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WO2024256870A1 - Ligands et aptamères à petites molécules - Google Patents

Ligands et aptamères à petites molécules Download PDF

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Publication number
WO2024256870A1
WO2024256870A1 PCT/IB2024/000314 IB2024000314W WO2024256870A1 WO 2024256870 A1 WO2024256870 A1 WO 2024256870A1 IB 2024000314 W IB2024000314 W IB 2024000314W WO 2024256870 A1 WO2024256870 A1 WO 2024256870A1
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alkyl
halo
och3
ch2f
chf2
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Kevin G. LIU
Ji-In Kim
Xuecui GUO
Alexandria FORBES
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MeiraGTx UK II Ltd
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MeiraGTx UK II Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Definitions

  • This application includes a Sequence Listing submitted electronically as an xml file named SeqList-162027-54076.xml, created on June 13, 2023, with a size of 9,477 bytes.
  • the Sequence Listing is incorporated herein by reference.
  • the present disclosure relates to small molecules that are modulators of target gene expression where the target gene contains a riboswitch comprising an aptamer as described herein. Also disclosed are riboswitches and polynucleotide cassettes for regulating the expression of a target gene, wherein the polynucleotide cassettes comprise the aptamer as disclosed herein.
  • RNA aptamers are oligonucleotides that bind to a target ligand with high affinity and specificity. These nucleic acid sequences have proven to be of high therapeutic and diagnostic value with recent FDA approval of the first aptamer drug and additional ones in the clinical pipelines. Their high degree of specificity and versatility have established RNA aptamers as one of the pivotal tools of the emerging RNA nanotechnology field in the fight against human diseases including cancer, viral infections and other diseases.
  • aptamers may be utilized as part of a riboswitch that has certain effects in the presence or absence of an aptamer ligand.
  • riboswitches may be used to regulate gene expression in response to the presence or absence of the aptamer ligand.
  • aptamers/ligands derived from prokaryotic sources or generated using in vitro selection methods often fail to demonstrate the functionality required for the expression of therapeutic targets genes in eukaryotic systems.
  • the ligand for the aptamer may be a cellular molecule that would not be appropriate for use in systems for regulating a therapeutic gene product, for example, because presence of the ligand would interfere in the regulation of target gene expression, or because the ligand is not otherwise appropriate for administration to cell or tissue.
  • new aptamer sequences, small molecule ligands, and aptamer/ligand combinations able to regulate gene expression in response to the presence or absence of the small molecule ligand are needed.
  • small molecules of Formula I are modulators of target gene expression where the target gene contains a riboswitch comprising an aptamer, also described herein. Also disclosed are riboswitches and polynucleotide cassettes for regulating the expression of a target gene, wherein the polynucleotide cassettes comprise the aptamers disclosed herein, where the aptamer binds one or more of the small molecules disclosed herein. Further provided are methods of using the small molecules with the aptamers, riboswitches, and/or polynucleotide cassettes for the regulation of target genes, including therapeutic genes.
  • the aptamer that binds to one or more small molecules disclosed herein is encoded by an aptamer encoding sequence disclosed in PCT/IB2022/000762 (WO2023/111686), incorporated herein by reference in its entirety.
  • the aptamer encoding sequence comprises a sequence that is at least 95% identical, or at least 99% identical, to an aptamer encoding sequence disclosed in PCT/IB2022/000762.
  • the aptamer encoding sequence (i) comprises a sequence that is at least 95% identical, or at least 99% identical, to SEQ ID NO: 1; or (ii) comprises SEQ ID NO: 1 (which is referred to herein as the 12C6-1 aptamer).
  • the aptamer sequence disclosed herein further comprises additional sequence at the 5' and 3' ends that is complementary and capable of forming part of the aptamer Pl stem.
  • this Pl stem of the aptamer is, comprises, or overlaps with the effector region of the riboswitches disclosed herein.
