Levels and/or Sustainability of DNA-based Gene Expression

• US 20090311786A1
• Filed: 01/29/2007
• Published: 12/17/2009
• Est. Priority Date: 01/27/2006
• Status: Active Grant

• Current Assignee: Carnegie Institution of Washington, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Carnegie Institution of Washington
• Inventors: Fire, Andrew, Lui, Weng-Onn, Gracey, Lia
• Granted Patent: US 8,716,462 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A61K 48/0066 Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 2310/18 acting by a non-sequence specific mechanism other than C12N2310/16 or C12N2310/17

Abstract

The invention encompasses methods for improving the level and/or sustainability of expression for a target nucleic acid in a eukaryotic cell comprising: (a) modifying the target nucleic acid to introduce or to comprise signals that limit or constrain the positions of nucleosome cores, and (b) introducing the modified target nucleic acid into the eukaryotic cell, wherein the modified target nucleic acid has improved levels and/or sustainability of expression compared to original unmodified nucleic acid.

Claims

1. A method for improving the level and/or sustainability of expression for a target nucleic acid in a eukaryotic cell comprising:

(a) modifying the target nucleic acid to introduce signals that limit or constrain the positions of nucleosome cores, and

(b) introducing the modified target nucleic acid into the eukaryotic cell,

wherein the modified target nucleic acid has improved levels and/or sustainability of expression compared to original unmodified nucleic acid.

2. The method of claim 1, wherein the nucleic acid is DNA.

3. The method of claim 1, in which the target nucleic acid is an expression vector.

4. The method of claim 3, in which the expression vector comprises a promoter and a coding region for a protein or RNA molecule whose synthesis is desired.

5. The method of claim 3, in which the expression vector comprises the coding region for a protein or RNA molecule that is desired.

10. The method of claim 1, in which the modifications to the target nucleic acid involve the mutation of at least 1 base pair in a manner expected to limit or constrain the positions of nucleosome cores.

11. The method of claim 1, in which the modifications to the target nucleic acid involve at least one insertion of at least 10 base pairs to said nucleic acid in a manner expected to limit or constrain the positions of nucleosome cores.

12. The method of claim 5, in which the modifications to the target nucleic acid involve at least one insertion of at least 10 base pairs upstream, downstream, or inside the RNA or protein coding region in a manner expected to limit or constrain the positions of nucleosome cores.

13. The method of claim 12, in which the inserted sequence acts as an intron sequence.

14. The method of claim 1, in which the modifications made to the target nucleic acid sequence reflect known biases for nucleosome positioning based on analysis of positioned nucleosomes from the biological literature.

15. The method of claim 1, in which the modifications made to the target nucleic acid sequence reflect experimental determination of nucleosome positions on unmodified and modified sequence.

18. The method of claim 1, in which the modifications to the target nucleic acid sequence result in at least 3 additional AA/TT dinucleotide junctions in a region of 1000 base pairs.

19. The method of claim 1, in which the modifications to the target nucleic acid involve at least one deletion of at least 1 base pair in a manner expected to limit or constrain the positions of nucleosome cores.

20. The method of claim 5, in which the modifications to the target nucleic acid involve at least one deletion of at least 1 base pair upstream, downstream, or inside the RNA or protein coding region in a manner expected to limit or constrain the positions of nucleosome

21. The method of claim 1, in which the target nucleic acid is introduced into the eukaryotic cell by transfection.

22. The method of claim 1, in which the target nucleic acid is a viral vector introduced into the eukaryotic cell by infection.

23. The method of claim 1, in which the target nucleic acid is introduced into the eukaryotic cell by injection of the cell, or by injection of the target nucleic acid into the organism in which the cell is contained.

24. The method of claim 1, in which the target nucleic acid is introduced into the eukaryotic cell by means of an aerosol.

25. The method of claim 5, in which the coding region encodes an endogenous protein.

26. The method of claim 5, in which the coding region encodes an exogenous protein.

27. The method of claim 5, in which the coding region encodes an RNA shorter than 100 bases.

30. The method of claim 5 in which expression of the modified target nucleic acid is at least 10% greater than that of the original unmodified nucleic acid.

33. The method of claim 5, in which expression of the modified target nucleic acid is at least 10% greater than that of the original unmodified nucleic acid after a time period of at least one day has been allowed to pass.

34. The method of claim 11, in which the inserted nucleic acid is one which has been shown to constrain nucleosome positions in vitro or in vivo.

35. The method of claim 11, in which the inserted nucleic acid is from a gene with known limitations to nucleosome mobility.

36. The method of claim 11, in which the inserted nucleic acid is from a nematode genome.

37. The method of claim 11, in which the inserted nucleic acid is from a nematode gene that is normally expressed in the nematode germline.

