# US 11,111,503 B2 · Method for expressing protein gene in response to expression of miRNA > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,111,503 B2 - Document ID: US11111503B2 - Status: Granted - Kind: B2 - Application number: 16/313,322 - Filed: 2017-06-27 - Published: 2019-08-22 - Granted: 2021-09-07 - Earliest claimed: 2016-06-27 - Assignees: Kyoto University - Applicants: Kyoto University - Inventors: Hirohide Saito; Yoshihiko Fujita - Web page: https://patentgrep.com/patent/US11111503B2 ## CPC - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - C12N 15/85: for animal cells - C12N 15/09: Recombinant DNA-technology - C12N 15/67: General methods for enhancing the expression - C12N 5/10: Cells modified by introduction of foreign genetic material - C12N 2840/102: inhibiting translation - C12Q 1/68: involving nucleic acids - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) 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. ### Claim 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. ### Claim 5 (depends on 4) 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. ### Claim 6 (depends on 5) 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. ### Claim 7 (depends on 4) 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. ### Claim 8 (depends on 4) 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. ### Claim 9 (depends on 8) 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. ### Claim 10 (depends on 2) 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. ### Claim 11 (depends on 5) 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. ### Claim 12 (depends on 6) 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. ### Claim 13 (depends on 5) 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. ### Claim 14 (depends on 6) 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. ### Claim 15 (depends on 7) 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. ### Claim 16 (depends on 5) 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. ### Claim 17 (depends on 6) 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. ### Claim 18 (depends on 7) 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. ### Claim 19 (depends on 8) 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.