  • the aptamer Pl stem comprises a 5' splice site sequence of a 3' intron and sequence complementary thereto.
  • the Pl stem may comprise all of, or the intronic portion of:AG
  • the aptamers disclosed (including referenced) herein bind to one or more of the small molecules of Formula I to XIV, including those listed in Table A.
  • the disclosure provides the RNA aptamer encoded by the aptamer encoding sequences disclosed herein.
  • the disclosure provides nucleic acid sequence encoding a recombinant riboswitch for the regulation of target gene expression in response to a small molecule, wherein the riboswitch comprises an aptamer disclosed herein.
  • the disclosure provides a polynucleotide cassette for regulating the expression of a target gene, wherein the polynucleotide cassette comprises a sequence encoding an aptamer that binds to a small molecule, wherein the aptamer encoding sequence comprises an aptamer encoding sequence disclosed herein.
  • the polynucleotide cassette comprises sequence encoding:
  • an alternatively-spliced exon flanked by a 5' intron and a 3' intron
  • the riboswitch comprises (i) an effector region comprising a stem forming sequence that includes the 5' splice site sequence of the 3' intron (and sequence complementary to the 5' splice site sequence of the 3' intron), and (ii) the aptamer comprises an aptamer sequence disclosed herein; and wherein the alternatively-spliced exon comprises a stop codon that is in-frame with the target gene when the alternatively-spliced exon is spliced into the target gene mRNA.
  • the effector stem is, or comprises, a Pl stem of the aptamers disclosed herein.
  • the effector stem comprises a first sequence that is linked to the 5' end of the aptamers disclosed herein and a second sequence that is linked to the 3' end of the aptamers disclosed herein, wherein the first and second sequence comprise sequence that is complementary and capable of forming a stem.
  • the polynucleotide cassette is located in the protein coding sequence of the target gene. In embodiments, the polynucleotide cassette is located in an untranslated region of the target gene or in an intron of the target gene.
  • the small molecule has the structure according to Formula I: or a pharmaceutically acceptable salt thereof, wherein
  • X 4 is selected from CH, CR d and N;
  • X 6 is selected from CH, CR d and N;
  • X 7 is selected from CH, CR d and N; wherein 0 or 1 of X 4 , X 6 or X 7 is N;
  • A is selected from the group consisting of:
  • X a is selected from N and CH;
  • X b is selected from O, NH, and NCH3; each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH; each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom, or alternatively two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3,
  • R bl is Ci to C3 alkyl or C3 to Ce cycloalkyl, each optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b2 is selected from H, Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b3 is Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2;
  • R b5 is selected from H and Ci to C3 alkyl; m is 1 or 2; n is 1 or 2; p is 1 or 2; r is 1 or 2; 5 is 1 or 2; x is 0, 1, 2 or 3; y is 0, 1, 2 or 3; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, -CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; and w is 0, 1 or 2.
  • the small molecule has a structure according to Formula II-XIV, including, e.g., a structure provided in Table A.
  • the small molecule ligands of the Formula I-XIV and Table A may provide one or more beneficial properties, including improved penetration of the blood-brain barrier, improved partitioning into the tissue of the eye, low toxicity; and good bioavailability.
  • the disclosure provides a vector comprising a polynucleotide cassette, an aptamer encoding sequence/aptamer sequence, and/or riboswitch disclosed herein.
  • the vector is a viral vector or a non-viral vector.
  • the viral vector is an adenoviral vector, an adeno-associated virus vector, and a lentiviral vector.
  • the disclosure provides a cell comprising a vector, a polynucleotide cassette, an aptamer encoding sequence/aptamer sequence, or riboswitch disclosed herein.
  • the disclosure also provides methods for modulating the expression of a target gene using a polynucleotide cassette, an aptamer encoding sequence/aptamer sequence, or riboswitch disclosed herein, by provided to a cell or tissue a small molecule of Formula I- XIV, including, e.g., a small molecule provided in Table A.
