METHOD FOR INTRODUCING SIRNA INTO CELLS BY PHOTOCHEMICAL INTERNALISATION
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
A61K-045/00
C12N-005/06
A61P-035/00
C12N-013/00
A61K-031/7088
출원번호
US-0304266
(2007-07-11)
공개번호
US-0297487
(2009-12-03)
우선권정보
GB-0613753.3(2006-07-11)
국제출원번호
PCT/GB07/002569
(2007-07-11)
발명자
/ 주소
Boe, Sigurd
Hovig, Eivind Johannes
출원인 / 주소
PCI BIOTECH AS
대리인 / 주소
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
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 photosensiting agent, and ii) irradiating the cell with light of a wavelength effective to activate the photo
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 photosensiting 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, and ii) irradiating the cell with light of a wavelength effective to activate the photosensitising agent, wherein s
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, and ii) 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 lipopolyamine in a non-liposomal formulation, (b) polyethyleneimine (PEI) having an Mn value of 500-20000 by GPC, (c) a betacyclodextrin amine polymer of formula wherein n is an integer from 1 to 1 inclusive and n is an integer from 4 to 10 inclusive, (d) an amide group containing dendrimer, and (e) a cationic peptide. 2. The method of claim 1 wherein the lipopolyamine contains primary or secondary amine groups, or a mixture thereof. 3. The method of claim 1, wherein the polyamine region of the lipopolyamine has at least 2 nitrogen atoms and a charge of at least +1 at physiological pH. 4. The method of claim 1 wherein at least one nitrogen containing group of said carrier is uncharged at physiological pH. 5. The method of claim 4 wherein the pKa at which the last amine of the lipopolyamine is protonated on decreasing pH is greater than or equal to 5.5. 6. The method of claim 1 wherein the polyamine region of the lipopolyamine has the formula (I) in which m is an integer greater than or equal to 2 and n is an integer greater than or equal to 1, wherein m may be the same or different where it appears in said formula, at each position R1 is a hydrogen or a linking group to the lipo portion of the lipopolyamine, or the lipo portion itself, and may be the same of different at each carbon atom, R2 is a hydrogen or a linking group to the lipo portion of the lipopolyamine or the lipo portion itself, wherein only one of R1 and R2 is a linking group to the lipo portion of the lipopolyamine, or the lipo portion itself and wherein when R1 or R2 is the linking group attached to said lipo portion or the lipo portion itself, formula (I) is said lipopolyamine. 7. The method of claim 6 wherein m is between 2 and 6 inclusive and n is between 1 and 5. 8. The method of claim 7 wherein n is 3. 9. The method of claim 6 wherein R2 is H. 10. The method of claim 6 wherein the polyamine region is represented by the following formula Wherein R1a to R1j are as R1 defined in claim 7. 11. The method of claim 10 wherein R1a is a linker and the remaining R1 groups are hydrogen. 12. The method of claim 6 wherein R1 or R2 represent a hydrogen atom or a radical of general formula II: in which R3 and R4, which may be identical or different, each represent a saturated aliphatic radical Cp H2p+2 or unsaturated aliphatic radical Cp H2p or Cp H2p-2, p being an integer between 12 and 22 inclusive, and R5 represents a hydrogen atom or an alkyl radical containing 1 to 4 carbon atoms optionally substituted with a phenyl radical, with the proviso that only one of R1 and R2 can represent a radical of general formula (II). 13. The method of claim 6 wherein R1 and R2 each represent a hydrogen atom or a radical of general formula III: in which X represents a methylene group (--CH2--) or a carbonyl group (--CO--), and R6 and R7, which may be identical or different, each represent a saturated aliphatic radical Cp' H2p'+2 or unsaturated aliphatic radical Cp' H2p' or Cp' H2p'-2, and p' is an integer between 11 and 21 inclusive, with the proviso that only one of R1 and R2 can represent a radical of general formula (III). 14. The method of claim 6 wherein n is an integer between 2 and 5 inclusive, and the values of m in the different fragments are identical. 15. The method of claim 6 wherein n is between 2 and 5 inclusive, and the values of m in the different fragments are different. 16. The method of claim 6 wherein n is equal to 3 and the values of m in the fragments are identical or different and represent 3 or 4, and either R1 or R2 represents: (i) a radical of general formula (II) in which R3 and R4 each represent an alkyl radical containing 12 to 22 carbon atoms and R5 represents a hydrogen atom, or (ii) a radical of general formula (III) in which R6--X--and R7--X--each represent an alkanoyl radical containing 12 to 22 carbon atoms. 17. The method of claim 1 wherein the lipophilic region of said lipopolyamine is any saturated or unsaturated hydrocarbon chain, cholesterol or other steroid, a natural lipid or a synthetic lipid capable of forming lamellar or hexagonal phases. 18. The method of claim 17 wherein the length of the hydrocarbon chain is from 10 to 30 carbons in length. 19. The method of claim 1 wherein the lipopolyamine is selected from 5-carboxyspermylglycinedioctadecylamide (DOGS), dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES), 2,3-dioleyl-oxy-N [2-sperminecarboxyyl-amido]ethyl-N,N-dimethyl-1-propanaminium trifluoracetate (DOS PA), 1,3-dioleoyloxy-2-(6-carboxyspermine) (DOSPER) and RPR-120535. 20. The method of claim 1 wherein the lipopolyamine is selected from 5-carboxyspermylglycinedioctadecylamide (DOGS), and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES). 21. The method of claim 1 wherein the carrier is JetSI® or JetSI-ENDO®, or Transfectam®. 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, preferably less than 25 kDa. 24. The method of claim 1 wherein said carrier molecule is a PAMAM dendrimer molecule, preferably a generation 2-6 PAMAM dendrimer molecule. 25. The method of claim 1 wherein said carrier molecule is Poly-L-Lysine, Poly-D-Lysine, Poly-Histidine, 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 a method as defined in 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 method of claim 1. 37. A composition containing an siRNA molecule and a carrier molecule as defined in claim 1, and optionally separately also a photosensitizing agent as defined in claim 1. 38. The composition of claim 37, containing 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 optionally a photosensitizing agent as defined in claim 1. 42. (canceled) 43. (canceled) 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 methods as defined in claim 35 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 as defined in claim 36 to said patient. 47. The method of claim 45 which is used to treat a disease which is typified by abnormal gene expression or which would benefit from suppression of one or more genes, preferably cancer.
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