Aptamer regulated nucleic acids and uses thereof

• US 20060088864A1
• Filed: 10/05/2005
• Published: 04/27/2006
• Est. Priority Date: 10/05/2004
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Smolke, Christina D., Bayer, Travis S.
• Granted Patent: US 9,315,862 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2525/205 Aptamer

Abstract

The invention relates to trans-acting ligand-responsive nucleic acids and uses thereof. In particular, a ligand responsive nucleic acid comprises an effector domain and an aptamer domain that is responsive to a ligand.

Claims

1. A nucleic acid comprising (i) a substrate sequence that can form a substrate for an extrinsic enzymatic activity, and (ii) an aptamer that binds to a ligand, wherein binding of the ligand to said aptamer causes a conformational change in the nucleic acid that alters the ability of said substrate sequence to form said substrate and/or alters the Km and/or Kcat of said substrate for the extrinsic enzymatic activity.

5. The nucleic acid of any of claim 1, wherein said nucleic acid is a ribonucleic acid (RNA).

6. The nucleic acid of claim 1, wherein the conformational change produces or removes an intramolecular double-stranded feature, including said substrate sequence, which double-stranded feature is said substrate for said extrinsic enzymatic activity.

7. The nucleic acid of claim 1, wherein said extrinsic enzymatic activity is an RNAse enzyme.

8. The nucleic acid of claim 7, wherein said substrate sequence produces siRNA, miRNA or a precursor or metabolite thereof in an RNA Interference pathway, as a product of reaction with said RNAse enzyme.

9. The nucleic acid of claim 1, wherein the extrinsic enzymatic activity is selected from the group consisting of a polymerase, a recombinase, a ligase, a methylase, a glycosylase, and a nuclease.

19. An expression construct comprising (i) a coding sequence which, when transcribed, produces the RNA of claim 5, and (ii) one or more transcriptional regulatory sequences that regulate transcription of said RNA in a cell containing said expression construct.

21. A recombinant cell engineered with one or more expression constructs of claim 19.

2. A nucleic acid comprising (i) an antisense sequence for inhibiting expression of a target gene, and (ii) an aptamer that binds to a ligand, wherein binding of the ligand to said aptamer causes a conformational change in the nucleic acid that alters the ability of said antisense sequence to inhibit expression of the target sequence.

10. The nucleic acid of claim 2, wherein ligand binding to said aptamer causes a conformational change that changes the availability of said antisense sequence for hybridization to said target gene.

11. The nucleic acid of claim 2, wherein said antisense sequence is selected to hybridize to an RNA transcript of said target gene or to a genomic sequence of said target gene.

3. A nucleic acid comprising (i) a hybridization sequence that hybridizes to a target sequence of a second nucleic acid, and (ii) an aptamer that binds to a ligand, wherein binding of the ligand to said aptamer causes a conformational change in the nucleic acid that alters the ability of said hybridization sequence to hybridize to said target sequence.

4. A trans-acting nucleic acid for altering expression of a target gene, comprising:

(i) a targeting sequence that is capable of hybridizing to said target gene and regulate expression of said target gene; and

(ii) an aptamer sequence that binds to a ligand,

wherein, binding of said ligand to said aptamer causes a conformational change in said nucleic acid, and said nucleic acid regulates expression of said target gene in a manner dependent on the presence of said ligand.

12. The nucleic acid of claim 4, wherein said targeting sequence is selected to hybridize to an RNA transcript of said target gene and (i) to reduce the amount of protein translated from said RNA transcript, and/or (ii) alter splicing of said RNA transcript.

13. The nucleic acid of claim 12, wherein said RNA transcript is an mRNA.

14. The nucleic acid of claim 4, wherein the ligand is a small molecule having a molecular weight less than 2500 amu.

15. The nucleic acid of claim 4, wherein the ligand is a metal ion.

16. The nucleic acid of claim 4, wherein the ligand is a natural product.

17. The nucleic acids of claim 4, wherein the ligand is selected from the group consisting of polypeptides, peptides, nucleic acids, carbohydrates, fatty acids and lipids, a non-peptide hormone (such as steroids) and metabolic precursors or products thereof.

18. The nucleic acids of claim 4, wherein the ligand is selected from the group consisting of enzyme co-factors, enzyme substrates and products of enzyme-mediated reactions.

20. A library of aptamer-regulated nucleic acids comprising a variegated population of nucleic acids of claim 4, or of expression constructs encoding said nucleic acids.

22. A cell comprising one or more nucleic acids of claim 4.

26. A pharmaceutical preparation composition comprising a nucleic acid of claim 4 or an expression construct for expressing said nucleic acids as a transcript thereof, and a pharmaceutically acceptable carrier suitable for use administration to a human or non-human patient.

23. A method for rendering expression of a target gene in a cell dependent on the presence or absence of a ligand, comprising introducing into the cell a trans-acting nucleic acid comprising:

(i) a targeting sequence that is capable of hybridizing to said target gene and regulate expression of said target gene; and

(ii) an aptamer sequence that binds to a ligand,

wherein binding of said ligand to said aptamer induces a conformational change in said trans-acting nucleic acid, and said trans-acting nucleic acid regulates expression of said target gene in a dose-dependent manner dependent on the concentration of said ligand.

24. The method of claim 23, wherein the ligand is produced by said cell.

25. The method of claim 23, wherein the ligand is a cell permeable agent that is contacted with said cell.