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EP-3589734-B1 - REGULATION OF GENE EXPRESSION BY APTAMER-MODULATED RNASE P CLEAVAGE

EP3589734B1EP 3589734 B1EP3589734 B1EP 3589734B1EP-3589734-B1

Inventors

  • GUO, Xuecui

Dates

Publication Date
20260506
Application Date
20180302

Claims (14)

  1. A polynucleotide cassette for the regulation of the expression of a target gene comprising an RNase P substrate sequence, comprising a leader sequence and an acceptor stem, linked to a riboswitch, wherein the riboswitch comprises an effector region and an aptamer sequence, wherein the effector region comprises sequence capable of forming a stem structure, wherein the effector region stem comprises some or all of the leader sequence and sequence that is complementary to said some or all of the leader sequence and wherein the leader sequence and the sequence that is complementary to the leader sequence are separated by the aptamer, wherein the RNase P substrate sequence comprises a sequence encoding a precursor tRNA, tRNA, mascRNA, MEN beta tRNA-like structure, viral tRNA-like structure, or RNase P model substrate; the aptamer specifically binds a small molecule ligand; and wherein, when the aptamer is not bound to its ligand, the effector region does not form a stem and provides access to the RNase P substrate sequence, thereby leading to cleavage by RNase P; and wherein when the aptamer is bound to its ligand, the effector region forms the stem, thereby preventing cleavage of the substrate sequence by RNase P.
  2. A polynucleotide cassette for the regulation of the expression of a target gene comprising an RNase P substrate sequence, comprising a leader sequence and an acceptor stem, linked to a riboswitch, wherein the riboswitch comprises an effector region and an aptamer sequence, wherein the effector region comprises sequence capable of forming a stem structure, wherein the effector region stem comprises some of the acceptor stem sequence and sequence that is complementary to said some of the acceptor stem sequence and wherein the acceptor stem sequence and the sequence that is complementary to the acceptor stem sequence are separated by the aptamer, wherein the RNase P substrate sequence comprises a sequence encoding a precursor tRNA, tRNA, mascRNA, MEN beta tRNA-like structure, viral tRNA-like structure, or RNase P model substrate; the aptamer specifically binds a small molecule ligand; and wherein, when the aptamer is not bound to its ligand, the effector region does not form a stem and provides access to the RNase P substrate sequence, thereby leading to cleavage by RNase P; and wherein when the aptamer is bound to its ligand, the effector region forms the stem, thereby preventing cleavage of the substrate sequence by RNase P.
  3. The polynucleotide cassette of claim 1, wherein the effector region sequence and the acceptor stem of the RNase P substrate are separated by 0, 1, 2, 3, or 4 nucleotides.
  4. The polynucleotide cassette of claim 1, wherein the effector region stem further comprises one or more nucleotides of the acceptor stem of the RNase P substrate, and sequence complementary to the one or more nucleotides of the acceptor stem.
  5. The polynucleotide cassette of claim 2, wherein the effector region stem comprises 1, 2, 3, 4, 5, or 6, nucleotides of the acceptor stem of the RNase P substrate and sequence complementary to said nucleotides.
  6. The polynucleotide cassette of any one of claims 1 to 5, wherein the effector region stem is 6 to 12 base pairs.
  7. A method of modulating the expression of a target gene comprising: (a) inserting the polynucleotide cassette of any one of claims 1 to 6 into an untranslated region (UTR) of the target gene, (b) introducing the target gene comprising the polynucleotide cassette into a cell ex vivo or in vitro, and (c) exposing the cell to a small molecule ligand that specifically binds the aptamer in an amount effective to increase expression of the target gene.
  8. The method of claim 7, wherein the polynucleotide cassette (a) is inserted into the 5' untranslated region of the target gene; or (b) is inserted into the 3' untranslated region of the target gene.
  9. The method of claim 7, wherein two or more of the polynucleotide cassettes are inserted into the target gene.
  10. The method of claim 9, wherein the two or more polynucleotide cassettes (a) comprise different aptamers that specifically bind to different small molecule ligands; or (b) comprise the same aptamer.
  11. The method of claim 7, wherein (a) the target gene further comprises a gene regulation cassette that modulates target gene expression by aptamer-mediated regulation of alternative splicing; or (b) the target gene comprising the polynucleotide cassette is incorporated in a vector for the expression of the target gene.
  12. A vector comprising a target gene that contains a polynucleotide cassette according to any one of claims 1-6, wherein the polynucleotide cassette is inserted into an untranslated region (UTR) of the target gene.
  13. The method of claim 11 or the vector of claim 12, wherein the vector is a viral vector, optionally wherein the viral vector is selected from the group consisting of adenoviral vector, adeno-associated virus vector, and lentiviral vector.
  14. The vector of claim 12, wherein the target gene further comprises a gene regulation cassette that modulates target gene expression by aptamer-mediated regulation of alternative splicing.

