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EP-4313168-B1 - MULTI-INPUT MIRNA SENSING WITH CONSTITUTIVE ERNS TO REGULATE MULTI-OUTPUT GENE EXPRESSION IN MAMMALIAN CELLS

EP4313168B1EP 4313168 B1EP4313168 B1EP 4313168B1EP-4313168-B1

Inventors

  • WEISS, RON
  • MISHRA, DEEPAK
  • PERY, EREZ
  • XU, WENLONG
  • WANG, LEI

Dates

Publication Date
20260506
Application Date
20220322

Claims (15)

  1. A sequestron comprising: (i) a sensor circuit comprising a first constitutive promoter operably linked to a nucleic acid sequence encoding: (a) a nucleic acid sequence encoding a repressor; and (b) one or more target sequences for a first set of one or more miRNAs; and (ii) a signal circuit comprising a second constitutive promoter operably linked to a nucleic acid sequence encoding: (a) a repressor recognition sequence that is capable of being cleaved by the repressor of (i)(a); and (b) a nucleic acid sequence encoding an output molecule, wherein the repressor is an endoribonuclease or a ribozyme.
  2. The sequestron of claim 1, (A) wherein the one or more target sequences for the first set of one or more miRNAs of (i)(b) are downstream from the nucleic acid sequence encoding the repressor of (i)(a); and/or (B) wherein the repressor recognition sequence of (ii)(a) is upstream from the nucleic acid sequence encoding the output molecule of (ii)(b); and/or (C) wherein the nucleic acid sequence encoded by the signal circuit of (ii) further comprises one or more target sequences for a second set of one or more miRNAs, wherein optionally the one or more target sequences for the second set of one or more miRNAs are downstream from the nucleic acid sequence encoding the output molecule; and/or (D) wherein the sequestron comprises a plurality of the signal circuit of (ii), wherein optionally each of the plurality of signal circuits comprises a target sequence for a different miRNA of the second set of miRNAs, wherein the target sequence is not present on the other signal circuits; and/or (E) wherein the sequestron comprises a plurality of the sensor circuit of (i), wherein optionally each of the plurality of sensor circuits comprises a target sequence for a different miRNA of the first set of miRNAs, wherein the target sequence is not present on the other sensor circuits.
  3. The sequestron of any one of claims 1-2, wherein the repressor is a CRISPR endoribonuclease, and the repressor recognition sequence is a CRISPR endoribonuclease recognition sequence, wherein optionally the CRISPR endoribonuclease is Cas6, Csy4, CasE, Cse3, LwaCas13a, PspCas13b, RanCas13b, PguCas13b, or RfxCas13d.
  4. The sequestron of any one of claims 1-3, wherein the first and/or second constitutive promoter is an hEF1-alpha promoter.
  5. A composition comprising a plurality of the sequestron of any one of claims 1-4, wherein: (A) the nucleic acid sequence of (i)(a) of each of the plurality of sequestrons encodes a different repressor; (B) the repressor recognition sequence of (ii)(a) of each of the plurality of sequestrons comprises a different nucleic acid sequence; (C) the repressor encoded by the sensor circuit of each of the plurality of sequestrons is capable of binding or cleaving the repressor recognition sequence of the signal circuit of the same sequestron; and (D) the repressor encoded by the sensor circuit of each sequestron is not capable of binding or cleaving the repressor recognition sequence of a different sequestron.
  6. A composition comprising the sequestron of any one of claims 1-4 or a plurality of the sequestron of any one of claims 1-4.
  7. The composition of claim 5 or 6, further comprising a pharmaceutically acceptable excipient.
  8. A cell comprising the sequestron of any one of claims 1-4 or a plurality of the sequestron of any one of claims 1-4.
  9. The cell of claim8, (i) wherein the cell is a prokaryotic cell, wherein optionally the prokaryotic cell is a bacterial cell, or (ii) wherein the cell is a eukaryotic cell, wherein optionally the eukaryotic cell is a plant cell, an insect cell, a mammalian cell, or a human cell.
  10. The cell of any one of claims 8-9, (a) wherein the cell is a diseased cell, wherein optionally the cell is a cancer cell, and/or (b) wherein the cell expresses any one of the first set of miRNAs.
  11. A method comprising maintaining the cell of any one of claims 8-10 in culture.
  12. A method comprising delivering the sequestron of any one of claims 1-4 or the composition of any one of claims 5-7 to a cell, wherein said method is not a method for treatment of the human or animal body by surgery or therapy.
  13. The method of any one of claims 11 or 12, further comprising detecting the output molecule, optionally further comprising classifying the cell.
  14. A sequestron according to any one of claims 1-4 or a composition according to any one of claims 5-7 for use in a method of treating a disease or disorder, the method comprising delivering the sequestron of any one of claims 1-4 or the composition of any one of claims 5-7 to a subject in need thereof, wherein the output molecule is a therapeutic molecule that treats the disease or disorder.
  15. A sequestron according to any one of claims 1-4 or a composition according to any one of claims 5-7 for use in a method of diagnosing a disease or disorder, the method comprising administering an effective amount of the sequestron of any one of claims 1-4 or the composition of any one of claims 5-7 to a subject, optionally further comprising detecting the output molecule.

