WO2004092388A1 - ベクター - Google Patents
ベクター Download PDFInfo
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- WO2004092388A1 WO2004092388A1 PCT/JP2004/005450 JP2004005450W WO2004092388A1 WO 2004092388 A1 WO2004092388 A1 WO 2004092388A1 JP 2004005450 W JP2004005450 W JP 2004005450W WO 2004092388 A1 WO2004092388 A1 WO 2004092388A1
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Classifications
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- 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
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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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
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- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Definitions
- the present invention relates to a vector according to a gene transfer technique.
- a cationic lipid dioleoyloxypropyltrimethylammonium, which is commercialized as Lipofectin (registered trademark) ( Natl. Acad. Sci. USA, 84, 7413-7417, 1987).
- Polyethyleneimine has also been commercialized as an exogen as a synthetic polymer for vectors.
- a nucleic acid is formed into an aggregate, and a cationic polymer is surrounded around the aggregate.
- the vector of the present invention comprises a cationic polymer having a branched chain.
- the vector of the present invention is administered in vivo by forming a complex with the nucleic acid aggregate.
- Devices that can be inserted into the body include those that percutaneously penetrate the tissue near the affected area, and those that are placed in the blood vessel, such as a vascular catheter or stent graft.
- Nucleic acids are generally not very stable in vivo and are degraded by certain enzymes.
- the nucleic acid is formed into an aggregate, and a vector containing a cationic polymer is arranged around the aggregate to protect the nucleic acid from the enzyme. It can function normally in the body. It is also possible to introduce a nucleic acid complex into a capsule micelle or the like having a micro or nano order size and administer it into a blood vessel.
- FIG. 1 is a graph showing the measurement results of the granular distribution of the polyion complex.
- FIG. 2 is a graph showing the change over time of the particle size of the polyion complex.
- FIG. 3 is a graph showing the results of a transfection experiment.
- FIG. 4 is a graph showing the measurement results of the relationship between the cation anion ratio and the gene expression efficiency.
- FIG. 5 is a TEM photograph of a gene complex stained with lintandustate.
- FIG. 6 is a TEM photograph of the gene complex stained with peran acetate.
- FIG. 7 is a graph showing the relationship between the molecular weight of a vector having one branch and the gene expression efficiency.
- FIG. 8 is a graph showing the relationship between the molecular weight of a vector having three branches and gene expression efficiency.
- FIG. 9 is a graph showing the relationship between the molecular weight of a vector having four branches and the gene expression efficiency.
- Fig. 10 shows a vector with a molecular weight of 10,000 having four branches, and a di-chain at the tip of the branch.
- 4 is a graph showing the relationship between the molecular weight of a block polymer and gene expression efficiency in block polymerized methylacrylamide.
- FIG. 11 is a graph showing the in-solution stability of the block polymer, branched product and PEI of this vector (time-dependent change when the initial value of the gene expression efficiency is 100%).
- the cationic polymer used in the present invention has a branched chain.
- the effect of the vector is enhanced in a specific molecular weight range, and the range differs depending on the number of branched chains.
- the vector has 3 polymer chains and a molecular weight (number average molecular weight) of 300000 to 600000.
- the vector has 4 polymer chains and a molecular weight of 300000 to 6500.
- the vector has six polymer chains and a molecular weight of 1,000 to 50,000.
- the vector has a nonionic molecular group bound to the leading end of the branched chain of the polymer.
- the vector is a vector comprising a polymer having a multifunctional 1 "raw compound as a nucleus and a plurality of polymer chains bonded to the nucleus as branch chains; A nonionic molecular group is bonded to the front end of the compound.
- the nonionic molecular group may be an aromatic compound, an aliphatic compound, a polyacrylate, or a polymethacrylate.
- a plurality of branched chains are bonded to a polyfunctional compound, preferably a benzene ring, naphthalene, anthracene or pyrene.
- a polyfunctional compound preferably a benzene ring, naphthalene, anthracene or pyrene.
- the number of bonds is from 2 to 6, especially 2, 3, 4 or 6, the more bonds the more effective.
