Method for introducing sirna into cells by photochemical internalisation

Abstract

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.

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 M n  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 R 1  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, 
         R 2  is a hydrogen or a linking group to the lipo portion of the lipopolyamine or the lipo portion itself, 
         wherein only one of R 1  and R 2  is a linking group to the lipo portion of the lipopolyamine, or the lipo portion itself and wherein when R 1  or R 2  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 R 2  is H. 
     
     
         10 . The method of  claim 6  wherein the polyamine region is represented by the following formula 
       
         
           
           
               
               
           
         
         Wherein R 1a  to R 1j  are as R 1  defined in  claim 7 . 
       
     
     
         11 . The method of  claim 10  wherein R 1a  is a linker and the remaining R 1  groups are hydrogen. 
     
     
         12 . The method of  claim 6  wherein R 1  or R 2  represent a hydrogen atom or a radical of general formula II: 
       
         
           
           
               
               
           
         
         in which 
         R 3  and R 4 , which may be identical or different, each represent a saturated aliphatic radical C p  H 2p+2  or unsaturated aliphatic radical C p  H 2p  or C p  H 2p−2 , p being an integer between 12 and 22 inclusive, and 
         R 5  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 R 1  and R 2  can represent a radical of general formula (II). 
       
     
     
         13 . The method of  claim 6  wherein R 1  and R 2  each represent a hydrogen atom or a radical of general formula III: 
       
         
           
           
               
               
           
         
         in which 
         X represents a methylene group (—CH 2 —) or a carbonyl group (—CO—), and R 6  and R 7 , which may be identical or different, each represent a saturated aliphatic radical C p′  H 2p′+2  or unsaturated aliphatic radical C p′  H 2p′  or C p′  H 2p′−2 , and p′ is an integer between 11 and 21 inclusive, 
         with the proviso that only one of R 1  and R 2  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 R 1  or R 2  represents: 
         (i) a radical of general formula (II) in which R 3  and R 4  each represent an alkyl radical containing 12 to 22 carbon atoms and R 5  represents a hydrogen atom, or 
         (ii) a radical of general formula (III) in which R 6 —X— and R 7 —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 TPPS 4 , TPPS 2a , AlPcS 2a , TPCS 2a , 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.