MODULAR APTAMAR-REGULATED RIBOZYMES

• US 20110002892A1
• Filed: 11/09/2007
• Published: 01/06/2011
• Est. Priority Date: 11/09/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Win, Maung Nyan, Galloway, Katie, Smolke, Christina D.
• Granted Patent: US 8,158,595 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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 having a first and 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, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes 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 binding of said ligand to said aptamer causes helix-slipping involving said information transmission domain.

5. The ribozyme of claim 1, wherein binding of said ligand to said aptamer causes strand-displacement involving said information transmission domain.

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

7. The ribozyme of claim 6, wherein said ligand and second ligand are different.

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

9. 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.

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

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

12. The ribozyme of claim 11, wherein the ligand is a signal transduction second messenger molecule.

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

14. 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.

15. 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.

16. An expression construct comprising (i) a coding sequence which, when transcribed to RNA, produces one or more 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.

17. The expression construct of claim 16 wherein said RNA is an mRNA including a coding sequence for a polypeptide, said one or more ribozymes being transcribed as part of said mRNA and regulates expression of said polypeptide in a manner dependent on said ligand.

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

UTR of said mRNA through said stem III.

19. A cell, engineered to include the expression construct of claim 16.

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

(i) providing a cell of claim 19,

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

50. 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 18, 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.

51. The method of claim 50, which is used to induce cell death in a manner dependent on the presence of said ligand.

52. The method of claim 50, which is used to prevent cell death in a manner dependent on the presence of said ligand.

53. The method of claim 50, which is used to induce differentiation in a manner dependent on the presence of said ligand.

54. The method of claim 50, which is used to inhibit differentiation in a manner dependent on the presence of said ligand.

55. The method of claim 50, which is used to prevent the growth of hyperplastic or tumor cells.

56. The method of claim 50, which is used to reduce fat cells in the patient.

57. The method of claim 50, which is used to regulate growth and differentiation of stem cells.

58. The method of claim 50, which is used to regulate activation of an immune response.

59. The method of claim 50, wherein said ribozyme, or an expression construct for transcribing said ribozyme, are introduced ex vivo into cells which are transplanted into said patient.

21. An aptamer-regulated ribozyme for cleaving a target nucleic acid, comprising

(a) a trans-acting hammerhead ribozyme comprising catalytic core, a 5′

targeting arm which hybridizes to a 3′

sequence of said target nucleic acid, a 3′

targeting arm which hybridizes to a 5′

sequence of said target nucleic acid, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;

(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop 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, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves a target nucleic acid at a rate dependent upon the presence or absence of said ligand.

22. The ribozyme of claim 21, wherein binding of said ligand to said aptamer alters the size of said loop, thereby altering the ability of said ribozyme to cleave said target nucleic acid in a manner dependent on said ligand.

23. The ribozyme of claim 21, wherein binding of said ligand to said aptamer alters the size of said catalytic core, thereby altering the ability of said ribozyme to cleave said target nucleic acid in a manner dependent on said ligand.

24. The ribozyme of claim 21, wherein binding of said ligand to said aptamer causes helix-slipping involving said information transmission domain.

25. The ribozyme of claim 21, wherein binding of said ligand to said aptamer causes strand-displacement involving said information transmission domain.

26. The ribozyme of claim 21, wherein the ligand is a small molecule having a molecular weight less than 2500 amu and/or is cell permeable.

27. The ribozyme of claim 21, wherein the ligand is a metal ion.

28. The ribozyme of claim 21, wherein the ligand is a natural product.

29. The ribozyme of claim 28, wherein the ligand is a signal transduction second messenger molecule.

30. The ribozyme of claim 28, wherein the ligand is a post-translationally modified protein.

31. The ribozyme of claim 21, 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.

32. The ribozyme of claim 21, wherein the ligand is selected from the group consisting of enzyme co-factors, enzyme substrates and products of enzyme-mediated reactions.

33. An expression construct comprising (i) a coding sequence which, when transcribed to RNA, produces said ribozyme of claim 21, and (ii) one or more transcriptional regulatory sequences that regulate transcription of said RNA in a cell containing said expression construct.