- the number of bonds is possible up to 8
- anthracene and pyrene are possible up to 10.
- biphenyls in which a plurality of aromatic rings that are not in the same plane are bonded in this case, the number of bonds in the branched chain can be up to 10
- cycloparaffinic hydrocarbons such as cyclohexane as a nucleus It is also possible.
- a benzene ring is preferably used as a nucleus for bonding a branched chain, and specific examples thereof include the following.
- 2,4,6-tris (bromomethyl) mesitylene and sodium N, N-getyldithiocarbamate can be obtained by addition reaction in ethanol to give 2,4, 6-Tris (N, N-Jetyldithiocarbamyl) Mesitylene; 4-branched chains are 1,2,4,5-tetrakis (promomethyl) benzene and sodium N, N-Jetyldi 1,2,4,5-tetrakis (N, N-getyldithiocarbamylmethyl) benzene obtained by the addition reaction of thiocarpamate in ethanol, and hexakis (bromomethyl) as the 6-branched chain. Hexakis (N, N-getyldithiocarbamylmethyl) benzene obtained by the addition reaction of benzene with sodium N, N-getyldithiocarbamate in ethanol.
- branched chain a homopolymer or a copolymer of a bullet monomer is preferable, and the length of each branched chain may be the same or different.
- the structure of the chain may be linear, linear or dendritic.
- the 3-N, N-dimethylaminopropylacrylamide and each of the above benzene derivatives are mixed as an alcohol solution such as methanol or a solution of a low-polar solvent such as chloroform in consideration of solubility.
- a force-thione polymer in which the polymer of the above-mentioned monomer is bonded to the benzene ring via one CH 2 derived from the above-mentioned benzene derivative is formed.
- the molecular weight of this vector is preferably 5,000 to 500,000, particularly preferably 5,000 to 100,000, and particularly preferably about 10,000 to 50,000.
- the branched chain When the branched chain is a copolymer, it may be either a random copolymer or a block copolymer. If the monomer contains a cationic functional monomer, it may be copolymerized with a nonionic or hydrophobic monomer. It may be a polymer.
- a nonionic hydrophilic monomer such as N, N-dimethylacrylamide, N-butylpyrrolidone, PEG- (meth) atalylate is preferable, and as the hydrophobic monomer, ethyl (meth) is used. Atarilate, styrene and the like are preferred.
- the vector composed of the cationic polymer thus generated surrounds the nucleic acid as a nucleic acid-containing complex, and the enzyme in vivo Can suppress inactivation and decomposition of nucleic acids.
- the polymer chain constituting the branched chain may have a quaternary amine at the base end and a tertiary amine at the end. If quaternary amine is used near the nucleus to which the branched chain is bound and tertiary amine is used around the nucleus, reduction of cytotoxicity and higher efficiency of gene transfer can be expected. Although quaternary amine is a cation which is an essential element as a vector, it is cytotoxic as used in germicides and antibacterial coats. Is advantageous.
- the hydrophilic polymer may have a property that its positive charge is deactivated with time. By inactivating the positive charges in the vector in this manner, a function of releasing the nucleic acid from the nucleic acid aggregate surrounded by the vector can be provided. Those skilled in the art may appropriately set conditions for releasing the nucleic acid after the vector has passed through the cell membrane.
- Examples of the cationic polymer having such a function include those in which a compound having a 4,4,1-benzylidenebis ( ⁇ , ⁇ -dimethylaniline) skeleton in a long-chain branched chain is supported.
- 4,4, Compounds having a benzylidenebis ( ⁇ ⁇ ⁇ ⁇ , ⁇ _dimethylaniline) skeleton include leucomalachite green, and the cationic polymer that carries the compound is converted into a cathonyl by light irradiation, Has the property of deactivating positive charges.
- a nucleic acid may be added to a dispersion of the vector at a concentration of about 1 to 100 ⁇ g / mL at room temperature and mixed. It is preferable to add an excess amount of the cationic polymer to the nucleic acid, and to conjugate the cationic polymer to the nucleic acid in a saturated state as a nucleic acid-containing complex.
