Methods for compositions for in vivo gene delivery

• US 6,806,084 B1
• Filed: 06/10/1998
• Issued: 10/19/2004
• Est. Priority Date: 06/04/1992
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Zhu, Ning, Debs, Robert James
• Primary Examiner(s): NGUYEN, DAVE TRONG
• Application Number:
  US09/090,030
• Time in Patent Office: 2,323 Days
• Field of Search: 424/93.21, 514/44, 435/69.1, 435/320.1, 435/325, 435/455, 435/458, 536/23.1
• US Class Current: 435/455
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4644 Cancer antigens
• CPC Class Codes: A61K 39/464838 Viral antigens
• 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 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: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

Novel methods and compositions are provided for introducing a gene capable of modulating the genotype and phenotype into two or more tissues following systemic administration. The gene can be introduced into a mammalian host by way of an expression vector either as naked DNA or complexed to lipid carriers, particularly cationic lipid carriers. Multiple individual tissues can be transfected using naked DNA. Using a DNA:lipid carrier complex, multiple tissues and cell types can be transfected. The techniques and compositions find use in the palliation or treatment of any of a variety of genetic-based disorders.

Claims

1. A method of introducing a selected nucleic acid into cells of a mammal, comprising:

administering a composition comprising a cationic lipid-nucleic acid complex comprising the selected nucleic acid to the peritoneal cavity of the mammal, wherein the comnposition is substantially free of macroaggregates of the complex, wherein the selected nucleic acid encodes an RNA or polypeptide, and wherein administration of the cationic lipid-nucleic acid complex to the peritoneal cavity of the mammal results in detectable expression of the RNA or protein in the cells of the mammal.

2. The method of claim 1, wherein the cationic lipid-nucleic acid complex further comprises a neutral lipid.

3. The method of claim 2, wherein the molar ratio of cationic lipid to neutral lipid in the complex is at least about 1:

20.

4. The method of claim 2, wherein the molar ratio of cationic lipid to neutral lipid in the complex is about 1:

1.

5. The method of claim 1, wherein the cationic lipid-nucleic acid complex comprises a steroid.

6. The method of claim 1, wherein the cationic lipid-nucleic acid complex comprises a lipid selected from the group consisting of DOTAP, E-PC, L-PE, cholesterol, DOPE, and DDAB.

7. The method of claim 1, wherein the cationic lipid-nucleic acid complex comprises a site-directing molecule.

8. The method of claim 7, wherein the site-directing molecule is selected from the group consisting of an antibody and a ligand.

9. The method of claim 1, wherein the cationic lipid-nucleic acid complex is introduced into the peritoneal cavity of the mammal by intraperitoneal injection.

10. The method of claim 1, wherein the cationic lipid-nucleic acid complex is suspended in a physiologically acceptable medium for administration.

11. The method of claim 10, wherein the medium is selected from the group consisting of deionized water, saline, phosphate buffered saline, and 5% dextrose in water.

12. The method of claim 10, wherein the medium comprises a buffer, a stabilizer, or a biocide.

13. The method of claim 1 whereby the selected nucleic acid is introduced into cells of at least two tissues in the mammal.

14. The method of claim 1 whereby the selected nucleic acid is introduced into cells of at least two tissues in the mammal and expressed in said at least two tissues.

15. The method of claim 1, wherein the selected nucleic acid comprises DNA.

16. The method of claim 1 wherein the selected nucleic acid encodes a molecule selected from the group consisting of a ribozyme, an anti-sense molecule, and a protein.

17. The method of claim 1 wherein the selected nucleic acid comprises a DNA plasmid.

18. The method of claim 1 wherein the selected nucleic acid comprises a DNA expression cassette.

19. The method of claim 1 wherein at least about 50 μ

g of the selected nucleic acid is introduced into the mammal.

20. The method of claim 1, wherein the cationic lipid does not comprise DOTMA.

21. A mammalian transfection complex comprising:

a cationic lipid and a non-cationic lipid forming a lipid carrier ranging in size from 100 nm to 10 microns in diameter;

combined with a selected nucleic acid in a ratio of less than 6;

1 micrograms selected nucleic acid to nanomoles cationic lipid;

a physiologically acceptable medium for in vivo systemic administration; and

,

wherein said non-cationic lipid comprises a steroid and said complex does not aggregate in vitro and wherein the complex transfects a cell in vivo following systemic administration.

22. The mammalian transfection complex of claim 21, wherein the steroid is cholesterol.

23. The mammalian transfection complex of claim 21, wherein the nucleic acid is DNA.

27. The method of claim 21 or 24, wherein the selected nucleic acid is a DNA expression cassette comprising:

a promoter, and a DNA subsequence encoding a gene product.

24. A method of making a nucleic acid:

lipid complex for systemic administration to a mammal comprising;

mixing a nucleic acid and a lipid carrier to provide a non-aggregating nucleic acid;

lipid carrier complex having a mean diameter of less than about 10 microns, wherein the lipid carrier comprises cationic lipids and a steroid and wherein the nucleic acid to lipid carrier ratio is less than 6;

1 micrograms DNA to nanomoles cationic lipid, wherein the complex is suspended in a physiologically acceptable medium.

25. The method of claim 24, wherein the medium is selected from the group consisting of deionized water, saline, phosphate buffered saline, and 5% dextrose in water.

26. The method of claim 24, wherein the steroid is cholesterol.

31. The method of claim 24 or 28, further comprising loading the nucleic acid carrier complex into a systemic delivery container.

32. The method of claim 24 or 28, further comprising loading the nucleic acid carrier complex into a syringe.

28. A method of making a nucleic acid:

lipid complex for systemic administration to a mammal comprising;

mixing a selected nucleic acid and a lipid carrier comprising cationic and, optionally, non cationic lipids, said carrier having a mean diameter of less than about 10 microns, wherein the molar ratio of cationic lipids to non cationic lipids ranges from 1;

20 to 1;

0, resulting in a non-aggregating nucleic acid lipid carrier complex, wherein the nucleic acid to lipid carrier ratio is less than 6;

1 micrograms selected nucleic acid to nanomoles cationic lipid wherein the non-aggregating nucleic acid lipid carrier complex is suspended in a physiologically acceptable medium.

29. The method of claim 28, wherein the non-cationic lipid is cholesterol.

30. The method of claim 28, wherein the molar ratio of cationic to non-cationic lipids is about 1:

1.