34. A cell, engineered to include the expression construct of claim 33.

48. A method for treating or preventing infection by a pathogenic agent, comprising administering to a patient a sufficient amount of a ribozyme of claim 21, and a wherein said ligand is produced as a consequence to pathogen infection.

49. A method for causing phenotypic regulation of cell growth, differentiation or viability in cells of a patient, comprising introducing into cells in said a patient a ribozyme of claim 21, where said aptamer binds to a ligand, the concentration of which is dependent on cellular phenotype, wherein binding of said ligand to said aptamer induces a conformational change between active and inactive forms of said trans-acting ribozyme, and said active form of said trans-acting ribozyme inhibits expression of said target gene to alter the regulation of cell growth, differentiation or viability in said cells.

60. A pharmaceutical preparation comprising a ribozyme of claim 21, or an expression construct which, when transcribed, produces an RNA including said ribozyme, and a pharmaceutically acceptable carrier suitable for use administration to a human or non-human patient.

35. A cell comprising:

a metabolic pathway of one or more reactions that are regulated at least in part by a target gene; and

one or more trans-acting hammerhead ribozymes that act as control elements on said metabolic pathway by regulating expression of said target gene, each trans-acting nucleic acid including

(a) a catalytic core, a 5′

targeting arm which hybridizes to a 3′

sequence of said target gene, a 3′

targeting arm which hybridizes to a 5′

sequence of said target gene, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;

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

(c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand selected from an enzyme co-factor, a reactant, a substrate or a product of a reaction in said metabolic pathway;

wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves mRNA transcribed from said target gene at a rate dependent upon the presence or absence of said ligand.

36. The cell of claim 35, wherein the metabolic pathway includes at least one reaction mediated by an enzyme, and at least one of said trans-acting ribozyme that regulates expression of said enzyme.

37. A cell comprising a metabolic pathway of one or more reactions that are regulated at least in part by a target gene, said target gene engineered to include, upon expression as an mRNA, one or more cis-acting ribozymes that act to control expression of said target gene, each cis-acting ribozyme including

(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 having a first and 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 selected from an enzyme co-factor, a reactant, a substrate or a product of a reaction in said metabolic pathway;

wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes 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.

38. The cell of claim 37, wherein the metabolic pathway includes at least one reaction mediated by an enzyme, and at least one of said cis-acting ribozyme that regulates expression of said enzyme.

39. 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 ribozyme comprising:

(a) a catalytic core, a 5′

targeting arm which hybridizes to a 3′

sequence of said target gene, a 3′

targeting arm which hybridizes to a 5′

sequence of said target gene, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;

(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop 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, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves mRNA transcribed by said target gene at a rate dependent upon the presence or absence of said ligand.

40. The method of claim 39, wherein the ligand is produced by said cell.

41. The method of claim 39, wherein the ligand is a cell permeable agent that is contacted with said cell.

42. 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 ribozymes in said mRNA that regulate expression of the target gene, said cis-acting 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 said catalytic core, and said stem II having a loop II single-stranded loop region opposite said catalytic core;

(b) an information transmission domain having a first and 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, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes 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.

43. The method of claim 42, wherein said one or more ribozymes are transcribed as part of a 3′

UTR of said mRNA.

44. The method of claim 42, wherein the ligand is produced by said cell.

45. The method of claim 42, wherein the ligand is a cell permeable agent that is contacted with said cell.

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

introducing into the cell a trans-acting ribozyme comprising

(a) a catalytic core, a 5′

targeting arm which hybridizes to a 3′

sequence of said reporter gene, a 3′

targeting arm which hybridizes to a 5′

sequence of said reporter gene, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;

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

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

wherein, binding of said analyte to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves mRNA transcribed from said reporter gene at a rate dependent upon the presence or absence of 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 analyte in the cell.

47. 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 that for the reporter gene, which when transcribed to an mRNA transcript, also includes one or more cis-acting ribozymes in said mRNA that regulate expression of the reporter gene, said cis-acting 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 said catalytic core, and said stem II having a loop II single-stranded loop region opposite said catalytic core; and

(b) an information transmission domain having a first and 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, binding of said analyte to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes 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.