- the nucleic acid is preferably a polynucleotide such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), particularly DNA, but may be a liponucleoprotein.
- Nucleic acids include simple herpesvirus thymidine kinase gene (HSV1-TK gene), p53 tumor suppressor gene and BRC A1 tumor suppressor gene and cytokine gene as TNF-H gene, IL-2 gene, and IL-4 gene.
- HLA-B7 IL-12 gene HLA-B7 / B2M gene, IL-7 gene, GM-CSF gene, I It may be a cytokine gene such as FN- ⁇ gene and IL-112 gene and a cancer antigen peptide gene such as gp-100, MART-1 and MAGE-1 and these can be used for cancer treatment.
- cytokine gene such as FN- ⁇ gene and IL-112 gene
- a cancer antigen peptide gene such as gp-100, MART-1 and MAGE-1 and these can be used for cancer treatment.
- cytodynamic genes such as VEGF gene, HGF gene and FGF gene, c-myc antisense, c-myb antisense, cdc2 kinase antisense, PCNA antisense, and £ 2 decoy 21 (3 (1 1-1) Genes can be used for vascular treatment Such a series of genes is well known to those skilled in the art.
- the particle size of the nucleic acid-containing complex is preferably about 50 to 400 nm. If it is smaller than this, the action of the enzyme may reach the nucleic acid inside the nucleic acid-containing complex, or may be filtered out by the kidney. If it is larger than this, it may be difficult to be introduced into cells.
- the nucleic acid is used in such a form that it can express a function in the cell when introduced into the cell.
- the DNA is used as a plasmid in which the DNA is transcribed in the introduced cells and functionally expressed through production of the polypeptide encoded thereby.
- a promoter region, an initiation codon, a DNA encoding a protein having a desired function, a stop codon, and a terminator region are continuously arranged.
- cells that are desirable as a target to which a nucleic acid is to be introduced are those that require the expression of the function of the nucleic acid.
- examples of such cells include various cells depending on the nucleic acid used (that is, the function). Selected, for example, cardiomyocytes, smooth muscle cells, fibroblasts, skeletal muscle cells, vascular endothelial cells, bone marrow cells, bone cells, blood cell stem cells, blood cell cells, tumor cells, hematopoietic stem cells, peripheral blood stem cells, SP cells, ES cells Cells, B cells, T cells, NK cells and the like.
- Gastrointestinal epithelial cells and renal tubules such as monocytes, dendritic cells, macrophages, histiocytes, cupper cells, osteoclasts, synovial A cells, microglia, Langerhans cells, epithelioid cells, and multinucleated giant cells Epithelial cells and the like.
- the nucleic acid-containing complex using the vector of the present invention can be administered to a living body by any method. Injection into a vein or artery is particularly preferred as the administration method, but intramuscular, adipose tissue, subcutaneous, intradermal, intralymphatic, intralymphatic, intracorporeal (pericardial cavity, thoracic cavity, abdominal cavity, cerebrospinal cord In addition to administration into the cavity and bone marrow, it can also be administered directly into the affected tissue, for example, by spraying into the alveoli using a tracheoscopy.
- nucleic acid-containing complex may further contain a pharmaceutically acceptable carrier (an osmotic pressure adjusting agent, a stabilizing agent, a preservative, a solubilizing agent, a pH adjusting agent, Etc.) can be mixed.
- a pharmaceutically acceptable carrier an osmotic pressure adjusting agent, a stabilizing agent, a preservative, a solubilizing agent, a pH adjusting agent, Etc.
- Known carriers can be used as these carriers.
- an indirect method of using the nucleic acid complex once outside the body and then administering it to the living body which is well known to those skilled in the art, can also be used.
- a nucleic acid complex is allowed to act on cells such as lymphocytes having high tumor cytotoxicity collected from a living body in vitro to impart drug sensitivity to the cells and cultured, and then administered to the living body. In this case, it is possible to injure the lymphocytes cultured with the tumor cells and kill the lymphocytes administered with the drug if
- the medicine containing the nucleic acid-containing complex as an active ingredient includes those containing two or more nucleic acid-containing complexes containing different types of nucleic acids. Such a drug having multiple therapeutic purposes is particularly useful in the field of diversifying gene therapy.
