Methods and composition for in vivo gene therapy

• US 5,827,703 A
• Filed: 05/19/1994
• Issued: 10/27/1998
• 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): RORIES, CHARLES
• Application Number:
  US08/246,376
• Time in Patent Office: 1,622 Days
• Field of Search: 435/172.3, 435/240.2, 435/69.1, 435/325, 435/354, 435/375, 435/6, 514/44, 800/2, 424/450, 424/417, 424/420, 935/54
• 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 47/54 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 47/544 Phospholipids
• CPC Class Codes: A61K 47/547 Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• 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 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 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 DNA expression cassette into cells of a mammal, said method comprising introducing the expression cassette into the manual systemically, wherein:

the DNA expression cassette comprises a promoter, and a DNA sequence encoding a gene product;

the DNA expression cassette is complexed to a lipid carrier comprising cationic lipids and cholesterol having a mean diameter of less than about 10 microns, resulting in a DNA expression cassette-lipid carrier complex;

the DNA expression cassette and lipid carrier does not aggregate in vitro; and

the DNA expression cassette to lipid carrier ratio is less than 6;

1 micrograms DNA to nanomoles cationic lipid.

5. The method of claim 1 or 2 wherein the lipid carrier is an MLV.

6. The method of claim 5, wherein the lipid carrier is an MLV with a mean diameter of at least about 500 nm.

7. The method of claim 1 or 2 wherein the lipid carrier comprises DOPE.

8. The method of claim 1 or 2, wherein the promoter is a HCMV promoter.

9. The method of claim 1 or 2, wherein the DNA:

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

10. The method of claim 1 or 2, wherein the DNA expression cassette does not comprise an intron.

11. The method of claim 1 or 2, wherein the DNA expression cassette comprises a 5'"'"' intron.

12. The method of claim 1 or 2, wherein at least about 50 μ

g of the DNA expression cassette is introduced into the mammal.

13. The method of claim 1 or 2, wherein the DNA of said expression cassette is purified without PEG prior to complexing to said lipid carrier.

14. The method of claim 1 or 2, wherein the size of the DNA expression vector:

lipid carrier complex is at least about 500 nm.

15. The method of claim 1 or 2, wherein the size of the DNA expression cassette:

lipid carrier complex is at least about 500 nm.

16. The method of claim 1 or 2 wherein the DNA expression cassette is linear.

17. The method of claim 1 or 2, wherein the DNA expression cassette is plasmid DNA.

18. The method of claim 1 or 2, wherein the DNA expression cassette is introduced into said mammal by an administration technique selected from the group consisting of intraperitoneal and intravenous.

19. The method of claim 1 or 2, wherein the DNA expression cassette is introduced into said mammal intravenously.

20. The method of claim 1 or 2, wherein the DNA expression cassette is introduced into said mammal intraperitoneally.

21. The method of claim 1 or 2, wherein the DNA:

cationic lipid carrier ratio is about 1;

4 μ

g DNA;

nmoles cationic lipid.

22. The method of claim 1 or 2, wherein the DNA:

cationic lipid carrier ratio is about 1;

3 μ

g DNA;

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

24. The method of claim 1 or 2, wherein the DNA:

cationic lipid carrier ratio is about 1;

1 μ

g DNA;

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

26. The method of claim 1 or 2, wherein the DNA:

cationic lipid carrier ratio is about 2;

1 μ

g DNA;

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

27. The method of claim 1 or 2, wherein a cell into which DNA 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.

35. The method of claim 1 or 2, wherein said lipid carriers have a mean diameter ranging in size from about 100 nm to 10 μ

m.

36. The method of claim 1 or 2, wherein the cationic lipid does not comprise DOTMA.

37. The method of claim 1 or 2, wherein said lipid carrier do not comprise LIPOFECTIN®

.

38. The method of claim 1 or 2, wherein the DNA expression cassette to lipid carrier ratio is greater than 1:

3 micrograms DNA to nanomoles cationic lipid.

2. A method of introducing a DNA expression cassette into a mammal, said method comprising introducing the expression cassette into the mammal systemically, wherein:

the DNA expression cassette comprises a promoter, and a DNA sequence encoding a gene product;

the DNA molecule is complexed to 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;

19 to 1;

0 resulting in a DNA lipid carrier complex;

the DNA expression cassette and lipid carrier does not aggregate in vitro;

the DNA expression cassette to lipid carrier ratio is less than 6;

1 micrograms DNA to nanomoles cationic lipid,

whereby the expression cassette is introduced into cells of at least two tissues in the mammal.

3. The method of claim 2, wherein the non-cationic lipid is cholesterol.

4. The method of claim 2, wherein the molar ratio of cationic to non-cationic lipids is about 1:

1.

23. The method of claim 2, wherein the DNA:

cationic lipid carrier ratio is about 1;

6 μ

g DNA;

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

25. The method of claim 2, wherein the DNA:

cationic lipid carrier ratio is about 1;

5 μ

g DNA;

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

28. A mammalian transformation complex comparing:

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

combined with

DNA in a ratio of less than 6;

1 micrograms DNA to nanomoles cationic lipid;

wherein said non-cationic lipid comprises cholesterol and said complex does not aggregate in vitro wherein the cationic lipid is L-PE.

29. The mammalian transformation complex of claim 28, wherein the non-cationic lipid is cholesterol.

30. The mammalian transformation complex of claim 28, wherein the ratio of cationic to non cationic lipid is about 1:

1.

31. The mammalian transformation complex of claim 28, wherein the cationic lipid is L-PE and the molar ratio of L-PE to cholesterol is about 6:

4.

32. The mammalian transformation complex of claim 28, wherein said cationic and non-cationic lipids comprise an MLV.

33. A kit comprising a cationic lipid, a non-cationic lipid comprising cholesterol, a container, and instructions in the use of the cationic lipid and non-cationic lipid for the transformation of a mammalian cell in vivo, wherein the cationic lipid is L-PE.

34. A method of determining a ratio of DNA to lipid carrier which provides for optimum transformation of a cell in a mammal in vivo, said method comprising the steps of:

systemically introducing a first DNA;

lipid carrier complex to a first mammal, wherein said first complex has a first ratio of DNA;

lipid carrier;

systemically introducing a second DNA;

lipid carrier complex to a second mammal, wherein said second complex has a second ratio of DNA;

lipid carrier and said second ratio is different than said first ratio;

selecting a target cell type in the first and second mammal for transformation, thereby providing a selected cell type in said mammals;

measuring the percentage of cells of the selected cell type in the first and second mammal which are transformed with the DNA, thereby providing a percentage of cells of the selected cell type which are transformed in the first mammal, and a percentage of cells of the selected cell type which are transformed in the second mammal; and

,

comparing the percentage of cells of the selected type which are transformed in the first mammal to the percentage of cells of the selected type transformed in the second mammal, thereby determining whether the first DNA;

lipid complex or the second DNA;

lipid carrier complex provides for optimum transformation

whereby at least two tissues in the mammal are transformed with the DNA.