Freeze-Dried Product for Introducing Nucleic Acid, Oligonucleic Acid or Derivative Thereof

Abstract

A freeze-dried product is provided which, together with demonstrating satisfactory ability to express function when used to introduce a gene or antisense nucleic acid and the like, enables concentration to be adjusted easily, offers easy handling and has superior storage performance. A freeze-dried product of a complex containing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and, an anionic polymer; a method for preparing the freeze-dried product, comprising a step of forming a complex by mixing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and an anionic polymer, and a step of freeze-drying the complex; a preparation, reagent or kit containing the freeze-dried product for introducing a nucleic acid, oligonucleic acid or derivative thereof; and, a method for introducing a nucleic acid, oligonucleic acid or derivative thereof into cells that uses the freeze-dried product.

1 . A freeze-dried product of a complex containing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and, an anionic polymer. 
   
   
       2 . The freeze-dried product according to  claim 1 , wherein the molar ratio of each charged group (ratio of negative charge to positive charge) of a complex containing a nucleic acid, oligonucleic acid or derivative thereof and the cationic polymer, or cationic lipid or aggregate containing the same is 1:0.8 to 1:100. 
   
   
       3 . The freeze-dried product according to  claim 1 , wherein the molar ratio of each charged group (ratio of negative charge to negative charge) of a nucleic acid, oligonucleic acid or derivative thereof and an anionic polymer is 1:0.2 to 1:1000. 
   
   
       4 . The freeze-dried product according to  claim 1 , wherein the cationic polymer is a positively charged, naturally-occurring or synthetic polymer having a molecular weight of about 1,000 to 3,000,000 and having a plurality of functional groups capable of forming a complex with DNA in water in a molecule. 
   
   
       5 . The freeze-dried product according to  claim 4 , wherein the cationic polymer is selected from positively charged proteins and polypeptides; positively charged dendrimers; positively charged synthetic polymers; and positively charged polysaccharide derivatives or salts and combinations thereof. 
   
   
       6 . The freeze-dried product according to  claim 1 , wherein the cationic lipid is DC-Chol (3β-(N—(N′,N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB (N,N-distearyl-N,N-dimethylammonium bromide), DMRI (N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide), DODAC (N,N-dioleyl-N,N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido) ethyl)-N,N-dimethylammonium trifluoroacetate), DOTAP (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride) or DOTMA (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride). 
   
   
       7 . The freeze-dried product according to  claim 1 , wherein the anionic polymer is a negatively charged, naturally-occurring or synthetic polymer containing an anionic group in a molecule thereof, having a molecular weight of about 500 to 4,000,000, and having a plurality of functional groups in a molecule thereof capable of forming a complex with a polycation in water. 
   
   
       8 . The freeze-dried product according to  claim 7 , wherein the anionic polymer is selected from polysaccharides and derivatives thereof having functional groups selected from a carboxyl group, —OSO 3 H group, —SO 3 H group, phosphate group and salts thereof; polyamino acids containing an amino acid residue having negatively charged side chains; PEG derivatives having carboxyl side chains; synthetic polymers having functional groups selected from a carboxyl group, —OSO 3 H group, —SO 3 H group, phosphate group and salts thereof; polymers having functional groups selected from a carboxyl group, —OSO 3 H group, —SO 3 H group, phosphate group and salts thereof, as well as optionally substituted amino groups, ammonium groups or salts thereof, and combinations thereof. 
   
   
       9 . The freeze-dried product according to  claim 1 , wherein the aggregate containing a cationic lipid is an aggregate containing DOSPA (N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate) and the anionic polymer is hyaluronic acid. 
   
   
       10 . The freeze-dried product according to  claim 1 , wherein the aggregate containing a cationic lipid is an aggregate containing DOSPA (N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate), and the anionic polymer is a PEG derivative having carboxyl side chains. 
   
   
       11 . The freeze-dried product according to  claim 1 , wherein the cationic polymer is polyethyleneimine; and the anionic polymer is hyaluronic acid. 
   
   
       12 . The freeze-dried product according to  claim 1 , wherein the cationic polymer is polyethyleneimine; and the anionic polymer is a PEG derivative having carboxyl side chains. 
   
   
       13 . The freeze-dried product according to  claim 11 , wherein the blending ratio of nucleic acid, oligonucleic acid or derivative thereof to polyethyleneimine to hyaluronic acid is 1:4 to 24:1 to 160 (molar ratio of each charged group). 
   
   
       14 . The freeze-dried product according to  claim 12 , wherein the blending ratio of nucleic acid, oligonucleic acid or derivative thereof to polyethyleneimine to PEG derivative having carboxyl side chains is 1:4 to 24:2 to 160 (molar ratio of each charged group). 
   
   
       15 . The freeze-dried product according to  claim 9 , wherein the blending ratio of nucleic acid, oligonucleic acid or derivative thereof to aggregate containing DOSPA to hyaluronic acid is 1:1.2 to 48:0.2 to 160 (molar ratio of each charged group). 
   
   
       16 . The freeze-dried product according to  claim 10 , wherein the blending ratio of nucleic acid, oligonucleic acid or derivative thereof to aggregate containing DOSPA to PEG derivative having carboxyl side chains is 1:1.2 to 48:0.2 to 160 (molar ratio of each charged group). 
   
   
       17 . A method for preparing the freeze-dried product according to  claim 1 , comprising a step of forming a complex by mixing a nucleic acid, oligonucleic acid or derivative thereof; a cationic polymer, or cationic lipid or aggregate containing the same; and an anionic polymer, and a step of freeze-drying the complex. 
   
   
       18 . A preparation or reagent containing the freeze-dried product according to  claim 1  for introducing a nucleic acid, oligonucleic acid or derivative thereof. 
   
   
       19 . A kit containing the freeze-dried product according to  claim 1  for introducing a nucleic acid, oligonucleic acid or derivative thereof. 
   
   
       20 . A method for introducing a nucleic acid, oligonucleic acid or derivative thereof into cells that uses the freeze-dried product according to  claim 1 . 
   
   
       21 . The method according to  claim 20 , comprising a step of rehydrating the freeze-dried product in a solvent before introducing a nucleic acid, oligonucleic acid or derivative thereof. 
   
   
       22 . The method according to  claim 20 , wherein the freeze-dried product is used without rehydrating.