- the dose to be administered to animals, particularly humans, varies depending on various factors such as the nucleic acid of interest, the method of administration, and the specific site to be treated. However, the dose should be sufficient to produce a therapeutic response.
- This nucleic acid-containing complex is preferably applied for gene therapy.
- Applicable diseases vary depending on the type of nucleic acid included in the complex.
- diseases in the circulatory region that cause lesions such as peripheral artery disease, coronary artery disease, and restenosis after arterial dilatation, Cancer (malignant melanoma, brain tumor, metastatic malignant tumor, breast cancer, etc.), infectious disease (HIV, etc.), single genetic disease (cystic fibrosis, chronic granuloma, ⁇ 1-antitrypsin deficiency, Gaucher disease, etc. ) And the like.
- Dimethylaminopropyl propyl acrylamide (3.9 g, 24.96 marl ol, M. 156.23) and benzyl N, N-getyldithiocarbamate (23.94 mg, 0.1 mmol, Mw. 239) 41) was diluted with methanol to prepare a solution having a total volume of 20 ml.
- the solution was stirred while blowing nitrogen gas, and irradiated with ultraviolet light (light quantity lmW m 2 ). After 30 minutes of irradiation with ultraviolet light, the polymerization solution was concentrated by an evaporator and dropped into a large amount of getyl ether to precipitate a polymer.
- the black-mouthed form layer was concentrated by an evaporator and dried in a desiccator under vacuum to obtain 2,4,6-tris (N, N-getyldithiocarbamylmethyl) mesitylene (white solid).
- the yield was 3.03 g (98.4% yield), ⁇ -NMR: ⁇ 4.447 ppm (s, 6H, Ar-CS), ⁇ 4.086 to 4.015 ppm (q, 6H, —N—C_), ⁇ 3.746 3.63.676 ppm ( ⁇ i, 6H, —N_C3 ⁇ 4-), ⁇ 2.421 ppm (s, 9H, Ar-CH 3 ), ⁇ 1.324 -11222 ppm (m, 18H, -CH 2 -C).
- This 2,4,6-tris (N, N-getyldithiocarbamylmethyl) mesitylene is polymerized with dimethylaminopropylacrylamide according to the following reaction formula (Formula 4) to form a three-branch type.
- the vector was synthesized.
- the polymerization solution was concentrated by an evaporator, and the polymer was precipitated by dripping into a large amount of getyl ether. After removing the supernatant by decantation, the polymer was dissolved in water and freeze-dried. After freeze-drying, the molecular weight of the obtained polymer was measured by GPC and found to be about 18,000.
- -NMR ⁇ 7.7 to 7.4 ppm (br, 1H, - ⁇ ), ⁇ 3.4 to 3.0 ppm (br, 2H, -NH-CH 2 -C3 ⁇ 4-), ⁇ 2.4 to 2.3 ppm (br, 2H , - CH 2 - CH 2 - NR 2), ⁇ 2 ⁇ 3 ⁇ 2 lppm (br, 6H, - N- C), ⁇ 1.8 ⁇ :. 1.5 P pm (br, 2H, -CH 2 - CH 2 - C _)Met.
- 1,2,4,5-tetrakis (bromomethyl) benzene ig, 2.22nd 1, Mw. 449.83
- ethanol 100 ml
- sodium N, N-getyldithiocarbamate trihydrate 4g, 17. 76mmol, Mw. 225. 31
- the precipitate was collected by filtration. The collected precipitate was dissolved in black-mouthed form and separated and washed with water.
- porphyrin form layer was concentrated by an evaporator and dried in a desiccator under vacuum to obtain 1,2,4,5-tetrakis (N, N-dithio-potassium rubamylmethyl) benzene (white solid). Yield 1.48 g (91.4% yield).
- 1,2,4,5-tetrakis ( ⁇ , ⁇ -Jetyldithiocarbamylmethy) Benzene) (18.08 mg, 1.25 ramol, Mw. 723.30) was dissolved in chloroform (300 ⁇ l), and dimethylaminopropyl acrylacrylamide (1.326 g, 8.49 g) was dissolved. Ol, Mw. 156. 23).