  • FIG. 1A schematic of an embodiment of a synthetic riboswitch cassette containing a riboswitch in the context of an intron-alternative exon- aptamer-intron.
  • Fig. IB In the presence of aptamer ligand, aptamer ligand binding facilitates the formation of the effector stem that sequesters the accessibility of the splice site sequence on the 3' end of the alternative exon (e.g., the 5' splice site sequence of the 3' intron), resulting in the exclusion of the stop codon containing alternative exon from the target gene mRNA and target gene expression.
  • Figures 2A and 2B show the exposure levels in the plasma (P), brain (B) and eye (E) for mice dosed with selected compounds at 100 mg/kg PO.
  • Fold induction of luciferase expression, in response to the compounds, in HEK293 cells from a luciferase gene containing the 12C6-1 gene regulation cassette (SEQ ID NO: 4) is also provided.
  • aptamer sequences that bind to, or otherwise respond to the presence of, small molecules of Formula I-XIV.
  • the aptamer sequences provided herein are useful for the regulation of the expression of a target gene in response to the presence or absence of the small molecule ligand.
  • recombinant riboswitches comprising the aptamer sequences disclosed herein, as well as recombinant polynucleotide cassettes for regulating the expression of a target gene, wherein the polynucleotide cassettes comprise sequences encoding the riboswitches disclosed herein.
  • methods of using the aptamers, riboswitches, and/or polynucleotide cassettes for the regulation of target genes, including therapeutic genes, and for the treatment of subjects in need thereof.
  • Aptamers are single-stranded nucleic acid molecules that non-covalently bind to specific ligands with high affinity and specificity by folding into three-dimensional structures.
  • Aptamer ligands include ions, small molecules, proteins, viruses, and cells.
  • Aptamer ligands can be, for example, an organic compound, amino acid, steroid, carbohydrate, or nucleotide.
  • Non-limiting examples of small molecule aptamer ligands include antibiotics, therapeutics, dyes, cofactors, metabolites, molecular markers, neurotransmitters, pollutants, toxins, food adulterants, carcinogens, drugs of abuse. As such, aptamers are useful for the detection of small molecules.
  • Application of small-molecule detection by aptamers include environmental monitoring, food safety, medicine (including diagnostics), microbiology, analytical chemistry, forensic science, agriculture, and basic biology research.
  • aptamer refers to an RNA polynucleotide (or DNA sequence encoding the RNA polynucleotide) that specifically binds to a ligand or class of ligands.
  • ligand refers to a molecule that is specifically bound by an aptamer. Aptamers have binding regions that are capable of forming complexes with an intended target molecule (i.e., the ligand).
  • An aptamer will typically be between about 15 and about 200 nucleotides in length. More commonly, an aptamer will be between about 30 and about 100 nucleotides in length, for example, 70 to 90 nucleotides in length.
  • Aptamers typically comprise multiple paired (P) regions in which the aptamer forms a stem and unpaired regions where the aptamer forms a joining (J) region or a loop (L) region.
  • the paired regions can be numbered sequentially starting at the 5' end (Pl) and numbering each stem sequentially (P2, P3, etc.).
  • the loops (LI, L2, etc.) are numbered based on the adjacent paired region and the joining regions are numbered according to the paired regions that they link.
  • the aptamer that binds to one or more small molecules disclosed herein is encoded by an aptamer encoding sequence disclosed in PCT/IB2022/000762 (WO2023/111686), incorporated herein by reference in its entirety.
  • the aptamer encoding sequence comprises a sequence that is at least 95% identical, or at least 99% identical, to an aptamer encoding sequence disclosed in PCT/IB2022/000762.
  • the aptamer encoding sequence (i) comprises a sequence that is at least 95% identical, or at least 99% identical to SEQ ID NO: 1; or (ii) comprises SEQ ID NO: 1 (which is referred to herein as the 12C6-1 aptamer).
