Synthetic genes and genetic constructs

• US 20040266005A1
• Filed: 04/08/2004
• Published: 12/30/2004
• Est. Priority Date: 03/20/1998
• Status: Active Grant

• Current Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Original Assignee: Benitec Australia Limited (Benitec Biopharma, Inc.)
• Inventors: Rice, Robert Norman, Graham, Michael Wayne
• Granted Patent: US 8,053,419 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• 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 15/69 Increasing the copy number of the vector
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8283 for virus resistance
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/38 being a stuffer
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/55 from bacteria
• CPC Class Codes: C12N 2830/60 from viruses
• CPC Class Codes: C12N 2840/102 inhibiting translation
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/127 RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
• CPC Class Codes: C12N 9/503 : derived from viruses

Abstract

The present invention relates generally to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention provides novel synthetic genes and genetic constructs which are capable of repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto.

Claims

1-33. -33. (cancel).

34. A method for delaying, repressing or otherwise reducing the expression of a target gene in an animal cell comprising introducing an RNA nucleic acid molecule to the cell, wherein the RNA nucleic acid molecule includes a transcription product of a genetic construct comprising at least two copies of a structural gene sequence, wherein said structural gene sequence comprises a nucleotide sequence which is at least 80% identical to at least a region of said target gene, wherein at least one copy of said structural gene sequence is placed operably in the sense orientation and wherein at least one other copy of said structural gene sequence is placed operably in the antisense orientation.

35. The method of claim 34 wherein said at least two copies are operably under the control of a single promoter.

36. The method of claim 34 wherein said at least one copy is operably under the control of a first promoter and said at least one other is operably under the control of a second promoter.

37. The method according to claim 34, wherein said RNA nucleic acid molecule consists essentially of ribonucleotides.

38. The method according to claim 34, wherein said RNA nucleic acid molecule is comprised at least partially of ribonucleotide analogues.

39. The method according to claim 34, wherein said structural gene sequence is about 20-30 nucleotides in length.

40. The method according to claim 34, wherein said structural gene sequence is 23 nucleotides in length.

41. The method according to claim 34, wherein said structural gene sequence is 22 nucleotides in length.

42. The method according to claim 34, wherein said structural gene sequence is 21 nucleotides in length.

43. The method according to claim 34, wherein said structural gene sequence is 20 nucleotides in length.

44. The method according to claim 34, wherein said structural gene sequence is 19 nucleotides in length.

45. The method according to claim 34, wherein said structural gene sequence is 18 nucleotides in length.

46. The method according to any one of claims 39-45, wherein said at least two copies are about the same length.

47. The method according to any one of claims 39-45, wherein said at least two copies are the same length.

48. The method according to claim 34, wherein said at least two copies are in the same nucleic acid strand.

49. The method according to claim 48, wherein said at least two copies are separated by at least one nucleic acid stuffer sequence.

50. The method according to claim 34, wherein said at least two copies are in separate nucleic acid strands.

51. The method according to claim 34, wherein said first RNA sequence is identical to the region of said target gene and second RNA sequence is identical to the complement of said region of said target gene.

52. An isolated nucleic acid molecule comprising:

a first RNA sequence, the sequence being at least 80% identical to a region of a transcription product of a target gene,

a second RNA sequence, the sequence being at least 80% identical to an antisense copy of the region of the transcription product of the target gene;

wherein said first RNA sequence is about 20-30 nucleotides in length, and

wherein said nucleic acid molecule is capable of reducing expression of the target gene in an animal cell when the nucleic acid molecule is introduced into said animal cell.

53. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is at least 90% identical to the region of the transcription product of the target gene and said second RNA sequence is at least 90% identical to the antisense copy of the region of the transcription product of the target gene.

54. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is at least 95% identical to the region of the transcription product of the target gene and said second RNA sequence is at least 95% identical to the antisense copy of the region of the transcription product of the target gene.

55. The nucleic acid molecule according to claim 52, wherein said first and second RNA sequences consist essentially of ribonucleotides.

56. The nucleic acid molecule according to claim 52, wherein at least one of said first and second RNA sequences is comprised at least partially of ribonucleotide analogues.

57. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is 24 nucleotides in length.

58. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is 23 nucleotides in length.

59. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is 22 nucleotides in length.

60. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is 21 nucleotides in length.

61. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is 20 nucleotides in length.

62. The nucleic acid molecule according to claim 52, wherein said first RNA sequence is 19 nucleotides in length.

63. The nucleic acid molecule according to claim 52, wherein said second RNA sequence is 18 nucleotides in length.

64. The nucleic acid molecule according to any one of claims 57-63, wherein said first RNA sequence is about the same length as said second RNA sequence.

65. The nucleic acid molecule according to any one of claims 57-63, wherein said first RNA sequence is the same length as the second RNA sequence.

66. The nucleic acid molecule according to claim 52, wherein the first and second RNA sequences are in the same nucleic acid strand.

67. The nucleic acid molecule of claim 66, wherein the first and second RNA sequences are separated by a nucleic acid stuffer sequence.

68. The nucleic acid molecule according to claim 52, wherein the first and second RNA sequences are in separate nucleic acid strands.

69. The nucleic acid molecule according to claim 52, wherein the first RNA sequences is identical to said region of said transcription product of said target gene and said second RNA sequence is identical to said antisense copy of said region of said transcription product of said target gene.