- This mixture was diluted with methanol to prepare a solution having a total volume of 20 ml. The solution was stirred while blowing nitrogen gas, and irradiated with ultraviolet light (light amount: lmW m 2 ).
- the polymerization solution was concentrated by an evaporator and dropped into a large amount of ethyl ether to precipitate a polymer. After removing the supernatant by decantation, the polymer was dissolved in water and freeze-dried. After freeze-drying, the molecular weight of the obtained polymer was measured by GPC and found to be about 18,000. Further, -NMR:. 6 7. 8 ⁇ 7 4ppm (br, 1H, one NH), ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 0ppm (br, 2H, - NH- CH 2 - CH 2 -), S 2. 4 ⁇ 2. 2ppm.
- Hexakis (bromomethyl) benzene (lg, 1.57 mmol, Mw. 635.68) was added to ethanol (200 ml) and sodium N, N-getyldithiocarbamate trihydrate.
- Drate (6.36 g, 28.23 mmol, Mw. 225.31) was added, and the mixture was stirred at room temperature. Four days after the start of stirring, the precipitate was collected by filtration. The recovered precipitate was dissolved in black-mouthed form and washed with water.
- the black-mouthed form layer was concentrated with an evaporator and dried in a desiccator under vacuum to obtain hexakis (N, N-getyldithiocarbamylmethyl) benzene (white solid). The yield was 1.48 g (yield 90.2%).
- Each vector (2.36 mg, M.w. 18,000) was dissolved in Tris-HCl-buffer (2 ml). This solution was collected in an amount of 50 ⁇ l, diluted with Tris-1 HC 1 -buffer to give a total volume of 500 ⁇ l of a vector solution.
- a 2X buffer solution (450 1) was added to pGL3 control plasmid (0.2 ⁇ g / ⁇ 1, 90 ⁇ ) to prepare a DNA solution having a total volume of 540 ⁇ l. Transfer the vector solution (67 ⁇ 1) to the DNA solution (100 ⁇ 1) (the ratio of the positive charge of the cationic polymer to the negative charge of the DNA is 1: 1), and let stand at 37 ° C for 24 hours.
- a nucleic acid-containing complex was formed.
- Figure 1 shows the results of measuring the particle size of the polyion complex by dynamic light scattering measurement.
- FIG. 2 shows the change over time in the average particle diameter.
- each cationic polymer formed a polyion complex immediately after mixing with DNA. According to cumulant analysis, nanoparticles with a particle size of about 250 nm were generated, and were found to be stable after 24 hours.
- COS- 1 cells on culture day 1 24Well_plate was added polyionic complex solution lwell per 25.mu. 1, were cultured in 5% C0 2 incubator. After culturing for 3 hours, the medium was removed, washed with PBS, and added 1ml of DMEM per Lwell, it was cultured in again 5% C0 2 I Nkyubeta. Two days after the culture, the medium was removed, and the cells were washed with PBS. Then, Luciferase cell culture lysis5 XReagent was added in an amount of 200 ⁇ l per well and allowed to stand. After 30 minutes, the cells were transferred to an Eppendorf tube and centrifuged (4 ° C, 15000rpm, lmin).
- the supernatant was collected on a microplate at 4 ⁇ 1 each, and the luciferase activity was measured using a luminometer. In addition, 5 ⁇ l of the supernatant after centrifugation was collected, and the protein was quantified.
- a vector capable of protecting a nucleic acid from an enzyme by using the nucleic acid as an aggregate and arranging a vector containing a cationic polymer around the aggregate.
- Figures 5 and 6 are TEM photographs of the gene complex obtained by mixing the PGL3 plasmid and the above-described 6-branch vector.
- Figure 5 shows the result of staining with phosphotungstic acid
- Figure 6 shows the result of staining with perlan acetate. Things.
- FIG. 7 is a graph showing the relationship between the molecular weight of a vector having one branch and the gene expression efficiency.
- FIG. 8 is a graph showing the relationship between the molecular weight of a three-branched setter and gene expression efficiency.
