Polynucleotide
View Patent ↗The present invention relates to a polynucleotide comprising a ubiquitous chromatin opening element (UCOE). 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.
1. An isolated polynucleotide comprising
a) an element comprising an extended methylation-free CpG-island;
b) an expressible gene, wherein said expressible gene is operably-linked to said CpG-island; and
c) a tissue-specific promoter, operably-linked to said gene, wherein said promoter is not naturally operably-linked to said CpG-island,
wherein said element facilitates activation of transcription of said gene.
2. 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 a tissue-specific promoter not naturally operably-linked to said CpG-island,
wherein said element facilitates activation of transcription of said gene.
3. The isolated polynucleotide of claim 2 , wherein the extended methylation-free CpG-island comprises endogenous
a) dual or
b) bi-directional promoters that transcribe divergently.
4. The isolated polynucleotide of claim 1 , 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.
5. The isolated polynucleotide of claim 1 , 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.
6. The isolated polynucleotide of claim 1 , wherein said element comprises SEQ ID NO:28, or a functional homologue or fragment thereof.
7. The isolated polynucleotide of claim 1 , wherein said element comprises nucleotides 1-6264 of SEQ ID NO:28 or a functional homologue or fragment thereof.
8. The isolated polynucleotide of claim 1 , 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 AFP promoter.
9. The isolated polynucleotide of claim 1 , wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:28, or a functional homologue or fragment thereof.
10. The isolated polynucleotide of claim 1 , wherein said element comprises nucleotides 1-7627 of SEQ ID NO:28, or a functional homologue or fragment thereof.
11. The isolated polynucleotide of claim 1 , wherein said element comprises nucleotides 1-9127 of SEQ ID NO:28, or a functional homologue or fragment thereof.
12. The isolated polynucleotide of claim 1 , 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.
13. The isolated polynucleotide of claim 1 , 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.
14. The isolated polynucleotide of claim 1 , wherein said element comprises SEQ ID NO:29, or a functional homologue or fragment thereof.
15. 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 a tissue-specific promoter not naturally operably-linked to said CpG-island.
16. The vector of claim 15 wherein the promoter is the AFP promoter.
17. The vector of claim 15 further comprising a polyadenylation site operably-linked to said multiple cloning site.
18. The vector of claim 15 wherein the element comprises nucleotides 1-7627 of SEQ ID NO:28.
19. 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 a tissue-specific promoter not naturally operably-linked to said CpG-island,
wherein said element facilitates activation of transcription of said gene.
20. A host cell transfected with the vector of claim 15 .
21. The polynucleotide of claim 1 , wherein any DNase I hypersensitive sites in said element are associated with a promoter.
22. The polynucleotide of claim 2 , wherein any DNase I hypersensitive sites in said element are associated with a promoter.
23. The isolated polynucleotide of claim 2 , 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.
24. The isolated polynucleotide of claim 2 , 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.
25. The isolated polynucleotide of claim 2 , wherein said element comprises SEQ ID NO:28, or a functional homologue or fragment thereof.
26. The isolated polynucleotide of claim 2 , wherein said element comprises nucleotides 1-6264 of SEQ ID NO:28, or a functional homologue or fragment thereof.
27. The isolated polynucleotide of claim 2 , 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 AFP promoter.
28. The isolated polynucleotide of claim 2 , wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:28, or a functional homologue or fragment thereof.
29. The isolated polynucleotide of claim 2 , wherein said element comprises nucleotides 1-7627 of SEQ ID NO:28, or a functional homologue or fragment thereof.
30. The isolated polynucleotide of claim 2 , wherein said element comprises nucleotides 1-9127 of SEQ ID NO:28, or a functional homologue or fragment thereof.
31. The isolated polynucleotide of claim 2 , 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.
32. The isolated polynucleotide of claim 2 , 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.
33. The isolated polynucleotide of claim 2 , wherein said element comprises SEQ ID NO:29, or a functional homologue or fragment thereof.
34. The polynucleotide of claim 19 , wherein any DNAse I hypersensitive sites in said element are associated with a promoter.
35. The isolated polynucleotide of claim 19 , 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.
36. The isolated polynucleotide of claim 19 , 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.
37. The isolated polynucleotide of claim 19 , wherein said element comprises SEQ ID NO:28, or a functional homologue or fragment thereof.
38. The isolated polynucleotide of claim 19 , wherein said element comprises nucleotides 1-6264 of SEQ ID NO:28, or a functional homologue or fragment thereof.
39. The isolated polynucleotide of claim 19 , 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 AFP promoter.
40. The isolated polynucleotide of claim 19 , wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:28, or a functional homologue or fragment thereof.
41. The isolated polynucleotide of claim 19 , wherein said element comprises nucleotides 1-7627 of SEQ ID NO:28, or a functional homologue or fragment thereof.
42. The isolated polynucleotide of claim 19 , wherein said element comprises nucleotides 1-9127 of SEQ ID NO:28, or a functional homologue or fragment thereof.
43. The isolated polynucleotide of claim 19 , 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.
44. The isolated polynucleotide of claim 19 , 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.
45. The isolated polynucleotide of claim 19 , wherein said element comprises SEQ ID NO:29, or a functional homologue or fragment thereof.
46. A vector comprising the polynucleotide of any of claims 1 , 2 , 8 , 19 , 21 , 22 , 27 , 34 , or 39 .
47. The vector of claim 46 , wherein the vector is an episomal vector.
48. The vector of claim 46 , wherein the vector is an integrating vector.
49. The vector of claim 46 , wherein the vector is a plasmid.
50. The vector of claim 46 , wherein said expressible gene is a therapeutic nucleic acid.
51. A host cell transfected with the vector of claim 46 .
52. A composition comprising the polynucleotide of any of claims 1 , 2 , 8 , 19 , 21 , 22 , 27 , or 34 .