Modular aptamer-regulated ribozymes

• US 8,158,595 B2
• Filed: 11/09/2007
• Issued: 04/17/2012
• Est. Priority Date: 11/09/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Galloway, Katie, Smolke, Christina D., Win, Maung Nyan
• Primary Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US11/938,220
• Publication Number:
  US 20110002892A1
• Time in Patent Office: 1,621 Days
• Field of Search: None
• US Class Current: 514/44
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid

Abstract

An extensible RNA-based framework for engineering ligand-controlled gene regulatory systems, called ribozyme switches, that exhibit tunable regulation, design modularity, and target specificity is provided. These switch platforms typically contain a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of a hammerhead ribozyme sequence. A variety of modes of standardized information transmission between these domains can be employed, and this application demonstrates a mechanism that allows for the reliable and modular assembly of functioning synthetic hammerhead ribozyme switches and regulation of ribozyme activity in response to various effectors. In some embodiments aptamer-regulated cis-acting hammerhead ribozymes are provided.

Claims

1. An aptamer-regulated ribozyme, comprising:

(a) a cis-acting hammerhead ribozyme comprising a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite to said catalytic core, and said stem II having a loop II single-stranded loop region opposite to said catalytic core;

(b) an information transmission domain (ITD) having a first end and a second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and

(c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand,

wherein said ITD is between the cis-acting hammerhead ribozyme and the aptamer, and comprises;

(i) a general transmission region,

(ii) a switching strand that hybridizes with the general transmission region in the absence of the ligand, and,

(iii) a competing strand that hybridizes with the general transmission region in the presence of the ligand,

wherein the switching strand and the competing strand compete to bind to the general transmission region through hybridization interactions,

wherein binding of said ligand to said aptamer favors a conformation change in the aptamer,

wherein said conformation change causes the ITD to favor the binding of the general transmission region to said competing strand via a strand-displacement mechanism; and

,

wherein hybridization between the general transmission region and the competing strand causes, via the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, said ribozyme to undergo self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said ligand.

2. The ribozyme of claim 1, wherein binding of said ligand to said aptamer alters the size of said loop I or loop II to which said information transmission domain and aptamer are coupled, thereby altering the ability of said ribozyme to undergo self-cleavage in a manner dependent on said ligand.

3. The ribozyme of claim 1, wherein binding of said ligand to said aptamer alters the size of said catalytic core, thereby altering the ability of said ribozyme to undergo self-cleavage in a manner dependent on said ligand.

4. The ribozyme of claim 1, wherein said aptamer is coupled to said loop I through said information transmission domain, and wherein said ribozyme further comprises a second aptamer coupled to said loop II through a second information transmission domain, said second aptamer binds to a second ligand.

5. The ribozyme of claim 4, wherein said ligand and second ligand are different.

6. The ribozyme of claim 4, wherein said ligand and second ligand are the same.

7. The ribozyme of claim 1, wherein the ligand is a small molecule having a molecular weight less than 2500 amu and/or is cell permeable.

8. The ribozyme of claim 1, wherein the ligand is a metal ion.

9. The ribozyme of claim 1, wherein the ligand is a natural product.

10. The ribozyme of claim 9, wherein the ligand is a signal transduction second messenger molecule.

11. The ribozyme of claim 9, wherein the ligand is a post-translationally modified protein.

12. The ribozyme of claim 1, 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.

13. The ribozyme of claim 1, wherein the ligand is selected from the group consisting of enzyme co-factors, enzyme substrates and products of enzyme-mediated reactions.

14. An expression construct comprising

(i) a coding sequence which, when transcribed to RNA, produces one or more aptamer-regulated ribozymes of claim 1, and

(ii) one or more transcriptional regulatory sequences that regulate transcription of said RNA in a cell containing said expression construct.

15. The expression construct of claim 14, wherein said RNA is an mRNA including a coding sequence for a polypeptide, and wherein said one or more ribozymes are transcribed as part of said mRNA and regulates expression of said polypeptide in a manner dependent on said ligand.

16. The expression construct of claim 14, wherein said one or more ribozymes are transcribed as part of a 3′

UTR of said mRNA, and each ribozyme is joined with said mRNA through said stem III of said ribozyme.

17. A cell, engineered to include the expression construct of claim 14.

18. A method for regulating expression of a recombinant gene, comprising

(i) providing a cell of claim 17,

(ii) contacting the cell with said ligand in an amount that alters the activity of said ribozyme.