- FIG. 9 is a graph showing the relationship between the molecular weight of a vector having four branches and the gene expression efficiency.
- Figure 10 shows the molecular weight and gene expression efficiency of a block polymer of a vector with four branches and a molecular weight of 10,000, which was obtained by block polymerization of dimethyl acrylamide at the tip of the branch.
- Fig. 11 shows the stability of the block polymer, branched product and PEI of this vector in solution (change over time when the initial value of gene expression efficiency is 100%).
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Abstract
Description
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007230896A (ja) * | 2006-02-28 | 2007-09-13 | National Cardiovascular Center | 遺伝子導入剤 |
JP2008289468A (ja) * | 2007-02-15 | 2008-12-04 | National Cardiovascular Center | 遺伝子導入剤及びその製造方法 |
JP2009274998A (ja) * | 2008-05-15 | 2009-11-26 | National Cardiovascular Center | 遺伝子導入剤及びその製造方法並びに核酸複合体 |
JP2009274997A (ja) * | 2008-05-15 | 2009-11-26 | National Cardiovascular Center | 遺伝子導入剤及びその製造方法並びに核酸複合体 |
JP2012217388A (ja) * | 2011-04-08 | 2012-11-12 | Bridgestone Corp | 遺伝子導入剤組成物 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5830948A (en) * | 1995-06-30 | 1998-11-03 | Cornell Research Foundation, Inc. | Polymerizable macromonomers and polymers prepared therefrom |
WO1999006055A1 (en) * | 1997-08-01 | 1999-02-11 | Supratek Pharma Inc. | Polynucleotide compositions |
JP2000509428A (ja) * | 1996-12-20 | 2000-07-25 | コノート ラボラトリーズ リミテッド | 生体内分解性標的指向性マイクロパーティクル送達システム |
WO2001000708A1 (de) * | 1999-06-25 | 2001-01-04 | Christian Plank | Kombinationen zur einführung von nucleinsäuren in zellen |
JP2003500469A (ja) * | 1999-05-28 | 2003-01-07 | バイカル インコーポレイテッド | サイトフェクチン二量体およびその使用方法 |
WO2004011527A1 (en) * | 2002-07-26 | 2004-02-05 | The University Of Sheffield | Hyperbranched polyamidoamine |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4558120A (en) * | 1983-01-07 | 1985-12-10 | The Dow Chemical Company | Dense star polymer |
US5610268A (en) * | 1992-01-13 | 1997-03-11 | Dsm N.V. | Dendritic macromolecule and the preparation thereof |
ES2104518T1 (es) * | 1994-03-07 | 1997-10-16 | Dow Chemical Co | Conjugados dendrimeros bioactivos y/o directores hacia diana. |
US5919422A (en) * | 1995-07-28 | 1999-07-06 | Toyoda Gosei Co., Ltd. | Titanium dioxide photo-catalyzer |
US6084030A (en) * | 1995-08-04 | 2000-07-04 | Dsm Copolymer, Inc. | Branched polymers with polyolefin arms |
US6127481A (en) * | 1995-08-04 | 2000-10-03 | Dsm Copolymer, Inc. | Branched polyolefin polymers as additives in fuel and lubricating oil compositions |
EP0859857B1 (en) * | 1995-10-25 | 2005-05-04 | OctoPlus B.V. | Cationic polyacrylates and poly(alkyl)acrylates or the corresponding acrylamides for use in synthetic transfection systems |
US20010046705A1 (en) * | 2000-04-25 | 2001-11-29 | Arnaud Debin | Particulate complex for adminstering nucleic acid into a cell |
FR2814370B1 (fr) * | 2000-09-22 | 2004-08-20 | Centre Nat Rech Scient | Utilisation d'un complexe acide nucleique/pei pour le ciblage de cellules souches du cerveau |