  • the first and the last nucleotide of the aptamer encoding sequence disclosed herein can be any nucleotide or no nucleotide.
  • the first two and the last two nucleotides of the aptamer encoding sequence disclosed herein can be any nucleotide or no nucleotide.
  • additional sequence that is 5' and 3' of the aptamer encoding sequence may be present and form part of the stem forming sequence of the riboswitch.
  • the disclosure provides the aptamer encoded by the aptamer encoding sequences disclosed herein.
  • RNA molecules may be ribonucleic acid (RNA) molecules.
  • the aptamers described herein are part of a longer RNA polynucleotide, including, for example, hnRNA, mRNA, siRNA, or miRNA.
  • an aptamer disclosed herein binds to, or otherwise responds to the presence or addition of, a small molecule (ligand) disclosed herein, including small molecules having the structure according to Formula I to XIV, including the small molecules in Table A.
  • the small molecule has a structure according to Formula I: or a pharmaceutically acceptable salt thereof, wherein
  • X 4 is selected from CH, CR d and N;
  • X 6 is selected from CH, CR d and N;
  • X 7 is selected from CH, CR d and N; wherein 0 or 1 of X 4 , X 6 or X 7 is N;
  • A is selected from the group consisting of:
  • X a is selected from N and CH;
  • X b is selected from O, NH, and NCH3; each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH; each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom, or alternatively two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3,
  • R bl is Ci to C3 alkyl or C3 to Ce cycloalkyl, each optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b2 is selected from H, Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b3 is Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2;
  • R b5 is selected from H and Ci to C3 alkyl; m is 1 or 2; n is 1 or 2; p is 1 or 2; r is 1 or 2; 5 is 1 or 2; x is 0, 1, 2 or 3; y is 0, 1, 2 or 3; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; and w is 0, 1 or 2.
  • x may be selected to be 1, 2 or 3; x may be selected to be 1 or 2; or x may be selected to be 1.
  • R al may be selected to be methyl, fluoro or chloro; or R al may be selected to be methyl.
  • x may be 0.
  • y may be selected to be 0 or 1.
  • R a3 may be selected from halo or methyl; or R a3 may be selected to be methyl.
  • y may be 0.
  • z may be selected to be 1, 2 or 3; z may be selected to be 1 or 2; or z may be selected to be 1.
  • R a2 may be selected to be methyl, fluoro or chloro; or R a2 may be selected to be methyl.
  • z may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • m may be 1.
  • n may be 1.
  • p may be 1.
  • p may be 2.
  • r and 5 may be independently selected from 1 or 2. In embodiments, one of r and 5 are selected to be 1 and the other is 2. In embodiments, both r and 5 are 2. In other embodiments, both r and .s are 1.
  • X a may be N.
  • X 6 may be O. Alternatively, X 6 may be NH. Alternatively, X 6 may be NCH3.
  • R bl may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 67 may be methyl.
  • R b2 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 62 may be H or methyl.
  • R b2 may be H.
  • R b3 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b3 may be - CH3, -CH2CH3, -CH2CH2OH, -CH2CH2F and the like.
  • R /,J may be methyl.
  • each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom; or each R a is methyl.
  • two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; or two R a taken together with the carbon to which they are attached may form a spiro-cyclopropyl group.
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2.
  • R b4 may be -NH2, -NH(CH3), -NH(CH2CH3), - N(CH 3 ) 2 , or N(CH 2 CH 3 )2.
  • R b5 may be Ci to C3 alkyl, such as CH3, CH2CH3, CH2CH2CH3, CH(CH 3 ) 2 .
  • R b5 may be H.
  • the substructure may be selected from the following substructures: wherein R b4 and R b5 are as described above.
