Chromosome-based platforms

• US 20050181506A1
• Filed: 12/06/2004
• Published: 08/18/2005
• Est. Priority Date: 05/30/2001
• Status: Active Grant

• Current Assignee: Glaxo Group Limited (GSK plc)
• Original Assignee: Glaxo Group Limited (GSK plc)
• Inventors: Perez, Carl, Lindenbaum, Michael, Stewart, Sandra, Fleming, Elena, Perkins, Edward, Greene, Amy, Leung, Josephine, Shellard, Joan
• Granted Patent: US 7,521,240 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 2795/10322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2800/208 of mammalian origin, e.g. minichromosome
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/40 being an insulator
• CPC Class Codes: C12N 2830/60 from viruses
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2500/10 involving cells

Abstract

Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes permit tractable, efficient, rational engineering of the chromosome for a variety of applications.

Claims

1. A method of introducing heterologous nucleic acid into a chromosome, comprising:

contacting a eukaryotic chromosome comprising one or a plurality of att site(s) with a nucleic acid molecule comprising both the heterologous nucleic acid and a recombination site, in the presence of a recombinase that promotes recombination between the sites in the chromosome and in the nucleic acid molecule, wherein;

the one or a plurality of att site(s) are heterologous to the chromosome; and

the one or a plurality of att site(s) permits site-directed integration in the presence of lambda integrase.

2. The method of claim 1, wherein the recombinase is selected from the group consisting of Cre, Gin, Cin, Pin, FLP, a phage integrase and R from the pSR1 plasmid.

3. The method of claim 1, wherein the nucleic acid molecule encodes a therapeutic protein, antisense nucleic acid, or comprises an artificial chromosome.

4. The method of claim 3, wherein the nucleic acid molecule comprises a yeast artificial chromosomes (YAC), a bacterial artificial chromosome (BAC) or an insect artificial chromosome (IAC).

45. The method of claim 1, wherein the nucleic acid molecule with a recombination site is a PCR product.

46. The method of claim 1, wherein the recombinase is a protein and the recombination event occurs in vitro.

5. A method for introducing heterologous nucleic acid into a platform artificial chromosome, comprising:

(a) mixing an artificial chromosome comprising at least a first recombination site and a vector comprising at least a second recombination site and the heterologous nucleic acid;

(b) incubating the resulting mixture in the presence of at least one recombination protein under conditions whereby recombination between the first and second recombination sites is effected, thereby introducing the heterologous nucleic acid into the artificial chromosome.

6. The method of claim 5, wherein the artificial chromosome is an ACes.

7. The method of claim 5, wherein said mixing step (a) is conducted in cells ex vivo.

8. The method of claim 5, wherein said mixing step (a) is conducted extracellularly in an in vitro reaction mixture.

9. The method of claim 5, wherein the at least one recombination protein is encoded by a bacteriophage selected from the group consisting of bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1.

10. The method of claim 9, wherein the at least one recombination protein is encoded by bacteriophage lambda, or mutants thereof.

11. The method of claim 5, wherein the at least one recombination protein is selected from the group consisting of Int, IHF, Xis, Fis, Cre, γ

δ

, Tn3 resolvase, Hin, Gin, Cin and Flp.

12. The method of claim 5, wherein the recombination sites are selected from the group consisting of att and lox P sites.

13. The method of claim 5, wherein the first and/or second recombination site contains at least one mutation that removes one or more stop codons.

14. The method of claim 5, wherein the first and/or second recombination site contains at least one mutation that avoids hairpin formation.

15. The method of claim 5, wherein the first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:

41-56;

16. The method of claim 5, wherein the first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a mutated att recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

17. The method of claim 5, wherein the vector comprising the second recombination site further encodes at least one selectable marker.

18. The method of claim 17, wherein the marker is a promoterless marker, which, upon recombination is under the control of a promoter and is thereby expressed.

19. The method of claim 18, wherein the first recombination site is attP and is in the sense orientation prior to recombination.

20. The method of claim 18, wherein the selectable marker is selected from the group consisting of a gene that provides a selective growth advantage, an antibiotic resistance gene, and a gene encoding a detectable protein, wherein the detectable protein is chromogenic, fluorescent, or capable of being bound by an antibody and FACs sorted.

21. The method of claim 20, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and E. coli histidinol dehydrogenase (hisD).

28. The method of claim 18 wherein the promoterless marker is transcriptionally downstream of the heterologous nucleic acid, wherein the heterologous nucleic acid encodes a heterologous protein, and

wherein the expression level of the selectable marker is transcriptionally linked to the expression level of the heterologous protein.

