄한 후 총단백질을 분리하였다. 분리된 총단백질량은 DC 단백질 분석킷트(Bio-Rad laboratories, USA)를 이용하여 측정하였다.
도 3을 통해, 본 발명에 따른 약학 조성물(실시예)을 투여한 경우 유전자만 투여한 경우 또는 대조군의 조성물을 투여한 경우에 비해 통계적으로 유의하게 높은 발현량을 나타내는 것을 알 수 있다. 구체적으로, 실시예 중에서 F8M-MP1을 투여한 군은 유전자만 투여한 군(pGP-F8M)에 비해 44% 높은 발현량을 나타내었고, F8M-MP2를 투여한 군은 유전자만 투여한 군에 비해 116% 높은 발현량을 나타내었다.
도 4를 통해, 비교예의 조성물을 투여한 경우에는 유전자만 투여한 경우에 비해 유전자 발현이 거의 증가하지 않음을 알 수 있다.
즉, 상기 도 3 및 도 4을 통해, 본 발명에 따른 조성물(실시예)을 투여한 경우에는 유전자만 투여한 경우, 또는 대조군이나 비교예의 조성물을 투여한 경우에 비해 현저히 높은 발현량을 나타내는 것을 알 수 있다.
Claims (15)
- [규칙 제91조에 의한 정정 18.04.2019]
코어-쉘 구조의 마이크로 입자를 유효성분으로 포함하는 혈액응고 인자 유전자 발현 증가용 조성물로서,상기 코어는 생적합성 기체로서 할로겐화 탄화수소, 할로겐화 황 또는 이들의 혼합물이고, 상기 쉘은 지질 또는 이의 유도체를 포함하여 구성되며,상기 혈액응고 인자 유전자는 인간 혈액응고 제8인자(Factor VIII) 유전자 또는 이의 변이체 유전자 중에서 선택되는 1종 이상의 유전자인 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물. - 제1항에 있어서,상기 생적합성 기체는 헥사플루오르화황, 옥타플루오로프로판, 브로모클로로디플루오로메탄, 클로로디플루오로메탄, 디클로로디플루오로메탄, 브로모트리플루오로메탄, 클로로트리플루오로메탄, 클로로펜타플루오로에탄, 디클로로테트라플루오로에탄 및 그것의 혼합물 중에서 선택되는 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물.
- [규칙 제91조에 의한 정정 18.04.2019]
제1항에 있어서,상기 할로겐화 탄화수소는 퍼플루오르화 탄화수소인 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물. - [규칙 제91조에 의한 정정 18.04.2019]
제3항에 있어서,상기 퍼플루오르화 탄화수소는 퍼플루오로메탄, 퍼플루오로에탄, 퍼플루오로프로판, 퍼플루오로부탄, 퍼플루오로펜탄, 퍼플루오로헥산, 퍼플루오로헵탄, 퍼플루오로프로펜, 퍼플루오로부텐, 퍼플루오로부타디엔, 퍼플루오로부트-2-인, 퍼플루오로시클로부탄, 퍼플루오로메틸시클로부탄, 퍼플루오로디메틸시클로부탄, 퍼플루오로트리메틸시클로부탄, 퍼플루오로시클로펜탄, 퍼플루오로메틸시클로펜탄, 퍼플루오로디메틸시클로펜탄, 퍼플루오로메틸시클로헥산, 퍼플루오로메틸시클로헥산, 퍼플루오로메틸시클로헥산 또는 그것의 혼합물인 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물. - [규칙 제91조에 의한 정정 18.04.2019]
제1항에 있어서,상기 지질은 단순지질, 인지질, 글리세로 당지질, 스핑고 당지질, 콜레스테롤 및 양이온 지질로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물. - 제5항에 있어서,상기 인지질은 포스파티딜콜린 유도체, 포스파티딜에탄올아민 유도체, 포스파티딜세린 유도체, 디아세틸레이티드 인지질, L-α-디올레일 포스파티딜에탄올아민, 디올레인, 포스파티딘산, 포스파티딜글리세롤, 포스파티딜이노시톨, 리소포스파티딜콜린, 스핑고미엘린, 폴리에틸렌 글리콜화 인지질, 난황 레시틴, 대두 레시틴 및 수소첨가 인지질로 이루어진 군에서 선택되는 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물.
- 제5항에 있어서,상기 글리세로 당지질은 설폭시 리보실 글리세라이드, 디글리코실 디글리세라이드, 디갈락토실 디글리세라이드, 갈락토실 디글리세라이드 및 글리코실 디글리세라이드로 이루어진 군에서 선택되는 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물.
