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 1 is a basic amino acid residue, X 2 is Q or P, X 3 is A or T, X 4 is an acidic amino acid residue and X 5 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 comprising an amino acid sequence SX 4 RSMNF (SEQ. ID NO.:16), in which X 4 is E or Q, wherein said amino acid sequence binds to a cell surface receptor.
2. The isolated peptide according to claim 1 , wherein the amino acid sequence is SERSMNF (SEQ ID NO:7).
3. The isolated peptide according to claim 1 , consisting of 7 to 20 amino acids.
4. The isolated peptide according to claim 3 , consisting of 7 to 12 amino acids.
5. The isolated peptide according to claim 4 , consisting of 7 amino acids.
6. The isolated peptide according to claim 1 , wherein said amino acid sequence is comprised within a cyclic region of amino acids.
7. The isolated peptide according to claim 6 , wherein the peptide comprises two or more cysteine residues capable of forming one or more disulphide bond(s).
8. The isolated peptide according to claim 1 , wherein the peptide is linked to a polycationic nucleic acid-binding component.
9. The isolated peptide according to claim 8 , wherein the polycationic nucleic acid-binding component is polyethylenimine.
10. The isolated peptide according to claim 8 , wherein the polycationic nucleic acid-binding component is an oligo-lysine molecule consisting of from 5 to 25 lysine moieties.
11. The isolated peptide according to claim 8 , wherein the peptide is linked to the polycationic nucleic acid-binding component via a spacer element.
12. The isolated peptide according to claim 11 , wherein the spacer element is GG or GA or is longer and/or more hydrophobic than the dipeptide spacers GG (glycine-glycine) and GA (glycine-alanine).
13. 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 .
14. 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 .
15. The complex according to claim 14 , wherein the nucleic acid component is a gene that is the target for gene therapy, gene vaccination or anti-sense therapy.
16. The complex according to claim 14 , 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.
17. The complex according to claim 14 , wherein the nucleic acid component is DNA or RNA.
18. The complex according to claim 14 , wherein the nucleic acid-binding component has from 3 to 100 cationic monomers.
19. The complex according to claim 18 , wherein the polycationic nucleic acid-binding component is an oligolysine.
20. The complex according to claim 19 , wherein the oligolysine has from 10 to 20 lysine residues.
21. The complex according to claim 18 , wherein the polycationic nucleic acid-binding component is polyethylenimine.
22. The complex according to claim 14 , wherein the lipid component is or is capable of forming a cationic liposome.
23. The complex according to claim 22 , wherein the lipid component comprises one or more lipids selected from cationic lipids and lipids having membrane destabilising or fusogenic properties.
24. The complex according to claim 23 , wherein the lipid component comprises the neutral lipid dioleyl phosphatidyl-ethanolamine (DOPE).
25. The complex according to claim 23 , wherein the lipid component comprises the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).
26. The complex according to claim 23 , wherein the lipid component comprises a mixture of DOPE and DOTMA.
27. The complex according to claim 26 , which comprises an equimolar mixture of DOPE and DOTMA as the lipid component, and [K] 16 as the polycationic nucleic acid-binding component.
28. The complex according to claim 26 , wherein the lipid component comprises 2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoridoacetate (DOSPA).
29. The complex according to claim 28 , wherein the lipid component comprises a mixture of DOPE and DOSPA.
30. The complex according to claim 29 , 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.
31. A process for the production of a complex according to claim 14 , which comprises admixing the components (i), (ii), (iii) and (iv).
32. The process according to claim 31 , wherein the components are admixed in the following order:
lipid component, cell surface receptor-binding component/polycationic nucleic acid-binding component, nucleic acid.
33. A process for the production of a complex according to claim 14 , which comprises admixing components (i), (iii) and (iv).
34. The process according to claim 33 , wherein the components are admixed in the following order: cell surface receptor-binding component/polycationic nucleic acid-binding component, nucleic acid.
35. 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 .
36. The mixture according to claim 35 , wherein said cell surface receptor-binding component is a peptide of 7 to 20 amino acids comprising an amino acid sequence selected from the group consisting of: SERSMNF (SEQ ID NO:7), SQRSMNF (SEQ ID NO:36), and cyclic peptides thereof.
37. The mixture according to claim 36 , 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.
38. The mixture according to claim 36 or claim 37 , 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.
39. The mixture according to claim 36 , which comprises an equimolar mixture of DOPE and DOTMA as the lipid component, and [K] 16 as the polycationic component nucleic acid-binding component.
40. 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 .
41. The mixture according to claim 40 , wherein said cell surface receptor-binding component is a peptide of 7 to 20 amino acids comprising an amino acid sequence selected from the group consisting of: SERSMNF (SEQ ID NO:7), SQRSMNF (SEQ ID NO:36), and cyclic peptides thereof.
42. The mixture according to claim 41 , 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.
43. A process for producing a nucleic acid-containing transfection vector complex, which comprises incorporating a nucleic acid with a mixture according to claim 35 .
44. A process for producing a nucleic acid-containing transfection vector complex, which comprises incorporating a nucleic acid with a mixture as claimed in claim 40 .
45. 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 14 .
46. A pharmaceutical composition which comprises a complex according to claim 14 , in admixture or conjunction with a pharmaceutically suitable carrier.
47. A method for expressing a gene in a human or in a non-human animal with a defect and/or a deficiency in a gene, which comprises administering a complex as defined in claim 14 to the human or to the non-human animal.
48. A method for inducing an immune response in a human or a non-human animal, which comprises administering a complex as defined in claim 14 to the human or to the non-human animal.
49. A method of inhibiting the expression of a gene, which comprises administering a complex as defined in claim 14 to a human or to a non-human animal, wherein the expression of the gene is inhibited by the expression of an antisense nucleic acid.
50. 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 .
51. The isolated peptide according to claim 1 , wherein the amino acid sequence is SQRSMNF (SEQ ID NO:36).