Lipids for transfection of Eukaryotic cells
Compositions and methods for improved delivery of macromolecules into eukaryotic cells are provided. Fusogenic peptides from fusion proteins of non-enveloped viruses enhance the efficiency of transfection of eukaryotic cells mediated by transfection agents such as cationic lipids, polycationic polymers such as PEI and dendrimers. These fusogenic peptides are used as part of a transfection complex that efficiently delivers a macromolecule, for example, a nucleic acid, into a eukaryotic cell. Novel cationic lipids and compositions of cationic lipids also are provided that may be used for the introduction of macromolecules such as nucleic acids, proteins and peptides into a variety of cells and tissues. The lipids can be used alone, in combination with other lipids and/or in combination with fusogenic peptides to prepare transfection complexes.
1. A compound having the formula:
wherein X 1 and X 2 independently are selected from the group consisting of (CH 2 ) n , (CHOH) n , and CONH;
X 5 and X 6 independently are (CH 2 ) 1-6 ;
W 1 and W 2 independently are selected from the group consisting of, hydrogen, —OH, —O—(C 1 -C 18 ) alkyl, —O—(C 1 -C 18 ) alkenyl, —O—(C 1 -C 18 ) alkynyl, —NH 2 , —NH(CH 2 ) s CH 3 , —N((CH 2 ) s CH 3 ), —SH, and —NH—NH 2 ;
R 3 and (R 6 ) q independently are selected from the group consisting of N, NH, CH, and N(CH 2 ) s CH 3 , and q=0-1;
R 4 and R 5 independently are selected from the group consisting of (CH 2 ) n , (CH 2 —CHOH—CH 2 ) n , (CHOH) n , HNCO, CONH, CO, —O—, —S—, —S—S—, polyamide and an ester linkage;
L 1 and L 2 independently are selected from the group consisting of —NH—, —O—, —NHCO—, —CONH—, —OCO—, —COO—, —CO—, —S—, —S—S—, —NHC(O)O—, —OC(O)NH—, —NHCONH—, —NHC(═NH)NH—, —NH—NH—, —S(O)— and —SO 2 —;
Y is a heterocyclic moiety containing at least one amine or amide moiety, wherein the points of attachment of Y are carbon and/or heteroatoms;
R 1 and R 2 independently are selected from the group consisting of hydrogen, primary alkylamine, secondary alkylamine, tertiary alkyl amine, quaternary alkylamine, alkenylamine, secondary alkenylamine, tertiary alkenyl amine, quaternary alkenylamine, alkynylamine, secondary alkynylamine, tertiary alkynylamine, quaternary alkynylamine amino alcohol, alkyl polyamine, alkenyl polyamine, alkynyl polyamine, spermidine, spermine, carboxy spermine, guanidinium, pyridinium, pyrollidinium, piperidinium, piperazinium, amino acyl, peptidyl, and protein;
Z 1 and Z 2 independently are selected from the group consisting of long chain straight alkyl, long chain branched alkyl, long chain cycloalkyl, long chain straight chain alkenyl, long chain branched alkenyl, long chain cycloalkenyl, long chain straight chain alkynyl, and long chain branched alkynyl,
m, n, p, and s independently are 0-6, with the proviso that when m, n, and p all are 0 then Y is eliminated and R 3 is bonded directly to X 2 .
2. A compound- according to claim 1 having the formula:
wherein X 1 , X 2 , W 1 , W 2 , R 3 , (R 6 ), R 4 , R 5 , Y, R 1 , R 2 , Z 1 , Z 2 m, n, p, and s are as defined above.
3. A compound according to claim 1 wherein Y is
wherein X 3 and X 4 are independently selected from N and CH and
wherein n 1 and n 2 independently are 1-10.
4. A compound according to claim 3 , wherein X 3 and X 4 are N and n 1 and n 2 independently are 1-10.
5. The compound according to claim 1 , wherein L 1 and L 2 independently are selected from the group consisting of —NH—, —O—, —NHCO—, —CONH—, —NHC(O)O—, —OC(O)NH—, —NHCONH—, —NHC(═NH)NH—, —S(O)— and —SO 2 —.
6. The compound according to claim 5 , wherein L 1 and L 2 independently are selected from the group consisting of —NH—, —NHCO—, —CONH—, —NHC(O)O—, and —OC(O)NH—.
7. The compound according to claim 2 , wherein Y is
8. The compound according to claim 7 , wherein R 4 and R 5 independently are (CH 2 ) n .
9. The compound according to claim 7 , wherein R 4 and R 5 independently are (CH 2 —CHOH—CH 2 ) n .
10. The compound according to claim 8 wherein R 4 and R 5 are (CH 2 ) 3 .
11. The compound according to claim 9 wherein R 4 and R 5 are (CH 2 —CHOH—CH 2 ).
12. The compound according to claim 10 ,
wherein R 1 and R 2 are H; X 1 and X 2 are CH 2 ; R 3 and R 6 are N; W 1 and W 2 are H or OH; q, p, and m are 1;
and Z 1 and Z 2 independently are selected from the group consisting of oleoyl, oleyl, palmityl, myristyl, stearyl, and lauryl.
13. The compound according to claim 11 ,
wherein R 1 and R 2 are H; X 1 and X 2 are CH 2 ; R 3 and R 6 are N; W 1 and W 2 are H or OH; q, p, and m are 1;
and Z 1 and Z 2 independently are selected from the group consisting of oleoyl, oleyl, palmityl, myristyl, stearyl, and lauryl.
14. A composition comprising a compound according to claim 1 and a co-lipid- that is neutral, positively charged or negatively charged.
15. The composition according to claim 14 wherein said co-lipid is DOPE or cholesterol.
16. The composition according to claim 14 , further comprising a macromolecule.
17. The composition according to claim 16 wherein said macromolecule is a nucleic acid.
18. The composition according to claim 17 , wherein said nucleic acid comprises a DNA molecule.
19. The composition according to claim 17 , wherein said DNA molecule is a double stranded DNA molecule encoding an RNA molecule that is self complementary and that forms a region of double stranded RNA.
20. The composition according to claim 17 , wherein said nucleic acid comprises a double stranded RNA molecule.
21. The composition according to claim 17 wherein said RNA molecule is an siRNA.
22. The composition according to claim 16 , further comprising a eukaryotic cell.
23. The composition according to claim 22 , wherein said cell is a mammalian cell.
24. A method of introducing a macromolecule into a cell, comprising contacting a eukaryotic cell with a composition according to claim 16 .