Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes

• US 20020160970A1
• Filed: 03/05/2001
• Published: 10/31/2002
• Est. Priority Date: 04/10/1996
• Status: Abandoned Application

• Current Assignee: Biological Research Center Of The Hungarian Academy Of Sciences, Chromos Molecular Systems Inc. (Calyx Bio Ventures, Inc. (Old))
• Original Assignee: Chromos Molecular Systems Inc. (Calyx Bio Ventures, Inc. (Old))
• Inventors: Szalay, Aladar A., Hadlaczky, Gyula
• Application Number:
  US09/799,462
• Publication Number:
  US 20020160970A1
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/30 Bird
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 2267/025 Animal producing cells or organs for transplantation
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. 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 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: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/82 for plant cells , e.g. plant artificial chromosomes (PACs)
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2800/20 Pseudochromosomes, minichrosomosomes
• CPC Class Codes: C12N 2800/204 of bacterial origin, e.g. BAC
• CPC Class Codes: C12N 2800/206 : of yeast origin, e.g. YAC, 2u
• CPC Class Codes: C12N 2800/208 : of mammalian origin, e.g. minichromosome

Abstract

Methods for preparing cell lines that contain artificial chromosomes, methods for preparation of artificial chromosomes, methods for purification of artificial chromosomes, methods for targeted insertion of heterologous DNA into artificial chromosomes, methods for amplification of nucleic acids and methods for delivery of the chromosomes to selected cells and tissues are provided. Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods. Methods for use of the artificial chromosomes are also provided.6

Claims

1. A method comprising:

introducing a mammalian artificial chromosome that comprises nucleic acid encoding a therapeutic product into a cell; and

introducing the resulting cell into a host animal;

wherein the therapeutic product is expressed in the cell.

2. The method of claim 1, wherein the cell is a lymphocyte stem cell, nerve cell, chicken cell or muscle cell.

3. The method of claim 1, wherein:

the artificial chromosome comprises the minichromosome present in the cell line EC3/7C5; and

the minichromosome comprises the nucleic acid encoding the therapeutic product.

4. The method of claim 1, wherein:

the artificial chromosome comprises the λ

neo-chromosome in the cell line KE12/4; and

the λ

neo-chromosome comprises the [DNA] nucleic acid encoding the therapeutic product.

5. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 20 Mb and about 200 Mb.

6. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 100 Mb and about 200 Mb.

7. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 250 Mb and about 400 Mb.

8. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 1 Mb and about 15 Mb.

36. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome.

37. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 150 Mb and about 200 Mb.

38. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 90 Mb and about 120 Mb.

39. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 60 Mb and about 100 Mb.

40. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 20 Mb and about 200 Mb.

41. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 100 Mb and about 200 Mb.

42. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 7.5 Mb and about 60 Mb.

43. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 30 Mb and about 50 Mb.

44. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 10 Mb and about 15 Mb.

45. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 15 Mb and about 50 Mb.

46. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 1 Mb and about 15 Mb.

47. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome produced by a method comprising:

introducing one or more nucleic acid fragments into a cell, wherein the nucleic acid fragment or fragments comprise a selectable marker;

growing the cell under selective conditions to produce cells that have incorporated the nucleic acid fragment or fragments into their genomic DNA;

selecting a cell that comprises a mammalian satellite artificial chromosome.

48. The method of claim 1, wherein the artificial chromosome is a mammalian satellite artificial chromosome comprises a human centromere.

49. The method of claim 1, wherein the artificial chromosome is a minichromosome.

50. The method of claim 1, wherein the minichromosome is a A neo-chromosome.

51. The method of claim 1, wherein the minichromosome comprises a neocentromere.

52. The method of claim 1, wherein the minichromosome is produced by a method comprising:

introducing one or more nucleic acid fragments into a cell, wherein the nucleic acid fragment or fragments comprise a selectable marker;

growing the cell under selective conditions to produce cells that have incorporated the nucleic acid fragment or fragments into their genomic DNA; and

selecting a cell that comprises a minichromosome.