38. The method of claim 1, in which the eukaryotic cell is grown in cell culture.

39. The method of claim 1, in which the eukaryotic cell is in a living tissue.

40. The method of claim 1, in which the eukaryotic cell is in a human host.

41. The method of claim 1, in which the eukaryotic cell is infected with a virus.

42. The method of claim 1, in which the eukaryotic cell is part of a tumor.

43. The method of claim 11, in which the coding region is missing or compromised in function in a genetic disease and the target eukaryotic cell is from a host carrying that disease.

44. The method of claim 43, in which introduction of the modified target nucleic acid is used to provide a therapy to patients carrying the genetic disease

45. The method of claim 11, in which expression of the coding region facilitates an assay for specific biological properties of the target cell or its host organism.

6-9. -9. (canceled)

16-17. -17. (canceled)

28-29. -29. (canceled)

31-32. -32. (canceled)

46. (canceled)

47. A method for improving the level and/or sustainability of expression for a target nucleic acid in a eukaryotic cell comprising:

(a) modification of the target nucleic acid to introduce at least 10 AA/TT dinucleotides in positions where a majority of distances in base pairs between adjacent AA/TT dinucleotides in the modified nucleic acid are in the range of 1-3, 10-13, 20-23, 30-33, 40-43, or 50-53 nucleotides, and

(b) introducing said modified target nucleic acid into a eukaryotic cell,

wherein the modified target nucleic acid has improved levels and/or sustainability of expression compared to original unmodified nucleic acid.

48. A method for improving the level and/or sustainability of expression for a target nucleic acid in a eukaryotic cell comprising:

(a) modification of the target nucleic acid to introduce at least 5 AA/TT dinucleotides in positions where 80% or greater of the adjacent AA/TT dinucleotide separations that are a result of the modification are in the range of 1-3, 7-13, 17-23, 27-33, 37-43, or 47-53 nucleotides, and

(b) introducing the modified target nucleic acid into a eukaryotic cell,

wherein the modified target nucleic acid has improved levels and/or sustainability of expression compared to original unmodified nucleic acid.

49-51. -51. (canceled)

52. A method for improving the level and/or sustainability of expression for a target nucleic acid in a eukaryotic cell comprising:

(a) modification of the target nucleic acid by inserting sequences derived from a nematode into said target nucleic acid, and

(b) introducing the modified target nucleic acid into a target eukaryotic cell,

wherein the modified target nucleic acid has improved levels and/or sustainability of expression compared to original unmodified nucleic acid.

53. The method of claim 52, in which the inserted sequence is from a gene that is normally expressed in the C. elegans germline.

54. A method for expressing an RNA or protein molecule of interest in a eukaryotic cell comprising:

(a) synthesizing a nucleic acid molecule that is interrupted by at least one intron sequence that has signals capable of constraining or limiting the positions of nucleosomes and which nucleic acid molecule carries information sufficient to encode the RNA or protein molecule of interest,

(b) introducing said nucleic acid molecule into an expression vector, and

(c) introducing the expression vector comprising said nucleic acid into the eukaryotic cell,

wherein the expression vector comprising said nucleic acid expresses the RNA or protein molecule of interest in the eukaryotic cell.

55. A method for expressing an RNA or protein molecule of interest in a eukaryotic cell comprising:

(a) synthesizing a nucleic acid molecule that is interrupted by heterologous intron sequences or flanked by sequences within 1000 base pairs where the intron or flanking nucleic acid sequence contains one or more synthetic or biologically-derived segment with at least one 50 base sub-segment with at least 10 AA/TT base pairs where the DNA helix can be modeled as a straight molecule with 10-11 base periodicity so that at least 70% of the AA/TT base pairs are visible by an observer looking laterally at the DNA helix and which nucleic acid carries information sufficient to encode the RNA or protein molecule of interest,

(b) introducing said nucleic acid molecule into an expression vector, and

(c) introducing the expression vector comprising said nucleic acid into the eukaryotic cell,

wherein the expression vector comprising said nucleic acid expresses the RNA or protein molecule of interest in the eukaryotic cell.

56. The method of claim 55, in which the synthetic nucleic acid molecule comprises at least one intron sequence selected from the group consisting of SEQ ID NO:

8-11 and 20-23.

57. The method of claim 55, in which the synthetic nucleic acid molecule comprises at least 30 consecutive base pairs of a nucleotide sequence selected from the group consisting of SEQ ID NO:

3-7 and 15-19.

61. The method of claim 55, in which the synthetic nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:

3-7 and 15-19.

58-60. -60. (canceled)

62. A method of positioning nucleosomes for a target nucleic acid in a eukaryotic cell comprising:

modifying the target nucleic acid to introduce signals that limit or constrain the positions of nucleosome cores, wherein the introduction of the signals positions nucleosomes on the target nucleic acid.