24. A method for causing phenotypic regulation of cell growth, differentiation or viability in cells transplanted into a patient, comprising introducing into cells ex vivo an expression construct of claim 16, where said aptamer binds to a ligand, the concentration of which is dependent on cellular phenotype, wherein ligand binding to said aptamer causes a change in said cis-acting ribozyme between two conformational states, in one of which said cis-acting ribozyme inhibits expression of said polypeptide and in the other of which said cis-acting ribozyme does not inhibit expression of said polypeptide, and wherein expression of said polypeptide alters regulation of cell growth, differentiation or viability in said cells.

25. The method of claim 24, which is used to induce cell death in a manner dependent on the presence of said ligand.

26. The method of claim 24, which is used to prevent cell death in a manner dependent on the presence of said ligand.

27. The method of claim 24, which is used to induce differentiation in a manner dependent on the presence of said ligand.

28. The method of claim 24, which is used to inhibit differentiation in a manner dependent on the presence of said ligand.

29. The method of claim 24, which is used to prevent the growth of hyperplastic or tumor cells.

30. The method of claim 24, which is used to reduce fat cells in the patient.

31. The method of claim 24, which is used to regulate growth and differentiation of stem cells.

32. The method of claim 24, which is used to regulate activation of an immune response.

33. The method of claim 24, wherein said ribozyme, or an expression construct for transcribing said ribozyme, are introduced ex vivo into cells which are transplanted into said patient.

19. 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 an expression construct including a coding sequence that for the target gene, which when transcribed to an mRNA transcript, also includes one or more cis-acting aptamer-regulated ribozymes in said mRNA that regulate expression of the target gene, said cis-acting aptamer-regulated ribozyme comprising:

(a) a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite to said catalytic core, and said stem II having a loop II single-stranded loop region opposite to said catalytic core;

(b) an information transmission domain having a first end and a second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and

(c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand,

wherein said ITD is between the cis-acting hammerhead ribozyme and the aptamer, and comprises;

(i) a general transmission region,

(ii) a switching strand that hybridizes with the general transmission region in the absence of the ligand, and,

(iii) a competing strand that hybridizes with the general transmission region in the presence of the ligand,

wherein the switching strand and the competing strand compete to bind to the general transmission region through hybridization interactions,

wherein binding of said ligand to said aptamer favors a conformation change in the aptamer,

wherein said conformation change causes the ITD to favor the binding of the general transmission region to said competing strand via a strand-displacement mechanism; and

,

wherein hybridization between the general transmission region and the competing strand causes, via the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, said ribozyme to undergo self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said ligand so as to render expression of target gene dependent on said ligand.

20. The method of claim 19, wherein said one or more ribozymes are transcribed as part of a 3′

UTR of said mRNA.

21. The method of claim 19, wherein the ligand is produced by said cell.

22. The method of claim 19, wherein the ligand is a cell permeable agent that is contacted with said cell.

23. A method of determining the amount of an analyte in a cell which expresses a reporter gene, comprising

(i) introducing into the cell an expression construct including a coding sequence for the reporter gene, which when transcribed to an mRNA transcript, also includes one or more cis-acting aptamer-regulated ribozymes in said mRNA that regulate expression of the reporter gene, said cis-acting aptamer-regulated ribozyme comprising:

(a) a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite to said catalytic core, and said stem II having a loop II single-stranded loop region opposite to said catalytic core; and

(b) an information transmission domain having a first end and a second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and

(c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to an analyte,

wherein said ITD is between the cis-acting hammerhead ribozyme and the aptamer, and comprises;

(i) a general transmission region,

(ii) a switching strand that hybridizes with the general transmission region in the absence of the analyte, and,

(iii) a competing strand that hybridizes with the general transmission region in the presence of the analyte, wherein the switching strand and the competing strand compete to bind to the general transmission region through hybridization interactions,

wherein binding of said analyte to said aptamer favors a conformation change in the aptamer,

wherein said conformation change causes the ITD to favor the binding of the general transmission region to said competing strand via a strand-displacement mechanism; and

,

wherein hybridization between the general transmission region and the competing strand causes, via the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, said ribozyme to undergo self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said analyte so as to render expression of reporter gene dependent on said analyte;

(ii) measuring the amount of expression of said reporter gene; and

(iii) correlating the amount of expression of said reporter gene with the amount of analyte, thereby determining the amount of the ligand in the cell.