Description

FIELD OF THE INVENTION The invention provides polynucleotide constructs for the modulation of gene expression by aptamer-mediated ribonuclease cleavage of the RNA and methods of using the constructs to modulate gene expression in response to the presence or absence of a ligand that binds the aptamer. The polynucleotide construct contains a ribonuclease substrate sequence and a riboswitch comprising an effector region and an aptamer such that when the aptamer binds a ligand, target gene expression occurs. BACKGROUND OF THE INVENTION Ribonuclease P ("RNase P") is a ribonucleoprotein complex and functions as an endoribonuclease that removes 5' leader sequences from precursor tRNAs to generate mature tRNAs by catalyzing the hydrolysis of a specific phosphodiester bond. RNase P is found in species across all kingdoms and is made up of one RNA subunit and one (Bacteria) or many (Archaea and Eukarya) proteins. Studies on RNase P substrate recognition have revealed that the enzyme recognizes the structure rather than the primary nucleotide sequence of the substrates, and can cleave a model substrate that contains a structure equivalent to the acceptor stem, the T-stem, the 3' trail sequence and the 5' leader sequence of a precursor tRNA. RNase P cleavage of a target mRNA has been accomplished by expressing an external guide sequence (EGS), which is a sequence designed to form a hybrid complex with the mRNA target sequence that resembles a precursor tRNA. The EGS binds to its mRNA target through base pairing interactions and guides RNase P cleavage of the mRNA target. Use of an EGS sequence to target RNase P cleavage requires expression of the exogenous EGS sequence. Wieland discloses a construct for theophylline-dependent tRNA activity in which a tRNA is connected to a hammerhead ribozyme (HHR) containing a theophylline aptamer (Wieland, Markus (2010) Design of artificial, ribozyme-based genetic switches in bacteria, Doctoral dissertation, University of Konstanz Repository, http://kops.uni-konstanz.de/bitstream/handle/123456789/9677/Markus_Wieland_ Dissertation.pdf?sequence=1 &isAllowed=y). SUMMARY OF THE INVENTION Thepresent invention provides a polynucleotide cassette for the regulation of the expression of a target gene comprising an RNase P substrate sequence, comprising a leader sequence and an acceptor stem, linked to a riboswitch, wherein the riboswitch comprises an effector region and an aptamer sequence, wherein the effector region comprises sequence capable of forming a stem structure, wherein the effector region stem comprises some or all of the leader sequence and sequence that is complementary to said some or all of the leader sequence and wherein the leader sequence and the sequence that is complementary to the leader sequence are separated by the aptamer, wherein the RNase P substrate sequence comprises a sequence encoding a precursor tRNA, tRNA, mascRNA, MEN beta tRNA-like structure, viral tRNA-like structure, or RNase P model substrate; the aptamer specifically binds a small molecule ligand; and wherein, when the aptamer is not bound to its ligand, the effector region does not form a stem and provides access to the RNase P substrate sequence, thereby leading to cleavage by RNase P; and wherein when the aptamer is bound to its ligand, the effector region forms the stem, thereby preventing cleavage of the substrate sequence by RNase P. In one embodiment, the effector region sequence and the acceptor stem of the RNase P substrate are separated by 0, 1, 2, 3, or 4 nucleotides. In one embodiment, the effector region stem further comprises one or more nucleotides of the acceptor stem of the RNase P substrate, and sequence complementary to the one or more nucleotides of the acceptor stem. The present invention also provides a polynucleotide cassette for the regulation of the expression of a target gene comprising an RNase P substrate sequence, comprising a leader sequence and an acceptor stem, linked to a riboswitch, wherein the riboswitch comprises an effector region and an aptamer sequence, wherein the effector region comprises sequence capable of forming a stem structure, wherein the effector region stem comprises some of the acceptor stem sequence and sequence that is complementary to said some of the acceptor stem sequence and wherein the acceptor stem sequence and the sequence that is complementary to the acceptor stem sequence are separated by the aptamer, wherein the RNase P substrate sequence comprises a sequence encoding a precursor tRNA, tRNA, mascRNA, MEN beta tRNA-like structure, viral tRNA-like structure, or RNase P model substrate; the aptamer specifically binds a small molecule ligand; and wherein, when the aptamer is not bound to its ligand, the effector region does not form a stem and provides access to the RNase P substrate sequence, thereby leading to cleavage by RNase P; and wherein when the aptamer is bound to its ligand, the effector region forms the stem, thereby preventing cleavag