Gene delivery system

• US 5,972,707 A
• Filed: 07/09/1997
• Issued: 10/26/1999
• Est. Priority Date: 06/27/1994
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Leong, Kam W., August, Thomas, Mao, Hai-Quan, Roy, Krishnendu, Truong, Vu L.
• Primary Examiner(s): Stanton, Brian R.
• Assistant Examiner(s): REYNOLDS, DEBORAH J
• Application Number:
  US08/890,599
• Time in Patent Office: 839 Days
• Field of Search: 514/44, 435/172.3, 435/455, 435/440, 435/320.1
• US Class Current: 435/455
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/6435 the peptide or protein in the drug conjugate being a connective tissue peptide, e.g. collagen, fibronectin or gelatin
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6927 the form being a solid microparticle having no hollow or gas-filled cores
• 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 9/167 with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface with further drug-free outer coating A61K9/5073
• CPC Class Codes: A61K 9/5161 Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
• CPC Class Codes: A61K 9/5169 Proteins, e.g. albumin, gelatin
• CPC Class Codes: A61K 9/5192 Processes
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: Y10S 977/808 Exterior attachment for targeting, e.g. drug targeting
• CPC Class Codes: Y10S 977/906 Drug delivery
• CPC Class Codes: Y10S 977/915 Therapeutic or pharmaceutical composition
• CPC Class Codes: Y10S 977/916 Gene therapy
• CPC Class Codes: Y10S 977/92 Detection of biochemical

Abstract

A gene delivery system is made of enzymatically degradable polymeric cation and nucleic acid (DNA or RNA) nanospheres optionally with a linking moiety or a targeting ligand attached to the surface. The delivery system can be made by a simple method of coacervation. Targeting ligands can be attached to the nanosphere directly or via a linking moiety. The linkage design allows the attachment of any molecule onto the nanosphere surface including antibodies, cell adhesion molecules, hormones and other cell-specific ligands.

Claims

1. A composition comprising solid nanospheres of less than 3 μ

m for gene delivery to cells, comprising a polymeric cation and a polyanion, wherein the polyanion consists of nucleic acids, wherein the polymeric cation is a carbohydrate.

2. The composition of claim 1 wherein the polymeric cation is chitosan.

3. The composition of claim 1 wherein said nanospheres comprises greater than 5% (w/w) nucleic acids.

4. The composition of claim 1 wherein said nanospheres comprises greater than 20% (w/w) nucleic acids.

5. The composition of claim 1 wherein said nucleic acids comprise a gene of 2-20 kb.

6. The composition of claim 1 wherein the nanospheres are between about 200 and 300 nm.

7. The composition of claim 1 wherein the nanospheres are less than 2 μ

m.

8. The composition of claim 1 wherein the nanospheres are less than 1 μ

m.

9. The composition of claim 1 wherein the nanospheres are less than 151 nm.

10. A method of forming solid nanospheres for gene delivery to specific target cells, comprising the step of:

forming nanospheres of less than 3 μ

m by coacervation of nucleic acids and a polymeric cation which is a carbohydrate.

11. The method of claim 10 wherein the coacervation is performed in the presence of sodium sulfate.

12. The method of claim 10 wherein the polymeric cation is chitosan.

13. The method of claim 10 wherein the polymeric cation is present at a concentration of about 0.01-7% in the step of coacervation.

14. The method of claim 10 wherein the nucleic acids are present in a concentration of 1 ng/ml to 500 μ

g/ml in the step of coacervation.

15. The method of claim 11 wherein the concentration of sodium sulfate is between about 5 and 100 mM in the step of coacervation.

16. The method of claim 10 wherein the nanospheres are between about 200 and 300 nm.

17. The method of claim 10 wherein the nanospheres are less than 2 μ

m.

18. The method of claim 10 wherein the nanospheres are less than 1 μ

m.

19. The method of claim 10 wherein the nanospheres are less than 151 nm.

20. A method for introducing genes into cells, comprising the steps of:

incubating (a) cells to be transfected, in vitro with (b) solid nanospheres of less than 3 μ

m comprising polymeric cationic molecules and nucleic acid molecules, whereby the cells are transfected with the nucleic acid molecules, wherein the polymeric cationic molecules are carbohydrates.

21. The method of claim 20 wherein the polymeric cationic molecules are chitosan.

22. The method of claim 20 wherein the nucleic acid is DNA.

23. The method of claim 20 wherein the nucleic acid is RNA.

24. The method of claim 20 wherein the nanospheres are between about 200 and 300 nm.

25. The method of claim 20 wherein the nanospheres are less than 2 μ

m.

26. The method of claim 20 wherein the nanospheres are less than 1μ

m.

27. The method of claim 20 wherein the nanospheres are less than 151 nm.