- 제5항에 있어서,상기 스핑고 당지질은 갈락토실 세레브로시드, 락토실 세레브로시드 또는 강글리오사이드인 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물.
- 제5항에 있어서,상기 양이온 지질은 1,2-디올레오일-3-트리메틸암모니오 프로판(DOTAP), N-(2,3-디올레일옥시 프로판-1-일)-N,N,N-트리메틸 염화암모늄(DOTMA), 2,3-디올레일옥시-N-[2-(스페르민카르복시아미드) 에틸]-N,N-디메틸-1-프로판아미늄트리플루오로 초산(DOSPA), 1,2-디미리스틸옥시 프로필-3-디메틸하이드록시에틸 브롬화암모늄(DMRIE), 1,2-디올레오일옥시 프로필-3-디에틸 하이드록시에틸 브롬화암모늄(DORIE) 및 3β-[N-(N'N'-디메틸아미노에틸) 카르바모일]콜레스테롤(DC-Chol)로 이루어진 군에서 선택되는 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물.
- 제1항에 있어서,상기 혈액응고 제8인자는 서열번호 1로 표시되는 아미노산 서열로 이루어지고, 상기 혈액응고 제8인자의 변이체는 서열번호 3으로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물.
- [규칙 제91조에 의한 정정 18.04.2019]
제1항에 있어서,상기 조성물은 상기 혈액응고 인자 유전자의 발현량을 30% 이상 증가시키는 것을 특징으로 하는 혈액응고 인자 유전자 발현 증가용 조성물. - [규칙 제91조에 의한 정정 18.04.2019]
제1항의 조성물; 및 인간 혈액응고 제8인자(Factor VIII) 유전자 또는 이의 변이체 유전자;를 포함하는 출혈 질환 또는 출혈의 예방 또는 치료용 약학적 조성물. - 제12항에 있어서,상기 출혈 질환은 A형 혈우병, 혈액응고 제8인자 또는 혈액응고 제8인자a에 대한 억제성 항체에 의하여 야기되거나 복합화된 혈우병 또는 B형 혈우병인 것을 특징으로 하는 출혈 질환 또는 출혈의 예방 또는 치료용 약학적 조성물.
- [규칙 제91조에 의한 정정 18.04.2019]
제12항에 있어서,상기 출혈 질환 또는 출혈은 신생 응고병증; 중증 간질환; 저혈소판증; 인자 V, VII, X 또는 XI의 선천성 결핍; 및 폰 빌레브란트 인자에 대한 저해제를 갖는 폰 빌레브란트병;으로 이루어진 군으로부터 선택된 하나인 것을 특징으로 하는 출혈 질환 또는 출혈의 예방 또는 치료용 약학적 조성물. - [규칙 제91조에 의한 정정 18.04.2019]
제12항에 있어서,상기 출혈은 고위험 외과 수술 과정의 혈액 손실, 외상성 혈액 손실, 골수 이식에 의한 혈액 손실 또는 뇌출혈과 연관된 혈액손실로 인한 것임을 특징으로 하는 출혈 질환 또는 출혈의 예방 또는 치료용 약학적 조성물.
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JP2020543162A JP7236446B2 (ja) | 2018-02-12 | 2019-02-12 | コア-シェル構造のマイクロ粒子を有効成分として含む血液凝固因子遺伝子発現増加用組成物 |
US16/969,040 US20210113480A1 (en) | 2018-02-12 | 2019-02-12 | Composition for increasing expression of blood coagulation factor gene, comprising core-shell structured microparticles as active ingredient |
EP19750424.4A EP3753550A4 (en) | 2018-02-12 | 2019-02-12 | COMPOSITION FOR INCREASING THE EXPRESSION OF THE BLOOD COAGULATION FACTOR GENE, CONSISTING OF HEART-SHELL STRUCTURED MICROPARTICLES AS ACTIVE INGREDIENT |
CN201980025173.XA CN112218622B (zh) | 2018-02-12 | 2019-02-12 | 包含核壳结构的微粒作为活性成分的用于增加凝血因子基因表达的组合物 |
US18/062,252 US20230390211A1 (en) | 2018-02-12 | 2022-12-06 | Composition for increasing expression of blood coagulation factor gene, comprising core-shell structured microparticles as active ingredient |
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CN112218622B (zh) | 2023-10-24 |
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