Peptide-enhanced transfections

• US 8,058,068 B2
• Filed: 07/23/2002
• Issued: 11/15/2011
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hawley-Nelson, Pamela, Lan, Jianqing, Shih, PoJen, Jessee, Joel A., Schifferli, Kevin P., Ciccarone, Valentina C., Evans, Krista L., Gebeyehu, Gulilat
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US10/200,879
• Publication Number:
  US 20030144230A1
• Time in Patent Office: 3,402 Days
• Field of Search: None
• US Class Current: 435/458
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 47/6911 the form being a liposome
• 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 2319/09 containing a nuclear localisation signal
• 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 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2740/16322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2810/405 Vectors comprising RGD peptide
• CPC Class Codes: C12N 2810/60 from viruses
• CPC Class Codes: C12N 2810/6018 Adenoviridae
• CPC Class Codes: C12N 2810/6054 : Retroviridae
• CPC Class Codes: C12N 2810/6081 : rhabdoviridae, e.g. VSV
• CPC Class Codes: Y10T 428/2984 : Microcapsule with fluid core [includes liposome]

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 purified peptide, wherein the peptide is Sp-NLSNLS.

2. A composition for transfecting a cell, the composition comprising one or more nucleic acid molecules, one or more peptides and one or more transfection agents, wherein each peptide in the composition comprises Sp-NLSNLS.

3. The composition of claim 2, wherein the composition comprises two or more transfection agents.

4. The composition of claim 2, wherein the transfection agent comprises one or more cationic lipids.

5. The composition of claim 4, wherein the transfection agent further comprises one or more neutral lipids.

6. The composition of claim 4, wherein the one or more cationic lipids comprise one or more monovalent cationic lipids.

7. The composition of claim 6, wherein the one or more monovalent cationic lipids are selected from the group consisting of N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane (DOTAP), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), and dimethyl dioctadecyl ammonium bromide (DDAB).

8. The composition of claim 4, wherein the one or more cationic lipids comprise one or more polyvalent cationic lipids.

9. The composition of claim 8, wherein the one or more polyvalent cationic lipids are selected from the group consisting of 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoro-acetate (DOSPA), 1,3-dioleoyloxy-2-(6-carboxy spermyl)-propylamide (DOSPER), 5-carboxyspermylglycine dioctadecyl-amide (DOGS), tetramethyltetrapalmitoyl spermine (TMTPS), tetramethyltetraoleyl spermine (TMTOS), tetramethlytetralauryl spermine (TMTLS), tetramethyltetramyristyl spermine (TMTMS), and tetramethyldioleyl spermine (TMDOS).

10. The composition of claim 5, wherein the one or more neutral lipids are selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), diphytanoylphosphatidylethanolamine (DPhPE), and cholesterol.

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

12. The composition of claim 2, wherein the composition can transfect a primary cell culture, a passaged cell culture or a cell line.

13. The composition of claim 12, wherein the cell line is a human cell line.

14. The composition of claim 12, wherein the cell line is an animal cell line.

15. The composition of claim 12, wherein the cell line is a fibroblast.

16. The composition of claim 2, wherein the one or more nucleic acid molecules is one or more DNA molecules or fragments thereof.

17. The composition of claim 2, wherein the one or more nucleic acid molecules is one or more RNA molecules or fragments thereof.

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

18. A transfection reagent kit which comprises a transfection agent and one or more peptides comprising Sp-NLSNLS.

19. The kit of claim 18, wherein the kit comprises a cationic lipid transfection agent.

20. The kit of claim 19, wherein the cationic lipid is a polyvalent cationic lipid.

21. The kit of claim 20, wherein the polyvalent cationic lipid is selected from the group consisting of 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoro-acetate (DOSPA), 1,3-dioleoyloxy-2-(6-carboxy spermyl)-propylamide (DOSPER), 5-carboxyspermylglycine dioctadecyl-amide (DOGS), tetramethyltetrapalmitoyl spermine (TMTPS), tetramethyltetraoleyl spermine (TMTOS), tetramethlytetralauryl spermine (TMTLS), tetramethyltetramyristyl spermine (TMTMS), and tetramethyldioleyl spermine (TMDOS).

22. The kit of claim 18, wherein the kit further comprises one or more neutral lipids.

23. The kit of claim 22, wherein the one or more neutral lipids are selected from dioleoylphosphatidylethanolamine (DOPE), diphytanoylphosphatidylethanolamine (DPhPE) and cholesterol.

24. The kit of claim 18, wherein the kit comprises a 3:

1 (w/w) mixture of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate and dioleoylphosphatidyl-ethanolamine.