RNA-based logic circuits with RNA binding proteins, aptamers and small molecules

Patent metadata

Field	Value
Publication number	US-11351271-B2
Application number	US-201515509258-A
Country	US
Kind code	B2
Filing date	Sep 8, 2015
Priority date	Sep 8, 2014
Publication date	Jun 7, 2022
Grant date	Jun 7, 2022

Abstract

Engineered synthetic RNA-based genetic circuits are provided that are regulated exclusively at the post-transcriptional level.

First claim

What is claimed is:

1. A synthetic RNA circuit comprising
(a) a first RNA molecule comprising a 5′ cap, at least one target site specific for a first microRNA molecule, a sequence encoding an RNA binding protein, and a poly(A) tail; and
(b) a second RNA molecule comprising a 5′ cap, at least one target site specific for a second microRNA molecule, at least one RNA motif that is capable of being bound by the RNA binding protein in (a), a sequence encoding an output molecule, and a poly(A) tail,
wherein the first microRNA molecule and the second microRNA molecule are different, wherein the first microRNA molecule is a microRNA that is present in a first cell type such that it reduces expression of the first RNA molecule in the first cell type and the second microRNA molecule is a microRNA that is present in a second cell type such that it reduces expression of the second RNA molecule in the second cell type but does not reduce expression of the second RNA molecule in the first cell type or reduces expression of the second RNA molecule less in the first cell type compared to the second cell type, wherein the first cell type and the second cell type are different, wherein the RNA binding protein inhibits production of the output molecule when bound to the at least one RNA motif, and wherein the synthetic RNA circuit is capable of being expressed in a eukaryotic cell.

2. The synthetic RNA circuit of claim 1 , wherein the output molecule is a protein.

3. The synthetic RNA circuit of claim 2 , wherein the output molecule protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator.

4. The synthetic RNA circuit of claim 1 , wherein the RNA binding protein comprises a L7Ae protein or a fusion protein of a MS2 protein and a protein that inhibits protein production.

5. The synthetic RNA circuit of claim 1 , wherein the second RNA molecule is capable of expressing the output molecule in the first cell type.

6. The synthetic RNA circuit of claim 1 , wherein the at least one first microRNA molecule is a microRNA that is expressed in a cancer cell, and the at least one second microRNA molecule is a microRNA that is not expressed or is expressed at a low level in a cancer cell compared to a non-cancer cell.

7. The synthetic RNA circuit of claim 1 , further comprising a sequence encoding a Csy4 protein and a Csy4 recognition site.

8. The synthetic RNA circuit of claim 1 , wherein the second RNA molecule further comprises a target site for a third microRNA molecule, a target site for a fourth microRNA molecule, or a target site for a fifth microRNA molecule.

9. The synthetic RNA circuit of claim 1 , which is present in a cell that expresses the at least one first microRNA molecule but does not express the at least one second microRNA molecule.

10. The synthetic RNA circuit of claim 1 , wherein the first microRNA molecule is miR21.

11. The synthetic RNA circuit of claim 1 , wherein the eukaryotic cell is a mammalian cell.

12. A synthetic RNA circuit comprising:
(a) a first RNA molecule comprising a 5′ cap, at least one first RNA motif, a sequence encoding a second RNA binding protein that is capable of binding to a second RNA motif and that is capable of inhibiting protein production, at least one target site specific for a first siRNA molecule or first microRNA molecule, and a poly(A) tail; and
(b) a second RNA molecule comprising a 5′ cap, at least one second RNA motif that is capable of being bound by the second RNA binding protein, a sequence encoding a first RNA binding protein that is capable of binding to the first RNA motif and inhibiting protein production, at least one target site specific for a second siRNA molecule or second microRNA molecule, and a poly(A) tail, wherein the first siRNA molecule or first microRNA molecule and the second siRNA molecule or second microRNA molecule are different, and wherein the first RNA motif and the second RNA motif are different wherein the first RNA binding protein, upon binding to the first RNA motif, inhibits expression of the second RNA binding protein, and wherein the second RNA binding protein, upon binding to the second RNA motif, inhibits expression of the first RNA binding protein, and wherein the synthetic RNA circuit is capable of being expressed in a eukaryotic cell.

13. The synthetic RNA circuit of claim 12 , wherein the first RNA molecule and/or the second RNA molecule further comprises a sequence encoding an output molecule.

14. The synthetic RNA circuit of claim 13 , wherein the output molecule comprises a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator.

15. The synthetic RNA circuit of claim 12 , wherein the eukaryotic cell is a mammalian cell.

Assignees

• Massachusetts Inst Technology
• Univ Kyoto

Inventors

• Weiss Ron
• Wroblewska Liliana
• Siciliano Velia
• Kitada Tasuku
• Hottelet Foley Maria
• Bodner Katie
• Saito Hirohide
• Endo Kei
• Irvine Darrell J
• Wagner Tyler
• Becraft Jacob

Classifications

• C12N15/63 Primary

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

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• A61K48/0066 Primary

  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

• C12N15/85

  for animal cells · CPC title

• C12N2840/102

  inhibiting translation · CPC title