Method for expressing protein gene in response to expression of miRNA

Patent metadata

Field	Value
Publication number	US-11111503-B2
Application number	US-201716313322-A
Country	US
Kind code	B2
Filing date	Jun 27, 2017
Priority date	Jun 27, 2016
Publication date	Sep 7, 2021
Grant date	Sep 7, 2021

Abstract

An mRNA forcibly expresses a protein gene in response to a miRNA, and a method for forcibly expressing the same, are provided. An artificial mRNA comprising a sequence encoding a protein gene, a miRNA target sequence linked to the 3′-terminal side of a Poly A sequence, and a translational repression sequence linked to the 3′-terminal side of the miRNA target sequence; and a method for expressing a protein gene in response to the expression of a miRNA, comprising a step of introducing the artificial mRNA into a cell.

First claim

The invention claimed is:

1. An artificial mRNA comprising:
a sequence encoding a protein,
a miRNA target sequence linked to the 3′-terminal side of a Poly A sequence of at least 50 nucleotides, and
a translational repression sequence linked to the 3′-terminal side of the miRNA target sequence.

2. The artificial mRNA according to claim 1 , wherein
the translational repression sequence comprises a sequence selected from
(i) a nucleotide sequence consisting of 20 or more nucleotides that binds the Poly A sequence in an intracellular environment,
(ii) a sequence that binds a 5′ UTR in the artificial mRNA in an intracellular environment, and
(iii) a sequence consisting of 100 or more nucleotides.

3. A method for expressing a protein in response to the expression of a miRNA, which comprises a step of introducing the artificial mRNA according to claim 1 into a cell.

4. A method for determining a desired cell type from a cell group comprising two or more cells, using the expression of a miRNA as an indicator,
the method comprising the following steps:
(1) a step of introducing a first artificial mRNA comprising a sequence encoding a first marker, a first miRNA target sequence linked to the 3′-terminal side of a Poly A sequence of at least 50 nucleotides, and a translational repression sequence linked to the 3′-terminal side of the first miRNA target sequence, into the cell group; and
(2) a step of determining the cell type, using the translation level of the first marker as an indicator.

5. The method according to claim 4 , wherein
the translational repression sequence comprises a sequence selected from
(i) a nucleotide sequence consisting of 20 or more nucleotides that binds the Poly A sequence in an intracellular environment,
(ii) a sequence that binds a 5′ UTR of the first artificial mRNA in an intracellular environment, and
(iii) a sequence consisting of 100 or more nucleotides.

6. The method according to claim 5 , wherein
the desired cell type is a cell type in which the expression level of the first miRNA greater than in other cell types in the cell group, and
the step (2) is a step of determining a cell type in which the translation level of the first marker is greater than in other cell types in the cell group.

7. The method according to claim 4 , wherein the first miRNA target sequence comprises a target sequence of miR-302a, and the desired cell type is a pluripotent stem cell.

8. The method according to claim 4 , which further comprises:
(3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA in the cell group.

9. The method according to claim 8 , which further comprises:
(4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the mi RNA in the cell group.

10. A method for expressing a protein in response to the expression of a miRNA, which comprises a step of introducing the artificial mRNA according to claim 2 into a cell.

11. The method according to claim 5 , wherein the first miRNA target sequence comprises a target sequence of miR-302a, and the desired cell type is a pluripotent stem cell.

12. The method according to claim 6 , wherein the first miRNA target sequence comprises a target sequence of miR-302a, and the desired cell type is a pluripotent stem cell.

13. The method according to claim 5 , which further comprises:
(3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA.

14. The method according to claim 6 , which further comprises:
(3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA in the cell group.

15. The method according to claim 7 , which further comprises:
(3) a step of introducing into the cell group a second artificial mRNA encoding a second marker that is different from the first marker and is operably linked to a second miRNA target sequence, wherein the second miRNA target sequence is a sequence that is bound by the same miRNA as that for the first miRNA target sequence in an intracellular environment, and translation of the second marker is inhibited in response to the expression level of the miRNA in the cell group.

16. The method according to claim 5 , which further comprises:
(4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.

17. The method according to claim 6 , which further comprises:
(4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.

18. The method according to claim 7 , which further comprises:
(4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.

19. The method according to claim 8 , which further comprises:
(4) a step of introducing into the cell group a third artificial mRNA that does not comprise a miRNA target sequence but encodes a third marker that is different from the first and second markers, wherein translation of the third marker is not influenced by the expression level of the miRNA in the cell group.

Assignees

• Univ Kyoto

Inventors

• Saito Hirohide
• Fujita Yoshihiko

Classifications

• C12N5/10

  Cells modified by introduction of foreign genetic material · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12N2840/102

  inhibiting translation · CPC title

• C12N15/67

  General methods for enhancing the expression · CPC title