Polynucleotide

• US 20030018986A1
• Filed: 08/21/2002
• Published: 01/23/2003
• Est. Priority Date: 07/21/1998
• Status: Abandoned Application

• Current Assignee: Millipore Corporation
• Original Assignee: Millipore Corporation
• Inventors: Antoniou, Michael, Crombie, Robert
• Application Number:
  US10/224,972
• Publication Number:
  US 20030018986A1
• Time in Patent Office: Days
• US Class Current: 800/8
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2799/021 for the expression of a heterologous nucleic acid
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/205 bidirectional
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/46 elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
• 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 2840/44 being a specific part of the splice mechanism, e.g. donor, acceptor

Abstract

The present invention relates to a polynucleotide comprising a ubiquitous chromatin opening element (UCOE) which is not derived from 13. The polynucleotide of any one of claims 1 to 7, wherein the UCOE comprises the sequence of FIG. 20 between nucleotides 1 to 7627 or a functional homologue or fragment thereof. an LCR. The present invention also relates to a vector comprising the polynucleotide sequence, a host cell comprising the vector, use of the polynucleotide, vector or host cell in therapy and in an assay, and a method of identifying UCOEs. The UCOE opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types.

Claims

45. An isolated polynucleotide comprising

a. an element comprising an extended methylation-free CpG-island;

b. an eexpessible gene, wherein said expressible gene is operably-linked to said CpG-island; and

c. a promoter, operably-linked to said gene, wherein said promoter is not naturally operably-linked to said CpG-island,

wherein said element facilitates reproducible activation of transcription of said gene in two or more tissue types.

46. An isolated polynucleotide comprising

a.an element comprising an extended methylation-free CpG-island comprising at least one endogenous promoter;

b.an expressible gene, wherein said expressible gene is operably-linked to said CpG-island, further wherein said expressible gene is not naturally linked to said CpG-island; and

c.a further promoter, external to said CpG-island, operably-linked to said gene, wherein said further promoter is not naturally operably-linked to said CpG-island,

wherein said element facilitates reproducible activation of transcription of said gene in two or more tissue types.

47. The isolated polynucleotide of claim 46, wherein the extended methylation-free CpG-island comprises endogenous 1) dual or 2) bidirectional promoters that transcribe divergently.

48. The isolated polynucleotide of claim 45, wherein said element comprises a 44 kb DNA fragment spanning the human TATA binding protein (TBP) gene and 12 kb from each of the 5′

and 3′

flanking sequences, or a functional homologue or fragment thereof.

49. The isolated polynucleotide of claim 45, wherein said element comprises a 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′

and 5 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

50. The isolated polynucleotide of claim 45, wherein said element comprises SEQ ID NO:

28, or a functional homologue or fragment thereof.

51. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-6264 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

52. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-5636 of SEQ ID NO:

28, or a functional homologue or fragment thereof, and wherein said promoter comprises the CMV promoter.

53. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

54. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-7627 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

55. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-9127 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

56. The isolated polynucleotide of claim 45, wherein said element comprises a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′

and 20 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

57. The isolated polynucleotide of claim 45, wherein said element comprises a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5 kb 5′

and 1.5 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

58. The isolated polynucleotide of claim 45, wherein said element comprises SEQ ID NO:

29, or a functional homologue or fragment thereof.

59. A vector comprising the polynucleotide of any of claims 45-58, 72, or 74-98.

60. The vector of claim 59, wherein the vector is an episomal vector.

61. The vector of claim 59, wherein the vector is an integrating vector.

62. The vector of claim 59, wherein the vector is a plasmid.

63. The vector of claim 59, wherein said expressible gene is a therapeutic nucleic acid.

64. A vector comprising an isolated polynucleotide comprising

a.an element comprising an extended methylation-free CpG-island, wherein any DNAse I hypersensitive sites in said element are associated with an endogenous promoter;

b.a multiple cloning site operably-linked to said CpG-island, into which an expressible gene can be cloned; and

c.a further promoter operably-linked to said multiple cloning site, wherein said further promoter is not naturally operably-linked to said CpG-island.