Description

RELATED APPLICATION This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional Application No. 63/164,282 filed March 22, 2021. BACKGROUND The ability to classify individual cell types in complex biological samples (e.g., in a tissue or during cellular differentiation) can be achieved using the unique expression patterns of microRNAs (miRNAs) in specific cell types or in individual cells. The miRNA profile of a given cell thus provides a signature of its phenotype and/or stage of development. SUMMARY Provided herein are sequestrons as claimed designed to express an output molecule (e.g., a detectable molecule, a transcription factor, or a therapeutic molecule) under desired conditions. Such desired conditions include the presence of one or more miRNAs indicative of a desired cell state, absence of one or more miRNAs indicative of an undesired cell state, and/or a specific miRNA profile indicative of a desired cell state. Sensor circuits of certain sequestrons provided herein encode repressors that are translationally regulated by the presence of a desired miRNA, such that the repressor is not produced in the presence of one or more miRNAs that indicate a desired cell state. Signal circuits of the sequestrons provided herein encode output molecules that are produced only in the absence of a repressor, and optionally the absence of one or more undesired miRNAs. The sequestrons provided herein are regulated translationally, with RNA being transcribed constitutively from both sensor circuits and signal circuits, but translated only in the absence of negative regulation by miRNA and/or repressor activity. Without wishing to be bound by theory, it is believed that translational regulation of both repressor production and output molecule expression allows sequestrons to respond efficiently to changes in the miRNA profile in a cell. Because constitutively transcribed mRNA encoding the output molecule is consistently available in sequestron-expressing cells, output molecule translation can be increased quickly in response to decreased abundance of inhibitory miRNA or repressor. Conversely, constitutively transcribed mRNA encoding the repressor is also consistently available for translation following relaxation of miRNA-mediated downregulation, and so the repressor can be quickly translated to repress output molecule translation in response to decreased abundance of undesired miRNA. Accordingly, the present disclosure provides, in some aspects, a sequestron comprising: (i) a sensor circuit comprising a first constitutive promoter operably linked to a nucleic acid sequence encoding: (a) a nucleic acid sequence encoding a repressor; and(b) one or more target sequences for a first set of one or more miRNAs; and(ii) a signal circuit comprising a second constitutive promoter operably linked to a nucleic acid sequence encoding: (a) a repressor recognition sequence that is capable of being cleaved by the repressor of (i)(a); and(b) a nucleic acid sequence encoding an output molecule, wherein the repressor is an endoribonuclease or a ribozyme. In some embodiments, the one or more target sequences for the first set of one or more miRNAs of (i)(b) are downstream from the nucleic acid sequence encoding the repressor of (i)(a). In some embodiments, the repressor recognition sequence of (ii)(a) is upstream from the nucleic acid sequence encoding the output molecule of (ii)(b). In some embodiments, the nucleic acid sequence encoded by the signal circuit of (ii) further comprises one or more target sequences for a second set of one or more miRNAs. In some embodiments, the one or more target sequences for the second set of one or more miRNAs are downstream from the nucleic acid sequence encoding the output molecule. In some embodiments, the sequestron comprises a plurality of the signal circuit of (ii). In some embodiments, each of the plurality of signal circuits comprises a target sequence for a different miRNA of the second set of miRNAs, wherein the target sequence is not present on the other signal circuits. In some embodiments, the sequestron comprises a plurality of the sensor circuit of (i). In some embodiments, each of the plurality of sensor circuits comprises a target sequence for a different miRNA of the first set of miRNAs, wherein the target sequence is not present on the other sensor circuits. In some embodiments, the repressor is a CRISPR endoribonuclease, and the repressor recognition sequence is a CRISPR endoribonuclease recognition sequence. In some embodiments, the CRISPR endoribonuclease is Cas6, Csy4, CasE, Cse3, LwaCas13a, PspCas13b, RanCas13b, PguCas13b, or RfxCas13d. In some embodiments, the first and/or second constitutive promoter is an hEF1-alpha promoter. In some aspects, the disclosure provides a composition comprising a plurality of the sequestrons provided herein, where: (A) the nucleic acid sequence of (i)(a) of each of the plurality of sequestrons encodes a different repressor;