  • the small molecule has a structure according to Formula IA or a pharmaceutically acceptable salt thereof, wherein X 4 is selected from CH, CR d and N;
  • X 6 is selected from CH, CR d and N;
  • X 7 is selected from CH, CR d and N; wherein 0 or 1 of X 4 , X 6 or X 7 is N;
  • A is selected from the group consisting of:
  • X a is selected from N and CH;
  • X b is selected from O, NH, and NCH3; each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH; each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom, or alternatively two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3,
  • R bl is Ci to C 3 alkyl
  • R b2 is selected from H and Ci to C3 alkyl
  • R b3 is Ci to C 3 alkyl; m is 1 or 2; n is 1 or 2; p is 1 or 2; x is 0, 1, 2 or 3; y is 0, 1, 2 or 3; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; and w is 0, 1 or 2.
  • X 4 may be CH
  • X 6 may be CH
  • X 7 may be CH
  • x may be selected to be 1, 2 or 3; x may be selected to be 1 or 2; or x may be selected to be 1.
  • R al may be selected to be methyl, fluoro or chloro; or R al may be selected to be methyl.
  • x may be 0.
  • y may be selected to be 0 or 1.
  • R a3 may be selected from halo or methyl; or R a3 may be selected to be methyl.
  • y may be 0.
  • z may be selected to be 1, 2 or 3; z may be selected to be 1 or 2; or z may be selected to be 1.
  • R a2 may be selected to be methyl, fluoro or chloro; or R a2 may be selected to be methyl.
  • z may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • m may be 1.
  • n may be 1.
  • p may be 1.
  • p may be
  • X a may be N.
  • X 6 may be O.
  • R 67 may be methyl.
  • R /,J may be methyl.
  • R 62 may be H or methyl.
  • each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom; or each R a is methyl.
  • two R a attached to the same ring carbon atom form a 3- to 5- membered carbocyclic ring; or two R a taken together with the carbon to which they are attached may form a spiro-cyclopropyl group.
  • the small molecule has a structure according to Formula II: or a pharmaceutically acceptable salt thereof, wherein
  • A is selected from the group consisting of:
  • X a is selected from N and CH;
  • X b is selected from O, NH, and NCH3; each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH; each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom, or alternatively two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3,
  • R bl is Ci to C3 alkyl or C3 to Ce cycloalkyl, each optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b2 is selected from H, Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b3 is Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2;
  • R b5 is selected from H and Ci to C3 alkyl; m is 1 or 2; n is 1 or 2; p is 1 or 2; r is 1 or 2; 5 is 1 or 2; x is 0, 1, 2 or 3; y is 0, 1, 2 or 3; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and v is 0, 1 or 2.
  • x may be selected to be 1, 2 or 3; x may be selected to be 1 or 2; or x may be selected to be 1.
  • R al may be selected to be methyl, fluoro or chloro; or R al may be selected to be methyl.
  • x may be 0.
  • y may be selected to be 0 or 1.
  • R a3 may be selected from halo or methyl; or R a3 may be selected to be methyl.
  • y may be 0.
  • z may be selected to be 1, 2 or 3; z may be selected to be 1 or 2; or z may be selected to be 1.
  • R a2 may be selected to be methyl, fluoro or chloro; or R a2 may be selected to be methyl.
  • z may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • m may be 1.
  • n may be 1.
  • p may be 1.
  • p may be
  • r and 5 may be independently selected from 1 or 2. In embodiments, one of r and 5 are selected to be 1 and the other is 2. In embodiments, both r and 5 are 2. In other embodiments, both r and .s are 1.
  • X a may be N.
  • X 6 may be O.
  • X 6 may be NH.
  • R bl may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 67 may be methyl.
  • R b2 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 62 may be H or methyl.
  • R b2 may be H.
  • R b3 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b3 may be - CH3, -CH2CH3, -CH2CH2OH, -CH2CH2F and the like.
  • R /,J may be methyl.
  • each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom; or each R a is methyl.
  • two R a attached to the same ring carbon atom form a 3- to 5- membered carbocyclic ring; or two R a taken together with the carbon to which they are attached may form a spiro-cyclopropyl group.
