Transfection complexes
The invention provides a peptide having at least 3 amino acids comprising an amino acid sequence selected from a) X 1 SM [SEQ.ID.NO.:1] b) LX 2 HK [SEQ.ID.NO.:2] c) PSGX 3 ARA [SEQ.ID.NO.:9] d) SX 4 RSMNF [SEQ.ID. NO.:16] e) LX 5 HKSMP [SEQ.ID.NO.:18] in which X is a basic amino acid residue, X 1 is Q or P, X 2 is A or T, X 3 is an acidic amino acid residue and X 4 is P or Q, the invention further provides non-viral cell-targeting vector complexes and methods associated therewith.
1. An isolated peptide of 7 to 100 amino acids, said peptide having cell surface receptor binding activity and comprising an amino acid sequence selected from the group consisting of VGLPHKF [SEQ ID NO:8] and YGLPHKF [SEQ ID NO:19].
2. The peptide according to claim 1 , consisting of 7 to 12 amino acids.
3. The peptide according to claim 2 , consisting of 7 amino acids.
4. The peptide according to claim 1 , wherein the peptide is comprised within a cyclic region of amino acids.
5. The peptide according to claim 4 , wherein the peptide comprises two or more cysteine residues capable of forming one or more disuiphide bond(s).
6. The peptide according to claim 1 , wherein the peptide is linked to a polycationic nucleic acid-binding component.
7. The peptide according to claim 6 , wherein the polycationic nucleic acid-binding component is polyethylemmine.
8. The peptide according to claim 6 , wherein the polycationic nucleic acid-binding component is an oligo-lysine molecule having from 5 to 25 lysine moieties.
9. The peptide according to claim 6 , wherein the peptide is linked to the polycationic nucleic acid-binding component via a spacer element.
10. The peptide according to claim 9 wherein the spacer element is GG (glycine-glycine) or GA (glycine-alanine) or is longer and/or more hydrophobic than the dipeptide spacers GG and GA.
11. A peptide derivative of formula A-B-C wherein
A is a polycationic nucleic acid-binding component,
B is a spacer element, and
C is a peptide according to claim 1 .
12. A non-viral transfection complex comprising
(i) a nucleic acid,
(ii) optionally, a lipid component,
(iii) a polycationic nucleic acid-binding component, and
(iv) a cell surface receptor binding component, comprising a peptide according to claim 1 .
13. The complex according to claim 12 , wherein the nucleic acid component is a therapeutic gene, a gene vaccine, or an anti-sense therapeutic.
14. The complex according to claim 12 , further comprising transcriptional and/or translational control elements for directing expression of the nucleic acid component and wherein the nucleic acid is optionally packed in a phage or vector.
15. The complex according to claim 12 , wherein the nucleic acid component is DNA or RNA.
16. The complex according to claim 12 , wherein the nucleic acid-binding component has from 3 to 100 cationic monomers.
17. The complex according to claim 16 , wherein the polycationic nucleic acid-binding component is an oligolysine.
18. The complex according to claim 17 , wherein the oligolysine has from 10 to 20 lysine residues.
19. The complex according to claim 16 , wherein the polycationic nucleic acid-binding component is polyethylenimine.
20. The complex according to claim 12 , wherein the lipid component is or is capable of forming a cationic liposome.
21. The complex according to claim 20 , wherein the lipid component comprises one or more lipids selected from cationic lipids and lipids having membrane destabilising or fusogenic properties.
22. The complex according to claim 21 , wherein the lipid component comprises the neutral lipid dioleyl phosphatidyl-ethanolamine (DOPE).
23. The complex according to claim 21 , wherein the lipid component comprises the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).
24. The complex according to claim 21 , wherein the lipid component comprises a mixture of DOPE and DOTMA.
25. The complex according to claim 24 , which comprises an equimolar mixture of DOPE and DOTMA as the lipid component, and [K] 16 as the polycationic nucleic acid-binding component.
26. The complex according to claim 24 , wherein the lipid component comprises 2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoridoacetate (DOSPA).
27. The complex according to claim 26 , wherein the lipid component comprises a mixture of DOPE and DOSPA.
28. The complex according to claim 27 , which comprises a 1:3 mixture by weight of DOPE and DOSPA as the lipid component, and [K] 16 as the polycationic nucleic acid-binding component.
29. A process for the production of a complex according to claim 12 , which comprises admixing the components (i), (ii), (iii) and (iv).
30. The process according to claim 29 , wherein the components are admixed in the following order:
lipid component, cell surface receptor-binding componentlpolycationic nucleic acid-binding component, nucleic acid.
31. A process for the production of a complex according to claim 12 , which comprises admixing components (i), (iii) and (iv).
32. The process according to claim 31 , wherein the components are admixed in the following order: cell surface receptor-binding component/polycationic nucleic acid-binding component, nucleic acid.
33. A mixture comprising a cell surface receptor-binding component, a polycationic nucleic acid-binding component, and a lipid component, the cell surface receptor-binding component being a peptide as defined in claim 1 .
34. The mixture according to claim 33 , wherein said cell surface receptor-binding component is an isolated peptide of 7 to 100 amino acids comprising an amino acid sequence selected from the group consisting of VGLPHKF: [SEQ ID NO:8] or YGLPHKF [SEQ ID NO:19], and cyclic peptides including either of the foregoing sequences.
35. The mixture according to claim 34 , wherein the polycationic nucleic acid-binding component is comprised of 3 to 100 cationic monomers and is selected from the group consisting of oligolysine, polyethyleneimine, and combinations thereof.
36. The mixture according to claim 34 , wherein the lipid component is selected from the group consisting of dioleyl phosphatidyl-ethanolamine (DOPE), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), or combinations thereof.
37. The mixture according to claim 34 , which comprises an equimolar mixture of DOPE and DOTMA as the lipid component, and [K] 16 as the polycationic component nucleic acid-binding component.
38. A mixture comprising a cell surface receptor-binding component and a polycationic nucleic acid-binding component, the cell surface receptor-binding component being a peptide as defined in claim 1 .
39. The mixture according to claim 38 , wherein said cell surface receptor-binding component is an isolated peptide of 7 to 100 amino acids comprising an amino acid sequence selected from the group consisting of VGLPHKF: [SEQ ID NO:8] or YGLPHKF [SEQ ID NO:19], and cyclic peptides including either of the foregoing sequences.
40. The mixture according to claim 39 , wherein the polycationic nucleic acid-binding component is comprised of 3 to 100 cationic monomers and is selected from the group consisting of oligolysine, polyethyleneimine, and combinations thereof.
41. A process for producing a nucleic acid-containing transfection vector complex, which comprises incorporating a nucleic acid with a mixture according to any one of claims 33 to 37 .
42. A process for producing a nucleic acid-containing transfection vector complex, which comprises incorporating a nucleic acid with a mixture as claimed in any one of claims 38 to 40 .
43. A method of transfecting a cell with a nucleic acid, which comprises contacting the cell in vitro or in vivo with a complex according to claim 12 .
44. A kit that comprises
(i) nucleic acid,
(ii) optionally, a lipid component,
(iii) a polycationic nucleic acid-binding component, and
(iv) a cell surface receptor binding component, comprising a peptide as defined in claim 1 .