Method of transfection and compositions therefor
The present invention relates to the targeted delivery of molecules to cells expressing toll-like receptors (TLRs). Aspects of the invention provide compounds comprising a positively charged group linked to a TLR ligand. These compounds are useful for in vitro and in vivo methods of transfection of TLR-expressing cells. Other aspects of the invention relate to the use of such compounds for repression of gene expression and DNA vaccination approaches.
1. A positively charged compound comprising a positively charged group linked to at least one TLR-2 or TLR-6 ligand, wherein the positively charged group comprises a branched peptide comprising at least four positively charged amino acid residues, wherein the branched peptide comprises:
wherein
each X is independently a lysine residue, an arginine residue or a histidine residue.
2. The compound of claim 1 , wherein the TLR-2 or TLR-6 ligand is selected from the group consisting of bacterial lipoproteins, diacylated bacterial lipids, peptidoglycan, yeast zyomosan, Pam 2 Cys, Pam 3 Cys, Ste 2 Cys, Lau 2 Cys and Oct 2 Cys, or wherein the TLR-2 or TLR-6 ligand comprises palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl or decanoyl.
3. A compound according to claim 2 , wherein the TLR-2 or TLR-6 ligand comprises palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl.
4. A compound according to claim 2 , wherein the TLR-2 or TLR-6 ligand is selected from the group consisting of: Pam 2 Cys, Pam 3 Cys, Ste 2 Cys, Lau 2 Cys, and Oct 2 Cys.
5. A compound according to claim 2 , wherein the TLR-2 or TLR-6 ligand is Pam 2 Cys.
6. A complex comprising a nucleic acid and a compound of claim 1 , wherein the nucleic acid is associated with the compound of claim 1 by electrostatic interaction between the nucleic acid and the positively charged group.
7. A complex according to claim 6 , wherein the TLR-2 or TLR-6 ligand binds either TLR-2 or TLR-6.
8. A complex according to claim 6 , wherein the TLR-2 or TLR-6 ligand comprises palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl.
9. A complex according to claim 6 , wherein the TLR-2 or TLR-6 ligand is selected from the group consisting of: Pam 2 Cys, Pam 3 Cys, Ste 2 Cys, Lau 2 Cys, and Oct 2 Cys.
10. A complex according to claim 6 , wherein the TLR-2 or TLR-6 ligand binds to TLR-2.
11. A complex according to claim 6 , wherein the branched peptide comprises at least four arginine residues or at least four lysine residues.
12. A method of transfection comprising contacting a cell expressing at least one TLR with a complex according to claim 6 .
13. A method of raising an immune response against an antigen, comprising administering to a subject a complex according to claim 6 , wherein the nucleic acid encodes the antigen or an epitope thereof.
14. A method of raising an immune response against an antigen, comprising administering to a subject cells transfected with a complex according to claim 6 , wherein the nucleic acid encodes the antigen or an epitope thereof.
15. A method of repressing expression of a gene in a cell expressing a TLR, comprising administering to a subject a complex according to claim 6 , wherein the nucleic acid is selected from the group consisting of: siRNA; shRNA; DNA encoding siRNA; and DNA encoding shRNA, and wherein the siRNA or shRNA is targeted against the gene.
16. A compound according to claim 1 wherein the TLR-2 or TLR-6 ligand binds to TLR-2.
17. A compound according to claim 1 wherein the branched peptide comprises at least one lysine or at least one arginine residue.
18. A compound according to claim 1 , wherein the branched peptide is R 4 , represented by the structure:
19. A compound according to claim 1 , wherein the branched peptide is K 4 , represented by the structure:
20. A compound according to claim 1 , wherein the TLR-2 or TLR-6 ligand is Pam 2 Cys and the branched peptide is R 4 , represented by the structure:
21. A compound according to claim 1 , wherein the TLR-2 or TLR-6 ligand is Pam 2 Cys and the branched peptide is K 4 , represented by the structure:
22. A compound of claim 1 , wherein the compound is a compound of formula I:
wherein the C-terminal lysine residue is coupled to serine through the epsilon amino group of said C-terminal lysine residue.
23. compound of claim 1 , wherein the compound is a compound of formula II:
wherein the C-terminal lysine residue is coupled to serine through the epsilon amino group of said C-terminal lysine residue.