RNA cleavage-induced transcript stabilizer and uses thereof

Patent metadata

Field	Value
Publication number	US-12480119-B2
Application number	US-202318466891-A
Country	US
Kind code	B2
Filing date	Sep 14, 2023
Priority date	Jul 31, 2017
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

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 cleavage-induced transcript stabilizer comprising:
(i) an RNA transcript comprising a ribonucleotide sequence encoding an output molecule followed, in order, by: an RNA stabilizer, a cleavage site for an RNA cleaver, and a degradation signal that leads to degradation of the RNA transcript; wherein the RNA cleaver is an endoribonuclease, a siRNA, a miRNA, or a ribozyme; and wherein the cleavage-induced transcript stabilizer comprises one or more recognition sites for an RNA repressor operably linked to the nucleotide sequence encoding the output molecule.

2 . The cleavage-induced transcript stabilizer of claim 1 , further comprising:
(ii) a promoter operably linked to a nucleotide sequence encoding an RNA cleaver that cleaves the RNA transcript at the cleavage site.

3 . The cleavage-induced transcript stabilizer of claim 2 , wherein the promoter of (ii) is an inducible promoter.

4 . The cleavage-induced transcript stabilizer of claim 1 , wherein the RNA transcript is degraded in the absence of the RNA cleaver.

5 . The cleavage-induced transcript stabilizer of claim 1 , wherein the RNA transcript is expressed in the presence of the RNA cleaver.

6 . The cleavage-induced transcript stabilizer of claim 5 , wherein the cleavage of the RNA transcript stabilizes the RNA transcript and results in expression of the output molecule.

7 . The cleavage-induced transcript stabilizer of claim 1 , wherein the output molecule is a detectable molecule.

8 . The cleavage-induced transcript stabilizer of claim 1 , wherein the output molecule is a therapeutic molecule.

9 . The cleavage-induced transcript stabilizer of claim 1 , wherein the output molecule is a functional molecule.

10 . The cleavage-induced transcript stabilizer of claim 9 , wherein the functional molecule is selected from the group consisting of: TetR, CNOT7, DDX6, PPR10, L7Ae, Csy4, Cas6, CasE, and Cse3.

11 . An isolated cell comprising the cleavage-induced transcript stabilizer of claim 1 .

12 . The isolated cell of claim 11 , wherein a genetic circuit that encodes the cleavage-induced transcript stabilizer is inserted into the genome of the cell.

13 . A method comprising maintaining the isolated cell of claim 11 .

14 . A method comprising delivering the cleavage-induced transcript stabilizer of claim 1 to a cell and detecting the output molecule.

15 . A method of detecting an RNA cleaver activity, comprising:
delivering the cleavage-induced transcript stabilizer of claim 1 to a cell and detecting the output molecule.

16 . A method of treating a disease or disorder comprising delivering the cleavage-induced transcript stabilizer of claim 1 to a cell, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder.

17 . A method of diagnosing a disease or disorder comprising delivering the cleavage-induced transcript stabilizer of claim 1 to a cell and detecting the output molecule.

18 . A method of treating a disease or disorder comprising administering an effective amount of a composition comprising the cleavage-induced transcript stabilizer of claim 1 to a subject in need thereof, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder.

19 . A method of diagnosing a disease or disorder comprising administering an effective amount of a composition comprising the cleavage-induced transcript stabilizer of claim 1 to a subject in need thereof and detecting the output molecule.

Assignees

• Massachusetts Inst Technology

Inventors

• Weiss Ron
• Diandreth Breanna E

Classifications

• C12N15/67 Primary

  General methods for enhancing the expression · CPC title

• C12N2840/105

  enhancing translation · CPC title

• C12N15/63

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

• C12N2310/122

  Hairpin · CPC title

• C12N2310/126

  involving RNAse P · CPC title