Genetic constructs for delaying or repressing the expression of a target gene

• US 6,573,099 B2
• Filed: 06/19/1998
• Issued: 06/03/2003
• Est. Priority Date: 03/20/1998
• Status: Expired due to Term

• 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: Graham, Michael Wayne
• Primary Examiner(s): Priebe, Scott D.
• Assistant Examiner(s): Kaushal, Sumesh
• Application Number:
  US09/100,812
• Publication Number:
  US 20020168707A1
• Time in Patent Office: 1,810 Days
• Field of Search: 435/69.1, 435/320.1, 435/325, 435/455, 536/23.1, 536/23.5, 536/24.5, 514/44, 800/8, 424/93.2, 424/93.21
• 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. An isolated genetic construct which is capable of delaying, repressing or otherwise reducing the expression of a target gene in an animal cell which is transfected with said genetic construct, wherein said genetic construct comprises at least two copies of a structural gene sequence, wherein said structural gene sequence comprises a nucleotide sequence which is substantially identical to at least a region of said target gene, and wherein said at least two copies of said structural gene sequence are placed operably under the control of a single promoter sequence which is operable in said cell, wherein at least one copy of said structural gene sequence is placed operably in the sense orientation under the control of said promoter sequence.

6. An animal cell comprising the genetic construct of any one of claims 1-2 or 3-5.

11. The isolated genetic construct according to any one of claims 1, 2 or 3-5 wherein said region of the target gene is 20 to 30 nucleotides long.

13. The isolated genetic construct according to any one of claims 1, 2 or 3-5, comprising two copies of said structural gene sequence.

14. The isolated genetic construct according to any one of claims 1, 2 or 3-5 wherein said region of the target gene is at least 30 nucleotides long.

15. The isolated genetic construct according to any one of claims 1, 2 or 3-5 wherein said structural gene sequence comprises a nucleotide sequence that is identical to said region of said target gene.

19. An animal cell comprising the genetic construct according to claim 11.

20. An animal cell comprising the genetic construct according to claim 13.

21. An animal cell comprising the genetic construct according to claim 14.

22. An animal cell comprising the genetic construct according to claim 15.

2. An isolated genetic construct which is capable of delaying, repressing or otherwise reducing the expression of a target gene in an animal cell which is transfected with said genetic construct, wherein said genetic construct comprises at least two copies of a structural gene sequence wherein each copy of said structural gene sequence is separately placed under the control of a promoter which is operable in said cell, and wherein said structural gene sequence comprises a nucleotide sequence which is substantially 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 under the control of an individual promoter sequence.

3. An isolated genetic construct which is capable of delaying, repressing or otherwise reducing the expression of a target gene in an animal cell which is transfected with said genetic construct, wherein said genetic construct comprises at least two copies of a structural gene sequence, wherein said structural gene sequence comprises a nucleotide sequence which is substantially identical to at least a region of said target gene, and wherein said at least two copies of said structural gene sequence are placed operably under the control of a single promoter sequence which is operable in said cell, wherein at least one copy of said structural gene sequence is placed operably in the sense orientation under the control of said promoter sequence and wherein at least one other copy of said structural gene sequence is placed operably in the antisense orientation under the control of said promoter sequence.

4. An isolated genetic construct which is capable of delaying, repressing or otherwise reducing the expression of a target gene in an animal cell which is transfected with said genetic construct, wherein said genetic construct comprises at least two copies of a structural gene sequence and each copy of said structural gene sequence is separately placed under the control of a promoter which is operable in said cell, and wherein said structural gene sequence comprises a nucleotide sequence which is substantially 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 under the control of an individual promoter sequence, and wherein at least one other copy of said structural gene sequence is placed operably in the antisense orientation under the control of another individual promoter sequence.

5. An isolated genetic construct which is capable of delaying, repressing or otherwise reducing the expression of a target gene in an animal cell which is transfected with said genetic construct, wherein said genetic construct comprises at least two copies of a structural gene sequence, wherein said structural gene sequence comprises a nucleotide sequence which is substantially identical to at least a region of said target gene, and wherein said at least two copies of said structural gene sequence are placed operably under the control of a single promoter sequence which is operable in said cell, wherein at least one copy of said structural gene sequence is placed operably in the sense orientation under the control of said promoter sequence, wherein at least one other copy of said structural gene sequence is placed operably in the antisense orientation under the control of said promoter sequence, and wherein said at least one copy of said structural gene sequence that is placed in the sense orientation relative to said promoter and said at least one copy of said structural gene sequence that is placed in the antisense orientation relative to said promoter are spaced from each other by a nucleic acid stuffer fragment.

7. A method of delaying or repressing the expression of a target gene in an animal cell, comprising transfecting said animal cell with a genetic construct, wherein said genetic construct comprises at least two copies of a structural gene sequence, wherein said structural gene sequence comprises a nucleotide sequence which is substantially identical to at least a region of said target gene, and wherein said at least two copies of said structural gene sequence are placed operably under the control of a single promoter sequence which is operable in said cell, wherein at least one copy of said structural gene sequence is placed operably in the sense orientation under the control of said promoter sequence.

8. The method according to claim 7, wherein at least one other copy of said structural gene sequence is placed operably in the antisense orientation under the control of said promoter sequence.

9. The method according to claim 8, wherein said copy of said structural gene sequence that is placed in the sense orientation relative to said promoter and said copy of said structural gene sequence that is placed in the antisense orientation relative to said promoter are spaced from each other by a nucleic acid stuffer fragment.

10. A method of delaying or repressing the expression of a target gene in an animal cell, comprising expressing in said animal cell a genetic construct, wherein said genetic construct comprises at least two copies of a structural gene sequence, wherein each copy of said structural gene sequence is separately placed under the control of a promoter which is operable in said cell, and wherein said structural gene sequence comprises a nucleotide sequence which is substantially 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 under the control of an individual promoter sequence.

12. The method according to any one of claims 7-9 or 10 wherein said region of the target gene is 20 to 30 nucleotides long.

16. The method according to any one of claims 7-9 or 10, wherein said genetic construct comprises two copies of said structural gene sequence.

17. The method according to any one of claims 7-9 or 10, wherein said region of the target gene is at least 30 nucleotides long.

18. The method according to any one of claims 7-9 or 10, wherein said structural gene sequence comprises a nucleotide sequence that is identical to said region of said target gene.