29. The method of claim 28, wherein the selectable marker and the heterologous nucleic acid are transcriptionally linked by the presence of an IRES between them.

30. The method of claim 29, wherein the selectable marker is selected from the group consisting of a gene that provides a selective growth advantage, an antibiotic resistance gene, and a gene encoding a detectable protein, wherein the detectable protein is chromogenic or fluorescent.

31. The method of claim 30, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and E. coli histidinol dehydrogenase.

32. The method of claim 28, further comprising expressing the heterologous protein and isolating the heterologous protein.

47. The method of claim 5, wherein the vector is a PCR product comprising a second recombination site.

22. A human mesenchymal cell comprising an artificial chromosome.

23. The human mesenchymal cell of claim 22, wherein said artificial chromosome is an ACes.

24. The human mesenchymal cell of claim 23, wherein the ACes is a platform-ACes.

25. A method for introducing heterologous nucleic acid into the mesenchymal cell of claim 24, comprising:

(a) introducing a platform-ACes into the cell of claim 24, wherein the platform-ACes has a first recombination site, a vector comprising at least a second recombination site and the heterologous nucleic acid;

(b) incubating the resulting mixture in the presence of at least one recombination protein under conditions whereby recombination between the first and second recombination sites is effected, thereby introducing the heterologous nucleic acid into the platform-ACes within the mesenchymal cell.

26. A lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR.

27. The lambda-intR mutein of claim 26, wherein the lambda-intR mutein comprises SEQ ID NO:

37.

48. The lambda-intR mutein of claim 26, wherein the mutein further comprises an amino acid signal for nuclear localization.

49. The lambda-intR mutein of claim 26, wherein the mutein further comprises an epitope tag for protein purification.

33. A method for producing a transgenic animal, comprising introducing a platform-ACes into an embryonic cell.

34. The method of claim 33, wherein the embryonic cell is a stem cell.

35. The method of claim 33, wherein the embryonic cell is in an embryo.

36. The method of claim 33, wherein the platform-ACes comprises heterologous nucleic acid that encodes a therapeutic product.

37. The method of claim 33, wherein the transgenic animal is a fish, insect, reptile, amphibians, arachnid or mammal.

38. The method of claim 33, wherein the ACes is introduced by cell fusion, lipid-mediated transfection by a carrier system, microinjection, microcell fusion, electroporation, microprojectile bombardment or direct DNA transfer.

39. A transgenic animal produced by the method of claim 33.

40. A cell line useful for making a library of ACes, comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes.

41. A method of making a library of ACes comprising random portions of a genome, comprising introducing one or more ACes into the cell line of claim 40, under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites within the cell'"'"'s chromosomal DNA;

and isolating said multiplicity of ACes, thereby producing a library of ACes whereby multiple ACes have different portions of the genome within.

42. A library of cells useful for genomic screening, said library comprising a multiplicity of cells, wherein each cell comprises an ACes having a mutually exclusive portion of a chromosomal nucleic acid therein.

43. The library of cells of claim 42, wherein the cells of the library are from a different species than the chromosomal nucleic acid within the ACes.

44. A method of making one or more cell lines, comprising

a) integrating into endogenous chromosomal DNA of a selected cell species, a multiplicity of heterologous recombination sites,

b) introducing a multiplicity of ACes under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites integrated within the cell'"'"'s endogenous chromosomal DNA;

c) isolating said multiplicity of ACes, thereby producing a library of ACes whereby a multiplicity of ACes have mutually exclusive portions of the endogenous chromosomal DNA therein;

d) introducing the isolated multiplicity of ACes of step c) into a multiplicity of cells, thereby creating a library of cells;

e) selecting different cells having mutually exclusive ACes therein and clonally expanding or differentiating said different cells into clonal cell cultures, thereby creating one or more cell lines.

50. A modified iron-induced promoter comprising SEQ ID NO:

128.

51. A plasmid or expression cassette comprising the promoter of claim 50.

52. A vector, comprising:

a recognition site for recombination; and

a sequence of nucleotides that targets the vector to an amplifiable region of a chromosome.

53. The vector of claim 52, wherein the amplifiable region comprises heterochromatic nucleic acid.

54. The vector of claim 52, wherein the amplifiable region comprises rDNA.

55. The vector of claim 54, wherein the rDNA comprises an intergenic spacer.

56. The vector of claim 52, further comprising nucleic acid encoding a selectable marker that is not operably associated with any promoter.

57. The vector of claim 52, wherein the chromosome is a mammalian chromosome.

58. The vector of claim 52, wherein the chromosome is a plant chromosome.

59. A method, comprising:

introducing a vector of claim 52 into a cell;

growing the cells; and

selecting a cell comprising an artificial chromosome that comprises one or more repeat regions.

