Methods and compositions for in vivo gene therapy

• US 6,627,615 B1
• Filed: 08/11/1998
• Issued: 09/30/2003
• Est. Priority Date: 12/17/1991
• 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 J.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US09/132,391
• Time in Patent Office: 1,876 Days
• Field of Search: 435/69.1, 435/172.3, 435/240.2, 435/325, 435/354, 435/375, 435/6, 435/458, 514/44, 424/450, 424/417, 424/420
• US Class Current: 514/44.00R
• 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 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/544 Phospholipids
• 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: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 associated with lipid carriers, particularly cationic lipid carriers. Multiple individual tisssues 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 nucleic acid into cells of a mammal, said method comprising introducing the nucleic acid into the mammal systemically, wherein:

the nucleic acid is complexed to a lipid carrier comprising cationic lipids and a steroid, the carrier having a mean diameter of less than about 10 microns, resulting in a nucleic acid-lipid carrier complex;

the nucleic acid-lipid carrier complexes are substantially free of macroaggregates prior to said systemic introduction;

the nucleic acid to lipid carrier ratio is less than 6;

1 micrograms nucleic acid to nanomoles cationic lipid; and

the nucleic acid comprises a DNA expression cassette comprising a promoter and a DNA subsequence encoding a protein.

2. The method of claim 1, wherein the steroid is cholesterol.

3. The method of claim 1, wherein the nucleic acid:

cationic lipid carrier ratio is about 1;

6 μ

g nucleic acid;

nmoles cationic lipid and the lipid carrier comprises DOTAP and cholesterol.

7. The method of claim 1, or 4, whereby the nucleic acid is introduced into cells of at least two tissues in the mammal.

8. The method of claim 1, or 4, whereby the nucleic acid is introduced into cells of at least two tissues in the mammal and expressed in said at least two tissues.

9. The method of claim 1, or 4, wherein the lipid carrier is an MLV.

10. The method of claim 1, or 4, wherein the lipid carrier is an MLV with a mean diameter of at least about 500 nm.

11. The method of claim 1, or 4, wherein the lipid carrier is an SUV.

12. The method of claim 1, wherein the lipid carrier comprises DOPE.

13. The method of claim 1, or 4, wherein the promoter is an HCMV promoter.

14. The method of claim 1, or 4, wherein the nucleic acid is a DNA plasmid.

15. The method of claim 1, or 4, wherein the nucleic acid:

lipid carrier does not aggregate in an aqueous solution comprising 5% dextrose.

16. The method of claim 1, or 4, wherein the nucleic acid does not comprise an intron.

17. The method of claim 1, or 4, wherein the nucleic acid is a DNA expression cassette comprising a 5′

intron.

18. The method of claim 1, or 4, wherein at least about 50 μ

g of the nucleic acid is introduced into the mammal.

19. The method of claim 1, or 4, wherein the nucleic acid is a DNA purified without PEG prior to complexing to said lipid carrier.

20. The method of claim 1, or 4, wherein the size of the nucleic acid:

lipid carrier complex is at least about 500 nm.

21. The method of claim 1, or 4, wherein the nucleic acid is linear.

22. The method of claim 1, or 4, wherein the nucleic acid is introduced into said mammal intravenously.

23. The method of claim 1, or 4, wherein the nucleic acid is introduced into said mammal intraperitoneally.

24. The method of claim 23, wherein the lipid carrier comprises cholesterol.

25. The method of claim 1, or 4, wherein the nucleic acid:

cationic lipid carrier ratio is between about 1;

1 and about 1;

6 μ

g nucleic acid;

nmoles cationic lipid.

26. The method of claim 1, or 4, wherein the nucleic acid:

cationic lipid carrier ratio is about 1;

1 to about 1;

5 μ

g nucleic acid;

nmoles cationic lipid and the cationic lipid carrier comprises DDAB.

27. The method of claim 1, or 4, wherein the nucleic acid:

cationic lipid carrier ratio is about 1;

1 μ

g nucleic acid;

nmoles cationic lipid and the lipid carrier comprises LPE and CEBA.

28. The method of claim 1, or 4, wherein the nucleic acid:

cationic lipid carrier ratio is about 1;

5 μ

g nucleic acid;

nmoles cationic lipid and the lipid carrier comprises DDAB and cholesterol.

29. The method of claim 1, or 4, wherein a cell into which nucleic acid is introduced is selected from the group consisting of a mammalian T cell, a lung cell, a liver cell, a vascular endothelial cell, and a cell of lymph node.

30. The method of claim 1, or 4, wherein said lipid carriers have a mean diameter ranging in size from about 100 nm to 10 μ

m.

31. The method of claim 1, or 4, wherein the cationic lipid comprises a lipid other than DOTMA.

32. The method of claim 1, or 4, wherein said lipid carriers do not comprise DOTMA.

33. The method of claim 1, or 4, wherein the DNA expression cassette to lipid carrier ratio is greater than 1:

3 micrograms DNA to nanomoles cationic lipid.

4. A method of introducing a nucleic acid into a mammal, said method comprising introducing the nucleic acid into the mammal systemically, wherein:

the nucleic acid is complexed to a lipid carrier consisting essentially of cationic lipids and steroids, said carrier having a mean diameter of less than about 10 microns, wherein the molar ratio of cationic lipids to steroids ranges from about 1;

19 to about 1;

1, resulting in a nucleic acid lipid carrier complex;

the nucleic acid-lipid carrier complexes are administered in a form substantially free of macroaggregates;

the nucleic acid to lipid carrier ratio is less than 6;

1 micrograms nucleic acid to nanomoles cationic lipid; and

the nucleic acid comprises a DNA expression cassette comprising a promoter and a DNA subsequence encoding a protein.

5. The method of claim 4, wherein the non-cationic lipid is a steroid.

6. The method of claim 4, wherein the molar ratio of cationic lipids to steroids is about 1:

1.

34. A mammalian transformation complex comprising:

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

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

1 micrograms nucleic acid to nanomoles cationic lipid;

an excipient for in vivo systemic administration; and

,

wherein said non-cationic lipid comprises cholesterol and said complex is substantially free of macroaggregates in vitro, the complex transforms a cell in vivo following systemic administration, and wherein the nucleic acid comprises a DNA expression cassette comprising a promoter and a DNA subsequence encoding a protein.

35. The mammalian transformation complex of claim 34, wherein the nucleic acid is DNA.

36. 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, the nucleic acid to lipid carrier ratio is less than 6;

1 micrograms DNA to nanomoles cationic lipid, and wherein the nucleic acid comprises a DNA expression cassette comprising a promoter and a DNA subsequence encoding a protein; and

,

diluting the complex with a pharmaceutically acceptable excipient.

37. The method of claim 36, wherein the steroid is cholesterol.

38. The method of claim 36, wherein the nucleic acid:

cationic lipid carrier ratio is about 1;

6 μ

g nucleic acid;

nmoles cationic lipid and the lipid carrier comprises DOTAP and cholesterol.