WO2002024232A2 (en) * | 2000-09-25 | 2002-03-28 | Board Of Regents, The University Of Texas System | Pei: dna vector formulations for in vitro and in vivo gene delivery |
EP1527096A2 (en) * | 2002-08-06 | 2005-05-04 | AplaGen GmbH | Binding molecules |
DE102004028151A1 (de) * | 2004-06-10 | 2005-12-29 | Mikron Comp-Tec Ag | Speicheranordnung für Bearbeitungsmaschinen |
-
2004
- 2004-04-16 WO PCT/JP2004/005450 patent/WO2004092388A1/ja active Application Filing
- 2004-04-16 EP EP04727984.9A patent/EP1616957B1/en not_active Expired - Lifetime
- 2004-04-16 JP JP2005505463A patent/JP5153072B2/ja not_active Expired - Fee Related
-
2005
- 2005-10-18 US US11/252,063 patent/US8298817B2/en not_active Expired - Fee Related
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5830948A (en) * | 1995-06-30 | 1998-11-03 | Cornell Research Foundation, Inc. | Polymerizable macromonomers and polymers prepared therefrom |
JP2000509428A (ja) * | 1996-12-20 | 2000-07-25 | コノート ラボラトリーズ リミテッド | 生体内分解性標的指向性マイクロパーティクル送達システム |
WO1999006055A1 (en) * | 1997-08-01 | 1999-02-11 | Supratek Pharma Inc. | Polynucleotide compositions |
JP2003500469A (ja) * | 1999-05-28 | 2003-01-07 | バイカル インコーポレイテッド | サイトフェクチン二量体およびその使用方法 |
WO2001000708A1 (de) * | 1999-06-25 | 2001-01-04 | Christian Plank | Kombinationen zur einführung von nucleinsäuren in zellen |
WO2004011527A1 (en) * | 2002-07-26 | 2004-02-05 | The University Of Sheffield | Hyperbranched polyamidoamine |
Non-Patent Citations (5)
Title |
---|
CHOI, YH ET AL.: "Lactose-poly (ethylene glycol)- grafted poly-L-Lysine as hepatoma cell-tapgeted gene carrier", BIOCONJUG CHEM., vol. 9, no. 6, 1998, pages 708 - 718, XP000786594 * |
HAN S.O. ET AL.: "Water-soluble lipopolymer for gene delivery", BIOCONJUG CHEM., vol. 12, no. 3, 2001, pages 337 - 345, XP002305803 * |
See also references of EP1616957A4 * |
TONCHEVA, V. ET AL.: "Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-Lysine) grafted with hydrophilic polymers", BIOCHIM BIOPHYS ACTA., vol. 1380, no. 3, 8 May 1988 (1988-05-08), pages 354 - 368, XP002083038 * |
UMEDA, M. ET AL.: "Hikari Cation Seiseigata Shinsuisei Kobunshi o Mochiita DNA tono Complex Keisei no Hikari seigyo", POLYMER PREPRINTS, JAPAN, vol. 51, no. 5, 10 May 2002 (2002-05-10), pages 976, XP002984682 * |
Cited By (5)
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JP2007230896A (ja) * | 2006-02-28 | 2007-09-13 | National Cardiovascular Center | 遺伝子導入剤 |
JP2008289468A (ja) * | 2007-02-15 | 2008-12-04 | National Cardiovascular Center | 遺伝子導入剤及びその製造方法 |
JP2009274998A (ja) * | 2008-05-15 | 2009-11-26 | National Cardiovascular Center | 遺伝子導入剤及びその製造方法並びに核酸複合体 |
JP2009274997A (ja) * | 2008-05-15 | 2009-11-26 | National Cardiovascular Center | 遺伝子導入剤及びその製造方法並びに核酸複合体 |
JP2012217388A (ja) * | 2011-04-08 | 2012-11-12 | Bridgestone Corp | 遺伝子導入剤組成物 |
Also Published As
Publication number | Publication date |
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JPWO2004092388A1 (ja) | 2006-07-06 |
US8298817B2 (en) | 2012-10-30 |
US20060057714A1 (en) | 2006-03-16 |
EP1616957A1 (en) | 2006-01-18 |
EP1616957A4 (en) | 2010-03-10 |
JP5153072B2 (ja) | 2013-02-27 |
EP1616957B1 (en) | 2017-05-17 |
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