60. The method of claim 59, wherein a sufficient portion of the vector integrates into a chromosome in the cell to result in amplification of chromosomal DNA.

61. The method of claim 59, wherein the artificial chromsome is an ACes.

62. A method for screening, comprising:

contacting a cell comprising a reporter ACes with test compounds or known compounds, wherein;

the reporter ACes comprises one or a plurality of reporter constructs;

a reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds; and

detecting any increase or decrease in signal output from the reporter gene, wherein a change in the signal is indicative of activity of the test or known compound on the regulatory region.

63. The method of claim 62, wherein the reporter gene is operatively linked to a promoter that controls expression of a gene in a signal transduction pathway, whereby activation or reduction in the signal indicates that the pathway is activated or down-regulated by the test compound.

64. The method of claim 62, wherein the reporter gene in the construct encodes drug resistance or encodes a fluorescent protein.

65. The method of claim 64, wherein the fluorescent protein is selected from the group consisting of red, green and blue fluorescent proteins.

66. The method of claim 62, wherein the ACes comprises a plurality of reporter constructs, each with a different reporter gene, whereby the pathway(s) affected by the test compounds can be elucidated.

67. The method of claim 62, wherein a reporter gene is operatively linked to a promoter that is transcriptionally regulated in response to DNA damage, and the test compounds are genotoxicants.

68. The method of claim 67, wherein the DNA damage is induced by apoptosis, necrosis or cell-cycle perturbations.

69. The method of claim 67, wherein unknown compounds are screened to assess whether they are genotoxicants.

70. The method of claim 67, wherein the promoter is a cytochrome P450-profiled promoter.

71. The method of claim 67, wherein the cell is in a transgenic animal and toxicity is assessed in the animal.

72. The method of claim 62, wherein:

the cell is a patient cell sample;

the patient has a disease;

the regulatory region is one targeted by a drug or drug regimen; and

the method assesses the effectiveness of a treatment for the disease for the particular patient.

73. The method of claim 72, wherein the cell is a tumor cell.

74. The method of claim 62, wherein the cell is a stem cell or a progenitor cell, whereby expression of the reporter gene is operatively linked to a regulatory region expressed in the cells to thereby identify stem cells or progenitor cells.

75. The method of claim 62, wherein the cell is in an animal;

and the method comprises whole-body imaging to monitor expression of the reporter gene in the animal.

76. A reporter ACes comprises one or a plurality of reporter constructs, wherein the reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds.

77. A method for obtaining site-specific recombination within a plant cell, comprising:

introducing into the genome of a plant cell a nucleic acid molecule containing one or more recombinase recognition sites;

introducing into the plant cell an artificial chromosome containing a recombinase recognition site; and

introducing into the plant cell a gene encoding a recombinase enzyme, whereby plant chromosomal DNA from the genome of the plant cell is introduced into the artificial chromosome.

78. The method of claim 77, further comprising isolating the resulting artificial chromosome.

79. The method of claim 77, wherein the nucleic acid molecule further comprises a promoterless marker or a selectable marker.

80. The method of claim 77, wherein the artificial chromosome is introduced into the plant cell by Agrobacterium-mediated transformation, protoplast transformation, PEG-mediated transformation, electroporation, protoplast fusion, microcell fusion, lipid-mediated delivery, liposomes, sonoporation, microinjection, particle bombardment or silicon carbide whisker-mediated transformation.

81. The method of claim 77, wherein the nucleic acid molecule comprising one or more recombinase recognition sites is introduced into the plant cell by Agrobacterium-mediated transformation, protoplast transformation, PEG-mediated transformation, electroporation, protoplast fusion, microcell fusion, lipid-mediated delivery, liposomes, sonoporation, microinjection, particle bombardment or silicon carbide whisker-mediated transformation.

82. The method of claim 77, wherein the recombinase enzyme is a wild-type lambda integrase or a mutant lambda integrase.

83. The method of claim 82, wherein the mutant lambda integrase is Int-h or Int-h/218.

84. The method of claim 82, wherein the mutant lambda integrase is a lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR.

85. A method of conferring a trait on a plant cell, comprising:

introducing into a first plant cell a nucleic acid molecule containing one or more recombinase recognition sites;

introducing into the first plant cell an artificial chromosome containing a recombinase recognition site;

introducing into the first plant cell a gene encoding a recombinase enzyme, whereby plant chromosomal DNA from the genome of the plant cell is introduced into the artificial chromosome;

isolating the resulting artificial chromosome; and

introducing the isolated artificial chromosome into a second plant cell, whereby a trait from the first plant cell is conferred on the second plant cell.