METHOD FOR INTRODUCING SIRNA INTO CELLS BY PHOTOCHEMICAL INTERNALISATION
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IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
A61K-031/713
C12N-005/071
C12N-013/00
A61P-035/00
C12N-015/00
출원번호
US-0349334
(2012-01-12)
공개번호
US-0264807
(2012-10-18)
우선권정보
GB-0613753.3 (2006-07-11)
발명자
/ 주소
Bøe, Sigurd
Hovig, Eivind Johannes
출원인 / 주소
PCI BIOTECH AS
인용정보
피인용 횟수 :
0인용 특허 :
0
초록▼
The present invention relates to a method for introducing an siRNA molecule into the cytosol of a cell, said method comprising i) contacting said cell with an siRNA molecule, a carrier and a photosensitising agent, and ii) irradiating the cell with light of a wavelength effective to activate the pho
The present invention relates to a method for introducing an siRNA molecule into the cytosol of a cell, said method comprising i) contacting said cell with an siRNA molecule, a carrier and a photosensitising agent, and ii) irradiating the cell with light of a wavelength effective to activate the photosensitising agent, wherein said carrier comprises a cationic polyamine such as a lipopolyamine in a non-liposomal formulation, polyethyleneimine (PEI), a betacyclodextrin amine polymer, an amine group containing dendrimer, and a cationic peptide. Cells or a population of cells obtainable by the method, a composition containing an siRNA molecule and the carrier molecule, kits and therapeutic uses of the above are also provided.
대표청구항▼
1. A method for introducing an siRNA molecule into the cytosol of a cell, said method comprising i) contacting said cell with an siRNA molecule, a carrier and a photosensitising agent, andii) irradiating the cell with light of a wavelength effective to activate the photosensitising agent,wherein sai
1. A method for introducing an siRNA molecule into the cytosol of a cell, said method comprising i) contacting said cell with an siRNA molecule, a carrier and a photosensitising agent, andii) irradiating the cell with light of a wavelength effective to activate the photosensitising agent,wherein said carrier comprises a cationic polyamine selected from(a) a polyethyleneimine (PEI),(b) an amide group containing dendrimer, and(c) a cationic peptide. 2.-21. (canceled) 22. The method of claim 1 wherein said carrier molecule is a branched PEI molecule. 23. The method of claim 22, wherein the molecular weight of the PEI is less than 50 kDa. 24. The method of claim 1 wherein said carrier molecule is a PAMAM dendrimer molecule. 25. The method of claim 1 wherein said carrier molecule is Poly-L-Lysine, Poly-D-Lysine, Poly-Histidine, Polyarginine, Histidylated poly-lysine and Poly-ornithine or a copolymer of L or D lysine, L or D arginine, L or D histidine and/or ornithine residues with one or more other amino acids. 26. The method of claim 1 wherein the siRNA molecule is 12-28 nucleotides long. 27. The method of claim 1 wherein said cell is a mammalian cell. 28. The method of claim 1 wherein the photosensitising agent is selected from a porphyrin, phthalocyanine, purpurin, chlorin, benzoporphyrin, lysomotropic weak base, naphthalocyanine, cationic dye, tetracycline or a derivative thereof, 5-aminolevulinic acid and/or esters thereof, preferably TPPS4, TPPS2a, AlPcS2a, TPCS2a, 5-aminolevulinic acid or esters of 5-aminolevulinic acids or pharmaceutically acceptable salts thereof. 29. The method of claim 1, further comprising the additional step of contacting said siRNA with said carrier. 30. The method of claim 1 wherein the siRNA molecule and the carrier molecule are contacted with one another for 20-40 minutes before being contacted with the cell. 31. The method of claim 1, wherein 10 nM-200 nM siRNA is used for transfection. 32. The method of claim 1 wherein a photosensitizer carrier selected from a polycation, polyethyleneimine, a dendrimer, a cationic lipid and a peptide is additionally present. 33. The method of claim 1, wherein the siRNA is mixed with the carrier so as to form a complex, which is then administered to the cell simultaneously or sequentially with the photosensitising agent. 34. The method of claim 1 wherein said method is performed by contacting said cell with a photosensitising agent, contacting said cell with the carrier and the siRNA molecule to be introduced and irradiating said cell with light of a wavelength effective to activate the photosensitising agent, wherein said irradiation is performed prior to the cellular uptake of said siRNA molecule and said carrier into an intracellular compartment containing said photosensitising agent, preferably prior to cellular uptake of said molecule and said carrier into any intracellular compartment. 35. A method of inhibiting the expression of a target gene by introducing an siRNA molecule into a cell containing said target gene by the method of claim 1, wherein said siRNA molecule specifically inhibits expression of said target gene. 36. A cell or a population of cells containing an siRNA molecule which has been internalised into the cytosol of said cell, which cell is obtainable by a the method of claim 1. 37. A composition containing (a) an siRNA molecule and (b) a carrier molecule wherein said carrier molecule comprises a cationic polyamine selected from (i) a polyethyleneimine (PEI),(ii) an amide group containing dendrimer, and(iii) a cationic peptide. 38. The composition of claim 37, further comprising a photosensitizing agent. 39. A composition comprising the cell or a population of cells according to claim 36. 40. The composition of claim 37 for use in therapy. 41. A kit comprising an siRNA molecule, a carrier molecule and a photosensitizing agent, wherein said carrier molecule comprises a cationic polyamine selected from (i) a polyethyleneimine (PEI),(ii) an amide group containing dendrimer, and(iii) a cationic peptide. 42.-44. (canceled) 45. A method of treating or preventing a disease, disorder or infection in a patient comprising introducing an siRNA molecule and carrier into one or more cells in vitro, in vivo or ex vivo according to the method of claim 1 and where necessary administering said cells to said patient. 46. A method of treating or preventing a disease, disorder or infection in a patient comprising introducing the cell or a population of cells according to claim 36 to said patient. 47. The method of claim 45 wherein the disease to be treated is typified by abnormal gene expression or which would benefit from suppression of one or more genes. 48. The method of claim 1 wherein said carrier is a PEI molecule, wherein the molecular weight of the PEI is less than 30 kDa. 49. The method of claim 48 wherein the molecular weight of the PEI is less than or equal to 25 kDa. 50. The method of claim 23 wherein the molecular weight of the PEI is less than or equal to 25 kDa. 51. The method of claim 1 wherein said PEI has an Mn (number average molecular weight) value of 500-20000 by gel permeation chromatography (GPC). 52. The method of claim 51 wherein said PEI has an Mn (number average molecular weight) value of 500-1500 by gel permeation chromatography (GPC). 53. The method of claim 45 wherein the siRNA is mixed with the carrier so as to form a complex, which complex is then introduced to the one or more cells simultaneously with the photosensitising agent. 54. The method of claim 45 wherein the siRNA is mixed with the carrier so as to form a complex, which complex is then introduced to the one or more cells sequentially with the photosensitising agent. 55. The kit of claim 41 wherein said siRNA molecule, carrier molecule and photosensitizing agent are formulated for simultaneous, separate, or sequential use in therapy. 56. The method of claim 24, wherein said PAMAM dendrimer molecule is a generation 2-6 PAMAM dendrimer molecule. 57. The method of claim 47, wherein the disease is cancer.
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