RNA cleavage-induced transcript stabilizer and uses thereof

Patent metadata

Field	Value
Publication number	US-11795455-B2
Application number	US-201816049042-A
Country	US
Kind code	B2
Filing date	Jul 30, 2018
Priority date	Jul 31, 2017
Publication date	Oct 24, 2023
Grant date	Oct 24, 2023

Abstract

Provided herein are genetic circuits and encoded RNA transcripts that produce an output molecule in response to an RNA cleavage event that removes a degradation signal. In some embodiments, the genetic circuits described herein may be used for detecting RNA cleaver activities (e.g., in a cell). Methods of using the genetic circuits described herein in diagnostic or therapeutic applications are also provided.

First claim

What is claimed is:

1. A composition comprising:
(i) a first genetic circuit encoding an RNA transcript, the genetic circuit comprising
a first promoter operably linked to a nucleotide sequence encoding an output molecule followed, from 5′ to 3′, by an RNA stabilizer, a cleavage site for an RNA cleaver, and a degradation signal, wherein the RNA transcript comprises one or more recognition sites for an RNA repressor operably linked to the nucleotide sequence encoding the output molecule; and
(ii) a second genetic circuit comprising a second promoter operably linked to a nucleotide sequence encoding the RNA repressor, and one or more cleavage sites for the RNA cleaver.

2. The composition of claim 1 , further comprising:
(iii) a third genetic circuit comprising a third promoter operably linked to a nucleotide sequence encoding the RNA cleaver.

3. The composition of claim 1 , wherein the RNA cleaver is selected from the group consisting of: endoribonucleases, RNAi molecules, and ribozymes.

4. The composition of claim 1 , wherein the RNA repressor is an RNA binding protein.

5. The composition of claim 4 , wherein the RNA binding protein is fused to a modifying domain.

6. The composition of claim 1 , wherein the RNA stabilizer is selected from the group consisting of: MALAT1 triplex, MENβ triplex, KSHV PAN triplex, histone stem loop, and a polyA signal.

7. A cell comprising the first and second genetic circuits of claim 1 .

8. The cell of claim 7 , wherein the first and second genetic circuits are inserted into the genome of the cell.

9. A method comprising maintaining the cell of claim 7 .

10. The method of claim 2 , further comprising detecting the output molecule.

11. The method of claim 10 , further comprising classifying the cell.

12. A method comprising delivering the RNA transcript encoded by the first genetic circuit of claim 1 to a cell and detecting the output molecule.

13. A method of treating a disease or disorder comprising administering an effective amount of a composition comprising the composition of claim 2 to a subject in need thereof, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder.

14. An RNA transcript encoded by the first genetic circuit of claim 1 .

Assignees

• Massachusetts Inst Technology

Inventors

• Weiss Ron
• Diandreth Breanna E

Classifications

• C12N15/113 Primary

  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 title

• A61K48/0066

  Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/67 Primary

  General methods for enhancing the expression · CPC title

• C12N2310/121

  Hammerhead · CPC title