56. The method of claim 1, wherein the artificial chromosome is islolated prior to introduction into the cell.

9. A method comprising:

introducing a mammalian artificial chromosome that comprises nucleic acid encoding a therapeutic product into a cell of a host animal, wherein the therapeutic product is expressed in the cell.

10. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome.

11. The method of claim 9, wherein the artificial chromosome is a satellite artificial chromosome that is between about 250 Mb and about 400 Mb.

12. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 150 Mb and about 200 Mb.

13. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 90 Mb and about 120 Mb.

14. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 60 Mb and about 100 Mb.

15. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 20 Mb and about 200 Mb.

16. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 100 Mb and about 200 Mb.

17. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 7.5 Mb and about 60 Mb.

18. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 30 Mb and about 50 Mb.

19. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 10 Mb and about 15 Mb.

20. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 15 Mb and about 50 Mb.

21. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome that is between about 1 Mb and about 15 Mb.

22. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome produced by a method comprising:

introducing one or more nucleic acid fragments into a cell, wherein the nucleic acid fragment or fragments comprise a selectable marker;

growing the cell under selective conditions to produce cells that have incorporated the nucleic acid fragment or fragments into their genomic DNA; and

selecting a cell that comprises a mammalian satellite artificial chromosome.

23. The method of claim 9, wherein the artificial chromosome is a mammalian satellite artificial chromosome comprises a human centromere.

24. The method of claim 9, wherein the artificial chromosome is a minichromosome.

25. The method of claim 24, wherein the minichromosome is a A neo-chromosome.

26. The method of claim 24, wherein the minichromosome comprises a neocentromere.

27. The method of claim 24, wherein the minichromosome is produced by a method comprising:

introducing one or more nucleic acid fragments into a cell, wherein the nucleic acid fragment or fragments comprise a selectable marker;

growing the cell under selective conditions to produce cells that have incorporated the nucleic acid fragment or fragments into their genomic DNA; and

selecting a cell that comprises a minichromosome.

29. The method of claim 28, wherein the artificial chromosome is a mammalian satellite artificial chromosome.

30. The method of claim 28, wherein the artificial chromosome is a mammalian satellite artificial chromosome produced by a method comprising:

introducing one or more nucleic acid fragments into a cell, wherein the nucleic acid fragment or fragments comprise a selectable marker;

growing the cell under selective conditions to produce cells that have incorporated the nucleic acid fragment or fragments into their genomic DNA;

selecting a cell that comprises a mammalian satellite artificial chromosome.

31. The method of claim 28, wherein the artificial chromosome is a mammalian satellite artificial chromosome comprises a human centromere.

32. The method of claim 28, wherein the artificial chromosome is a minichromosome.

33. The method of claim 28, wherein the minichromosome is a A neo-chromosome.

34. The method of claim 28, wherein the minichromosome comprises a neocentromere.

35. The method of claim 28, wherein the minichromosome is produced by a method comprising:

introducing one or more nucleic acid fragments into a cell, wherein the nucleic acid fragment or fragments comprise a selectable marker;

growing the cell under selective conditions to produce cells that have incorporated the nucleic acid fragment or fragments into their genomic DNA; and

selecting a cell that comprises a minichromosome.

28. A method comprising:

introducing a mammalian artificial chromosome that comprises nucleic acid encoding a therapeutic product into tissue of a host animal, wherein the therapeutic product is expressed in cells of the tissue.

53. A method comprising:

introducing a mammalian artificial chromosome that comprises nucleic acid encoding a gene product into a cell of a host animal, whereby the gene product is expressed in the cell.

54. The method of claim 53, wherein the mammalian artificial chromosome is a mammalian satellite artificial chromosome.

55. The method of claim 54, wherein the satellite artificial chromosome is isolated prior to introduction into the cell.

57. The method of claim 56, wherein the mammalian artificial chromosome is a mammalian satellite artificial chromosome.