REGULATABLE FUSION PROMOTERS
First Claim
Patent Images
1. A nucleic acid construct comprising a Pol III/Pol II fusion promoter comprisingan RNA Polymerase III-binding basal promoter region;
- andcis-acting regulatory regions from a Pol II promoter, operably linked with said basal promoter region;
wherein said cis-acting regulatory regions provide specific regulation of expression from said construct.
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Abstract
Fusion promoters are described that combine a RNA polymerase III basal promoter and regulatory elements from RNA polymerase II regulatory regions, and which provide specific regulation of expression from the promoter. Such fusion promoters are useful, for example, for expressing RNAi agents in vivo.
29 Citations
91 Claims
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1. A nucleic acid construct comprising a Pol III/Pol II fusion promoter comprising
an RNA Polymerase III-binding basal promoter region; - and
cis-acting regulatory regions from a Pol II promoter, operably linked with said basal promoter region; wherein said cis-acting regulatory regions provide specific regulation of expression from said construct. - View Dependent Claims (2, 8, 10, 15, 56, 57, 60)
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3-7. -7. (canceled)
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9. (canceled)
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11-14. -14. (canceled)
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16-17. -17. (canceled)
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18. A vector comprising a Pol III/Pol II fusion promoter comprising
a RNA Polymerase III-binding basal promoter region; - and
cis-acting regulatory regions from a Pol II promoter operably linked with said basal promoter region; wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter. - View Dependent Claims (19, 22)
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20-21. -21. (canceled)
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23-25. -25. (canceled)
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26. A cell comprising a Pol III/Pol II fusion promoter operably linked with a coding sequence, wherein said fusion promoter comprises
a RNA Polymerase III-binding basal promoter region; - and
cis-acting regulatory regions from a Pol II promoter operably linked with said basal promoter region; wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter. - View Dependent Claims (27, 30, 31, 33, 34, 38, 39)
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28-29. -29. (canceled)
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32. (canceled)
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35-37. -37. (canceled)
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40-47. -47. (canceled)
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48. A non-human transgenic organism, comprising
a plurality of cells comprising a genetic construct comprising a Pol III/Pol II fusion promoter operably linked with a coding sequence, wherein said Pol III/Pol II fusion promoter comprises a RNA Polymerase III-binding basal promoter region; - and
cis-acting regulatory regions from a Pol II promoter operably linked with said basal promoter region; wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter. - View Dependent Claims (49, 50)
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51-55. -55. (canceled)
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58-59. -59. (canceled)
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61-64. -64. (canceled)
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65. A method for expressing an RNAi agent in a cell, comprising
maintaining a cell under expression conditions, wherein said cell comprises a genetic construct comprising a Pol III/Pol II fusion promoter operably linked with a RNAi agent encoding sequence, wherein said Pol III/Pol II fusion promoter comprises a RNA Polymerase III-binding basal promoter region; - and
cis-acting regulatory regions from a Pol II promoter, operably linked with said basal promoter region; wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter. - View Dependent Claims (66)
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67. (canceled)
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68. A method for inhibiting expression of a target gene in a cell, comprising
transfecting said cell with a vector comprising a genetic construct, wherein said construct comprises a Pol III/Pol II fusion promoter operably linked with a nucleic acid sequence encoding an RNAi agent targeted to said target gene, wherein said fusion promoter comprises a RNA Polymerase III-binding basal promoter region; - and cis-acting regulatory regions from a Pol II promoter, operably linked with said basal promoter region, wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter; and
maintaining said cell under expression conditions. - View Dependent Claims (69, 70, 72)
- and cis-acting regulatory regions from a Pol II promoter, operably linked with said basal promoter region, wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter; and
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71. (canceled)
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73-79. -79. (canceled)
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80. A method for validating a target as a therapeutic target, comprising
inhibiting expression of a putative therapeutic target gene in said cell, wherein said inhibiting is due to expression of an RNAi agent from a genetic construct comprising a Pol III/Pol II fusion promoter operably linked with a nucleic acid sequence encoding said RNAi agent, wherein said fusion promoter comprises a RNA Polymerase III-binding basal promoter region; - and cis-acting regulatory regions from a Pol II promoter operably linked with said basal promoter region, wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter; and
determining whether a biological change in said cell following said inhibiting corresponds with a therapeutic effect, wherein correspondence of said biological change with said therapeutic effect is indicative that said gene is a therapeutic target gene.
- and cis-acting regulatory regions from a Pol II promoter operably linked with said basal promoter region, wherein said cis-acting regulatory regions allow specific regulation of expression from said fusion promoter; and
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81-83. -83. (canceled)
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84. A method for treating a disease or condition wherein inhibition of a target gene provides a beneficial effect, comprising
administering a pharmacologically effective amount of a vector comprising a genetic construct comprising a Pol III/Pol II fusion promoter providing specific regulation of expression, operably linked with a sequence encoding an RNAi agent, to a subject suffering from or at risk of said disease or condition, wherein said fusion promoter comprises a RNA Polymerase III-binding basal promoter region; - and cis-acting regulatory regions from a Pol II promoter operably linked with said basal promoter region, wherein said cis-acting regulatory regions provide specific regulation of expression from said fusion promoter.
- View Dependent Claims (85, 87, 90)
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86. (canceled)
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88-89. -89. (canceled)
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91-101. -101. (canceled)
Specification