Compositions and methods for delivering nucleic acid to a cell

Abstract

Provided are novel compositions useful for delivering nucleic acids to cells. Also provided are methods for making and using such compositions.

1 . A method for preparing a liposome comprising a nucleic acid and a lipid component, the method comprising:
 combining a lipid component and a nucleic acid component in a mixture comprising:
 water; 
 a water-miscible organic solvent; and 
 a polyamine; 
   under conditions such that a liposome comprising the nucleic acid and the lipid component is formed.   
     
     
         2 . The method of  claim 1 , wherein the polyamine is an oligoethyleneimine, a polyethyleneimine, or a polyaminoC 2 -C 10 alkane. 
     
     
         3 . The method of  claim 2 , wherein the polyamine is selected from the group consisting of spermine, spermidine, and putrescine. 
     
     
         4 . The method of  claim 3 , wherein the polyamine is spermine. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the lipid component comprises a non-cationic lipid. 
     
     
         10 . The method of  claim 1 , wherein the non-cationic lipid is a neutral lipid. 
     
     
         11 . The method of  claim 10  wherein the neutral lipid comprises DOPC, DOPE, cholesterol, or PEG-DSG. 
     
     
         12 . The method of  claim 9 , wherein the liposome further comprises a cationic lipid. 
     
     
         13 . The method of  claim 12 , wherein the cationic lipid comprises DOTAP or DOSPA. 
     
     
         14 . The method of  claim 1 , wherein the water-miscible organic solvent comprises methanol, ethanol, 1-propanol, or 2-propanol. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 18 , wherein the ratio of polyamine nitrogen to nucleic acid phosphate groups (N/P) is between about 0.8 and about 1.5. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . A composition comprising a liposome in an aqueous medium, the liposome having an interior and an exterior, wherein the liposome comprises:
 a nucleic acid;   a polyamine; and   a lipid component;   wherein the lipid component comprises a neutral phospholipid and essentially no cationic lipid, and wherein the nucleic acid and the lipid component are present at a ratio of from 5 nmol lipid per microgram of the nucleic acid to 20, 30, 40, 50, 60, 70, 80, 90, or 100 (optionally 5-20 or 30, preferably 10) nmol lipid per microgram of the nucleic acid, and wherein the liposome is from 30 to 500 nanometers in diameter.   
     
     
         28 . The composition of  claim 27 , wherein the polyamine is oligoethyleneimine, polyethyleneimine, or a polyaminoC 2 -C 10 alkane. 
     
     
         29 . The composition of  claim 27 , wherein the polyamine is selected from the group consisting of spermine, spermidine, and putrescine. 
     
     
         30 . The composition of  claim 29 , wherein the polyamine is spermine. 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . The composition of  claim 27 , wherein the non-cationic lipid comprises DOPC, DOPE, cholesterol, or PEG-DSG. 
     
     
         36 . (canceled) 
     
     
         37 . The composition of  claim 27 , wherein the lipid component comprises a cationic lipid and a non-cationic lipid. 
     
     
         38 . The composition of  claim 37 , wherein the cationic lipid comprises DOTAP or DOSPA. 
     
     
         39 . The composition of  claim 27 , wherein the liposome is from 70 to 300 nanometers in diameter. 
     
     
         40 . A method of using of a liposome of  claim 27 , the method comprising:
 attaching an internalizing antibody or a fragment thereof, which antibody or fragment binds to a specific cell surface antigen, to the exterior of the liposome, wherein the liposome with the antibody attached is internalized by a cell expressing at least 100,000 or at least 1,000,000 molecules of the antigen when contacted and incubated with the cell under internalizing conditions.   
     
     
         41 . (canceled) 
     
     
         42 . The composition of  claim 27 , wherein the liposome further comprises an internalizing antibody or a fragment thereof attached to the exterior of the liposome, wherein the antibody or fragment binds to a specific cell surface antigen. 
     
     
         43 . A method of delivering a nucleic acid to a cell, the method comprising:
 contacting the cell with the composition of  claim 42 ;   wherein the liposome with the internalizing antibody or a fragment thereof attached is internalized by a cell expressing at least 100,000 or at least 1,000,000 molecules of the antigen when contacted and incubated with the cell under internalizing conditions.   
     
     
         44 . A method of treating a patient in need thereof with a nucleic acid, the method comprising administering to the patient an effective amount of a composition of  claim 42  under conditions such that the patient is treated for a condition responsive to nucleic acid therapy. 
     
     
         45 . A composition prepared by the method of  claim 1 , the composition comprising a liposome in an aqueous medium, wherein the liposome comprises:
 a nucleic acid;   a polyamine; and   a lipid component.   
     
     
         46 . The composition of  claim 27  or  claim 45 , wherein the composition comprises no more than about 5 mol % or 1 mol % DOPE. 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . (canceled)