Peptide-enhanced transfections

• US 6,376,248 B1
• Filed: 03/16/1998
• Issued: 04/23/2002
• Est. Priority Date: 03/14/1997
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Gebeyehu, Gulilat, Jessee, Joel A., Hawley-Nelson, Pamela, Evans, Krista L., Shih, PoJen, Schifferli, Kevin P., Ciccarone, Valentina C., Lan, Jianqing
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US09/039,780
• Time in Patent Office: 1,499 Days
• Field of Search: 435/235.1, 435/320.1, 435/458, 536/23.1
• US Class Current: 435/458
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: A61K 47/6949 inclusion complexes, e.g. clathrates, cavitates or fullerenes
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0025 wherein the non-active part clearly interacts with the delivered nucleic acid
• CPC Class Codes: A61K 48/0041 the non-active part being polymeric
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2740/16322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The present invention provides compositions useful for transfecting eukaryotic cells comprising nucleic acid complexes with peptides, wherein the peptide is optionally covalently coupled to a nucleic acid-binding group, and cationic lipids or dendrimers as transfection agents. The invention also provides transfection compositions in which a peptide is covalently linked to the transfection agent (lipid, cationic lipid or dendrimer). Inclusion of peptides or modified-peptides in transfection compositions or covalent attachment of peptides to transfection agents results in enhanced transfection efficiency. Methods for the preparation of transfection compositions and methods of using these transfection compositions as intracellular delivery agents and extracellular targeting agents are also disclosed.

Claims

1. A peptide comprising a Tat sequence modified by covalent bonding to a nucleic acid-binding group which is not a Tat sequence.

2. A composition for transfecting a cell which comprises one or more nucleic acid molecules, a peptide of claim 1 and one or more transfection agents.

3. A method for transfecting a cell with a nucleic acid, the method comprising the steps:

(a) admixing a peptide of claim 1 with a nucleic acid to form a peptide-nucleic acid complex;

(b) adding a cationic lipid transfection agent to the complex from step (a) to obtain an aggregate of the transfection agent and said complex; and

(c) contacting said cell with the aggregate from step (b).

4. The composition of claim 1, wherein said composition comprises a peptide-nucleic acid complex.

5. The composition of claim 2, wherein said transfection agents comprise one or more cationic lipids.

6. The composition of claim 5, wherein said transfection agents further comprise one or more neutral lipids.

7. The composition of claim 5, wherein said cationic lipids comprise one or more monovalent cationic lipids.

8. The composition of claim 7, wherein said monovalent cationic lipids are selected from the group consisting of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide.

9. The composition of claim 5, wherein said cationic lipids comprise one or more polyvalent cationic lipids.

10. The composition of claim 9, wherein said polyvalent cationic lipids are selected from the group consisting of 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoracetate, 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamid, 5-carboxyspermylglycine dioctadecylamide, tetramethyltetrapalmitoyl spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl spermine, tetramethyltetramyristyl spermine and tetramethyldioleyl spermine.

11. The composition of claim 4, wherein said neutral lipids are selected from the group consisting of dioleoylphosphatidylethanolamine, diphytanoylphosphatidylethanolamine, and cholesterol.

12. The composition of claim 2, wherein one or more of said transfection agents are linked to said peptide.

13. The composition of claim 5, wherein one or more of said cationic lipids are covalently linked to said peptide.

14. The composition of claim 6, wherein one or more of said neutral lipids are covalently linked to said peptide.

15. The composition of claim 2, wherein said peptide is conjugated to one or more nucleic acid binding groups.

16. The composition of claim 15, wherein said nucleic acid binding groups comprise at least one polyamine.

17. The composition of claim 16, wherein said nucleic acid binding group comprises at least one spermine.

18. The composition of claim 2, further comprising DEAE-dextran, chloroquine or combinations thereof.

19. The composition of claim 2, wherein said peptide comprises a dimer or a multimer of a Tat sequence.

20. The composition of claim 2 for transfecting a cell obtained by first forming a peptide-nucleic acid complex followed by addition of a transfection agent that aggregates the peptide-nucleic acid complex.

21. The composition of claim 20 wherein after the peptide-nucleic acid complex is formed, said complex is added to a mixture of a cationic lipid and a neutral lipid.

22. A method for transfecting a cell with a nucleic acid, the method comprising the step of contacting the cell with the transfection composition of claim 2.

23. A method for transfecting a cell with a nucleic acid, the method comprising the step of contacting the cell with the transfection composition of claim 13.

24. The method of claim 3 wherein the nucleic acid binding group is spermine.

25. The method of claim 3 wherein the peptide is Sp-Tat.

26. The modified peptide of claim 1 which comprises a dimer or multimer of a Tat sequence.

27. The modified peptide of claim 26 wherein the nucleic acid-binding group is a spermine.

28. A transfection reagent kit which comprises a cationic lipid transfection agent and a modified peptide capable of enhancing transfection of the cationic lipid transfection agent wherein the modified peptide is Sp-Tat.

29. The kit of claim 28 which comprises a cationic lipid transfection agent.

30. The kit of claim 28 wherein the cationic lipid transfection agent is a 3:

1 (w/w) mixture of the polycationic lipid, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate and dioleoylphosphatidylethanolamine, a 1;

1 (w/w) mixture of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidylethanolamine, or a 1;

2.5 (w/w) mixture of dimethyldioctadecylammonium bromide and dioleoylphosphatidylethanolamine.