65. The vector of claim 64 wherein the promoter is the CMV promoter.

66. The vector of claim 64 further comprising a polyadenylation site operably-linked to said multiple cloning site.

67. The vector of claim 64 wherein the element comprises nucleotides 1-7627 of SEQ ID NO:

28.

68. The vector CET 200.

69. The vector CET 210.

70. A host cell transfected with the vector of claim 59.

71. A composition comprising the polynucleotide of any of claims 45-58, 72, or 74-98.

72. An isolated polynucleotide comprising

a.an element comprising an extended methylation-free CpG-island comprising at least one endogenous promoter;

b.an expressible gene, wherein said expressible gene is operably-linked to said CpG-island, further wherein said expressible gene is not naturally linked to said CpG-island; and

c.a further promoter, external to said CpG-island, operably-linked to said gene,

wherein said further promoter is not naturally operably-linked to said CpG-island,

wherein said element facilitates reproducible activation of transcription of said gene.

73. A host cell transfected with the vector of claim 64.

74. The polynucleotide of claim 45, wherein any DNAse I hypersensitive sites in said element are associated with a promoter.

75. The polynucleotide of claim 46, wherein any DNAse I hypersensitive sites in said element are associated with a promoter.

76. The isolated polynucleotide of claim 46, wherein said element comprises a 44 kb DNA fragment spanning the human TBP gene and 12 kb from each of the 5′

and 3′

flanking sequences, or a functional homologue or fragment thereof.

77. The isolated polynucleotide of claim 46, wherein said element comprises a 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′

and 5 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

78. The isolated polynucleotide of claim 46, wherein said element comprises SEQ ID NO:

28, or a functional homologue or fragment thereof.

79. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-6264 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

80. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-5636 of SEQ ID NO:

28, or a functional homologue or fragment thereof, and wherein said promoter comprises the CMV promoter.

81. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

82. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-7627 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

83. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-9127 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

84. The isolated polynucleotide of claim 46, wherein said element comprises a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′

and 20 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

85. The isolated polynucleotide of claim 46, wherein said element comprises a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5 kb 5′

and 1.5 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

86. The isolated polynucleotide of claim 46, wherein said element comprises SEQ ID NO:

29, or a functional homologue or fragment thereof.

87. The polynucleotide of claim 72, wherein any DNAse I hypersensitive sites in said element are associated with a promoter.

88. The isolated polynucleotide of claim 72, wherein said element comprises a 44 kb DNA fragment spanning the human TBP gene and 12 kb from each of the 5′

and 3′

flanking sequences, or a functional homologue or fragment thereof.

89. The isolated polynucleotide of claim 72, wherein said element comprises a 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′

and 5 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

90. The isolated polynucleotide of claim 72, wherein said element comprises SEQ ID NO:

28, or a functional homologue or fragment thereof.

91. The isolated polynucleotide of claim 72, wherein said element comprises nucleotides 1-6264 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

92. The isolated polynucleotide of claim 72, wherein said element comprises nucleotides 1-5636 of SEQ ID NO:

28, or a functional homologue or fragment thereof, and wherein said promoter comprises the CMV promoter.

93. The isolated polynucleotide of claim 72, wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

94. The isolated polynucleotide of claim 72, wherein said element comprises nucleotides 1-7627 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

95. The isolated polynucleotide of claim 72, wherein said element comprises nucleotides 1-9127 of SEQ ID NO:

28, or a functional homologue or fragment thereof.

96. The isolated polynucleotide of claim 72, wherein said element comprises a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′

and 20 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

97. The isolated polynucleotide of claim 72, wherein said element comprises a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5 kb 5′

and 1.5 kb 3′

flanking sequences, or a functional homologue or fragment thereof.

98. The isolated polynucleotide of claim 72, wherein said element comprises SEQ ID NO:

29, or a functional homologue or fragment thereof.