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US 2026/0295089 A1Published application
Programmable miRNA-Dependent Gene Expression
AssigneeNew York University
InventorBenjamin TenOever
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Abstract
The present invention provides miRNA-dependent polynucleotide circuits which can be programmed to express a target polypeptide in a target cell type or tissue, or a cell in a specific state, or a target species. The present invention further provides vectors, host cells, and pharmaceutical compositions comprising the miRNA-dependent polynucleotide circuits. The present invention further provides a method of selectively expressing a target polypeptide in a target cell type or tissue, or a cell in a specific state.
Claims280
A polynucleotide comprising:
- a) a nucleotide sequence encoding an RNA-binding protein;
- b) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the RNA-binding protein to allow expression of the RNA-binding protein in the absence of the respective miRNA targeting said MSE sequence;
- c) a first RNA element to which the RNA-binding protein specifically binds;
- d) a nucleotide sequence encoding a first target polypeptide, wherein the first RNA element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in either the presence or absence of the RNA-binding protein;
- and
- e) optionally one or more additional combinations of an RNA element to which the RNA-binding protein specifically binds and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the RNA element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in either the presence or absence of the RNA-binding protein, wherein translation of each additional target polypeptide is controlled by its closest RNA element in the resulting mRNA.
3 more elements
A polynucleotide comprising:
- a) a nucleotide sequence encoding a ribosomal frameshift stimulator;
- b) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the ribosomal frameshift stimulator to allow expression of the ribosomal frameshift stimulator in the absence of the respective miRNA targeting said MSE sequence;
- c) a first ribosomal frameshift element;
- d) a nucleotide sequence encoding a first target polypeptide, wherein the first ribosomal frameshift element is positioned 5′ to the nucleotide sequence encoding the first target polypeptide, and wherein translation of the first target polypeptide is controlled by the first ribosomal frameshift element and requires either the presence or absence of the ribosomal frameshift stimulator;
- and
- e) optionally one or more additional combinations of a ribosomal frameshift element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the ribosomal frameshift element is positioned 5′ to the nucleotide sequence encoding the target polypeptide, wherein translation of each additional target polypeptide is controlled by its closest 5′ ribosomal frameshift element in the resulting mRNA and independently requires either the presence or absence of the ribosomal frameshift stimulator.
3 more elements
A polynucleotide comprising from 5′ to 3′:
- a) a first promoter;
- b) a 5′ donor splice site;
- c) a nucleotide sequence encoding a ribosomal frameshift stimulator;
- d) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the ribosomal frameshift stimulator to allow expression of the ribosomal frameshift stimulator in the absence of the respective miRNA targeting said MSE sequence;
- e) a 3′ splice acceptor site;
- f) a first ribosomal frameshift element;
- and
- g) a nucleotide sequence encoding a first target polypeptide,
- wherein the first ribosomal frameshift element is positioned 5′ to the nucleotide sequence encoding the first target polypeptide,
- wherein translation of the first target polypeptide is controlled by the first ribosomal frameshift element and requires either the presence or absence of the ribosomal frameshift stimulator, and
- wherein the nucleotide sequence encoding the first target polypeptide is positioned in frame or in-1 translation frame in relation to the first ribosomal frameshift element.
8 more elements
A polynucleotide comprising from 5′ to 3′:
- a) a first promoter;
- b) a nucleotide sequence encoding a ribosomal frameshift stimulator;
- c) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the ribosomal frameshift stimulator to allow expression of the ribosomal frameshift stimulator in the absence of the respective miRNA targeting said MSE sequence,
- d) a second promoter;
- e) a first ribosomal frameshift element, and
- f) a nucleotide sequence encoding a first target polypeptide,
- wherein the first ribosomal frameshift element is positioned 5′ to the nucleotide sequence encoding the first target polypeptide,
- wherein translation of the first target polypeptide is controlled by the first ribosomal frameshift element and requires either the presence or absence of the ribosomal frameshift stimulator, and
- wherein the nucleotide sequence encoding the first target polypeptide is positioned in frame or in-1 translation frame in relation to the first ribosomal frameshift element.
6 more elements
A polynucleotide comprising from 5′ to 3′:
- a) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA,
- b) a nucleotide sequence encoding a ribosomal frameshift stimulator, wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the ribosomal frameshift stimulator to allow expression of the ribosomal frameshift stimulator in the absence of the respective miRNA targeting said MSE sequence;
- c) a bi-directional promoter;
- d) a first ribosomal frameshift element;
- e) a nucleotide sequence encoding a first target polypeptide, wherein the first ribosomal frameshift element is positioned 5′ to the nucleotide sequence encoding the first target polypeptide, wherein translation of the first target polypeptide is controlled by the first ribosomal frameshift element and requires either the presence or absence of the ribosomal frameshift stimulator, and wherein the bi-directional promoter drives expression of the ribosomal frameshift stimulator in one direction and the first target polypeptide in the opposite direction;
- and
- f) optionally one or more additional combinations of a ribosomal frameshift element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the ribosomal frameshift element is positioned 5′ to the nucleotide sequence encoding the target polypeptide, wherein translation of each additional target polypeptide is controlled by its closest 5′ ribosomal frameshift element in the resulting mRNA and independently requires either the presence or absence of the ribosomal frameshift stimulator.
