Polynucleotide
View Patent ↗The present invention relates to a polynucleotide comprising a ubiquitous chromatin opening element (UCOE) that does not occur in nature. The present invention also relates to a vector comprising the polynucleotide sequence, a host cell comprising the vector and use of the polynucleotide, vector or host cell in therapy, or for in vitro protein expression applications.
1. A polynucleotide comprising a ubiquitous chromatin opening element (UCOE) comprising an extended methylation free CpG-island, which 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, wherein the nucleotide sequence of the UCOE does not occur in nature.
2. The polynucleotide of claim 1 , which facilitates reproducible expression of an operably-linked gene non-tissue specifically.
3. The polynucleotide of claim 1 , which facilitates reproducible expression of an operably-linked gene in all tissue types where active gene expression occurs.
4. The polynucleotide of claim 1 , which facilitates expression of an operably-linked gene at a physiological level.
5. The polynucleotide of claim 1 , wherein the UCOE comprises an extended methylation-free, CpG-island.
6. The polynucleotide of claim 1 , wherein the UCOE comprises at least one naturally-occurring sequence associated with the control of gene expression.
7. The polynucleotide of claim 1 , wherein the UCOE comprises at least one naturally-occurring promoter.
8. The polynucleotide of claim 1 , wherein the UCOE comprises dual or bi-directional promoters that transcribe divergently.
9. The polynucleotide of claim 1 , wherein the UCOE comprises the human β-actin CpG island/promoter region or a fragment thereof.
10. The polynucleotide of claim 9 , wherein the UCOE comprises a DNA fragment within the range of 100 bp to 3.0 kb spanning the human β-actin CpG island/promoter region or a fragment thereof.
11. The polynucleotide of claim 1 , wherein the UCOE comprises the human PCDC2 CpG island/promoter region or a fragment thereof.
12. The polynucleotide of claim 11 , wherein the UCOE comprises a DNA fragment within the range of 100 bp to 3.0 kb spanning the human PCDC2 CpG island/promoter region or a fragment thereof.
13. The polynucleotide of claim 1 , wherein the UCOE comprises a DNA fragment within the range of 100 bp to 3.0 kb spanning the human β-actin CpG island/promoter region and a DNA fragment within the range of 100 bp to 3.0 kb spanning the human PDCD2 CpG island/promoter region.
14. The polynucleotide of claim 1 , wherein the UCOE comprises a 2.0 kb DNA fragment spanning the human β-actin CpG island/promoter region and a 1.8 kb DNA fragment spanning the human PDCD2 CpG island/promoter region.
15. The polynucleotide of claim 13 or claim 14 wherein the promoters are orientated divergently.
16. The polynucleotide of claim 1 , wherein the UCOE comprises SEQ ID NO:1 or a fragment thereof in either orientation.
17. The polynucleotide of claim 1 , wherein the UCOE comprises the human RNP CpG island/promoter region or a fragment thereof.
18. The polynucleotide of claim 17 , wherein the UCOE comprises a 4 kb DNA fragment spanning the human RNP CpG island/promoter region.
19. The polynucleotide of claim 1 , comprising an extended methylation-free CpG island containing bidivergent promoters adjacent to at least one further sequence comprising a methylation-free CpG island.
20. The polynucleotide of claim 1 , wherein the UCOE comprises the human RNP CpG island/promoter region and a DNA fragment in the range 100 bp to 3.0 kb spanning the human β-actin CpG island/promoter region.
21. The polynucleotide of claim 20 , wherein the UCOE comprises SEQ ID NO:2 or fragment thereof in either orientation.
22. The polynucleotide of claim 1 , further comprising a promoter.
23. The polynucleotide of claim 1 , wherein the UCOE comprises at least one promoter sequence mutated to be incapable of initiating transcription.
24. The polynucleotide of claim 22 or claim 23 wherein the promoter is the CMV promoter.
25. The polynucleotide of claim 24 wherein the promoter is the mouse CMV promoter.
26. The polynucleotide of claim 1 , wherein the UCOE comprises at least one sequence which is artificially synthesized.
27. A vector comprising the polynucleotide of claim 1 .
28. The vector of claim 27 , further comprising an expressible gene operably-linked to a promoter and the polynucleotide.
29. The vector of claim 27 or claim 28 wherein the vector is an episomal or integrating vector.
30. The vector according to claim 27 , wherein the vector is a plasmid.
31. The vector according to claim 27 , wherein the vector is a virus.
32. The vector according to claim 28 , wherein the operably-linked gene is a therapeutic nucleic acid sequence.
33. The vector of claim 27 , wherein the UCOE is effective in both orientations, said vector further comprising
a) a first and a second promoter, said arranged to transcribe divergently; and
b) a first and a second site for insertion of open reading frames to be expressed, wherein each of said first and second promoters is operably-linked to the UCOE,
wherein the first site for insertion is under the control of the first promoter, and the second site for insertion is under the control of the second promoter, and further wherein the UCOE is situated between the first and second promoter.
34. The vector of claim 33 , further comprising a nucleic acid encoding an immunoglobulin heavy chain inserted into the first site for insertion, and a nucleic acid encoding an immunoglobulin light chain inserted into the second site for insertion.
35. The vector of claim 27 , comprising SEQ ID NO:1, the CMV promoter, a multiple cloning site, a polyadenylation sequence and genes encoding selectable markers under suitable control elements.
36. The vector of claim 27 , comprising SEQ ID NO:2, the CMV promoter, a multiple cloning site, a polyadenylation sequence and genes encoding selectable markers under suitable control elements.
37. The vector of claim 35 or claim 36 , wherein the orientation of the UCOE is reversed.
38. The vector CET 500.
39. The vector CET 501.
40. The vector CET 600.
41. The vector CET 601.
42. A host cell transfected with the vector of any one of claims 27 or 38 - 41 .
43. A composition comprising the polynucleotide of claim 1 .
44. A composition comprising the host cell of claim 42 , in combination with a pharmaceutically acceptable excipient.
45. A method of obtaining a desired gene product comprising using the polynucleotide of claim 1 .
46. A method of obtaining a desired gene product comprising using the host cell of claim 42 in a cell culture system in order to obtain a desired gene product.
47. A nucleic acid molecule comprising a DNA sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.
48. A method for preparing a polypeptide comprising:
providing a cell transformed/transfected with the nucleic acid molecule according to claim 47 ;
growing said cell in conditions conducive to the production of said polypeptide; and
purifying said polypeptide from said cell, or its growth environment.
49. A composition comprising the vector of any one of claims 27 or 38 - 41 , in combination with a pharmaceutically acceptable excipient.
50. A method of obtaining a desired gene product comprising using the vector of any one of claims 27 or 38 - 41 in a cell culture system in order to obtain a desired gene product.