4 more elements
A polynucleotide comprising:
- a) a nucleotide sequence encoding an iron regulatory protein;
- b) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the iron regulatory protein to allow expression of the iron regulatory protein in the absence of the respective miRNA targeting said MSE sequence;
- c) a first iron response element;
- d) a nucleotide sequence encoding a first target polypeptide, wherein the first iron response element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of the iron regulatory protein;
- and
- e) optionally one or more additional combinations of an iron response element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the iron response element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of the iron regulatory protein, wherein translation of each additional target polypeptide is controlled by its closest iron response element in the resulting mRNA.
3 more elements
A polynucleotide comprising from 5′ to 3′:
- a) a first promoter;
- b) a 5′ donor splice site;
- c) a nucleotide sequence encoding an iron regulatory protein;
- d) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the iron regulatory protein to allow expression of the iron regulatory protein in the absence of the respective miRNA targeting said MSE sequence;
- e) a 3′ splice acceptor site;
- f) a first iron response element and a nucleotide sequence encoding a first target polypeptide, wherein the first iron response element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of the iron regulatory protein;
- and
- g) optionally one or more additional combinations of an iron response element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the iron response element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of the iron regulatory protein, wherein translation of each additional target polypeptide is controlled by its closest iron response element in the resulting mRNA.
5 more elements
A polynucleotide comprising from 5′ to 3′:
- a) a first promoter;
- b) a nucleotide sequence encoding an iron regulatory protein;
- c) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the iron regulatory protein to allow expression of the iron regulatory protein in the absence of the respective miRNA targeting said MSE sequence,
- d) a second promoter;
- e) a first iron response element and a nucleotide sequence encoding a first target polypeptide, wherein the first iron response element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of the iron regulatory protein;
- and
- f) optionally one or more additional combinations of an iron response element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the iron response element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of the iron regulatory protein, wherein translation of each additional target polypeptide is controlled by its closest iron response element in the resulting mRNA.
4 more elements
A polynucleotide comprising from 5′ to 3′:
- a) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA;
- b) a nucleotide sequence encoding an iron regulatory protein, wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the iron regulatory protein to allow expression of the iron regulatory protein in the absence of the respective miRNA targeting said MSE sequence;
- c) a bi-directional promoter;
- d) a first iron response element and a nucleotide sequence encoding a first target polypeptide, wherein the first iron response element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of the iron regulatory protein;
- and
- e) optionally one or more additional combinations of an iron response element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the iron response element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of the iron regulatory protein, wherein translation of each additional target polypeptide is controlled by its closest iron response element in the resulting mRNA.
3 more elements
A polynucleotide comprising:
- a) a nucleotide sequence encoding Lin28;
- b) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding Lin28 to allow expression of Lin28 in the absence of the respective miRNA targeting said MSE sequence;
- c) a first pre-let-7 element;
- d) a nucleotide sequence encoding a first target polypeptide, wherein the first pre-let-7 element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of Lin28;
- and
- e) optionally one or more additional combinations of a pre-let-7 element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the pre-let-7 element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of Lin28, wherein translation of each additional target polypeptide is controlled by its closest pre-let-7 element in the resulting mRNA.
3 more elements
A polynucleotide comprising:
- a) a nucleotide sequence encoding an MS2 bacteriophage coat protein;
- b) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the MS2 bacteriophage coat protein to allow expression of the MS2 bacteriophage coat protein in the absence of the respective miRNA targeting said MSE sequence;
- c) a first MS2 responsive RNA element;
- d) a nucleotide sequence encoding a first target polypeptide, wherein the first MS2 responsive RNA element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of the MS2 bacteriophage coat protein;
- and
- e) optionally one or more additional combinations of an MS2 responsive RNA element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the MS2 responsive RNA element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of the MS2 bacteriophage coat protein, wherein translation of each additional target polypeptide is controlled by its closest MS2 responsive RNA element in the resulting mRNA.
3 more elements
A polynucleotide comprising:
- a) a nucleotide sequence encoding a PP7 bacteriophage coat protein;
- b) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the PP7 bacteriophage coat protein to allow expression of the PP7 bacteriophage coat protein in the absence of the respective miRNA targeting said MSE sequence;
- c) a first PP7 responsive RNA element;
- d) a nucleotide sequence encoding a first target polypeptide, wherein the first PP7 responsive RNA element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of the PP7 bacteriophage coat protein;
- and
- e) optionally one or more additional combinations of a PP7 responsive RNA element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the PP7 responsive RNA element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of the PP7 bacteriophage coat protein, wherein translation of each additional target polypeptide is controlled by its closest PP7 responsive RNA element in the resulting mRNA.
3 more elements
A polynucleotide comprising:
- a) a nucleotide sequence encoding a U1 small nuclear ribonucleoprotein A (U1A);
- b) one or more microRNA (miRNA) Silencing Element (MSE) sequences, wherein each of said one or more MSE sequences is targeted by a miRNA, and wherein each of said one or more MSE sequences is positioned with respect to the nucleotide sequence encoding the U1A to allow expression of the ULA in the absence of the respective miRNA targeting said MSE sequence;
- c) a first U1A responsive RNA element;
- d) a nucleotide sequence encoding a first target polypeptide, wherein the first U1A responsive RNA element is positioned with respect to the nucleotide sequence encoding the first target polypeptide to allow translation of the first target polypeptide in the absence of the ULA;
- and
- e) optionally one or more additional combinations of a U1A responsive RNA element and a nucleotide sequence encoding a target polypeptide, wherein within each combination, the ULA responsive RNA element is positioned with respect to the nucleotide sequence encoding the target polypeptide to allow translation of the target polypeptide in the absence of the U1A, wherein translation of each additional target polypeptide is controlled by its closest U1A responsive RNA element in the resulting mRNA.
3 more elements
Dependencies are inferred from the claim text.