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2.
  • R b4 may be -NH2, -NH(CH3), -NH(CH2CH3), - N(CH 3 ) 2 , or N(CH 2 CH 3 )2.
  • R b5 may be Ci to C3 alkyl, such as CH3, CH2CH3, CH2CH2CH3, CH(CH 3 ) 2 .
  • R b5 may be H.
  • the substructure (Vv may be selected to from the following substructures: wherein R b4 and R b5 are as described above.
  • the small molecule has a structure according to Formula III: or a pharmaceutically acceptable salt thereof, wherein
  • A is selected from the group consisting of: each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH; each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom, or alternatively two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -
  • R bl is Ci to C3 alkyl or C3 to Ce cycloalkyl, each optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b2 is selected from H, Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl;
  • R b3 is Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl; m is 1 or 2; n is 1 or 2; p is 1 or 2; x is 0, 1, 2 or 3; y is 0, 1, 2 or 3; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and v is 0, 1 or 2.
  • x may be selected to be 1, 2 or 3; x may be selected to be 1 or 2; or x may be selected to be 1.
  • R al may be selected to be methyl, fluoro or chloro; or R al may be selected to be methyl.
  • x may be 0.
  • y may be selected to be 0 or 1.
  • R a3 may be selected from halo or methyl; or R a3 may be selected to be methyl.
  • y may be 0.
  • z may be selected to be 1, 2 or 3; z may be selected to be 1 or 2; or z may be selected to be 1.
  • R a2 may be selected to be methyl, fluoro or chloro; or R a2 may be selected to be methyl.
  • z may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • m may be 1.
  • n may be 1.
  • p may be 1.
  • p may be
  • X a may be N.
  • X 6 may be O.
  • X 6 may be NH.
  • R bl may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 67 may be methyl.
  • R b2 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 62 may be H or methyl.
  • R b2 may be H.
  • R b3 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b3 may be -CH3, -CH2CH3, -CH2CH2OH, -CH2CH2F and the like.
  • R /,J may be methyl.
  • each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom; or each R a is methyl.
  • two R a attached to the same ring carbon atom form a 3- to 5- membered carbocyclic ring; or two R a taken together with the carbon to which they are attached may form a spiro-cyclopropyl group.
  • the small molecule has a structure according to Formula IV: or a pharmaceutically acceptable salt thereof, wherein
  • X 4 is selected from CH, CR d and N;
  • X 6 is selected from CH, CR d and N;
  • X 7 is selected from CH, CR d and N; wherein 0 or 1 of X 4 , X 6 or X 7 is N;
  • X a is selected from N and CH; each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH;
  • R bl is Ci to C3 alkyl or C3 to Ce cycloalkyl, each optionally substituted with halo, OH, or O- C1-3 alkyl; m is 1 or 2; x is 0, 1, 2 or 3; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; and w is 0, 1 or 2.
  • x may be selected to be 1, 2 or 3; x may be selected to be 1 or 2; or x may be selected to be 1.
  • R al may be selected to be methyl, fluoro or chloro; or R al may be selected to be methyl.
  • x may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • m may be 1.
  • X a may be N.
  • R bl may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 67 may be methyl.
  • the small molecule has a structure according to Formula V: or a pharmaceutically acceptable salt thereof, wherein each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH;
  • R bl is Ci to C3 alkyl or C3 to Ce cycloalkyl, each optionally substituted with halo, OH, or O- C1-3 alkyl; x is 0, 1, 2 or 3; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and v is 0, 1 or 2.
  • x may be selected to be 1, 2 or 3; x may be selected to be 1 or 2; or x may be selected to be 1.
  • R al may be selected to be methyl, fluoro or chloro; or R al may be selected to be methyl.
  • x may be 0.
  • R bl may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R bl may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 67 may be methyl.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl. Alternatively, w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • the small molecule has a structure according to Formula Va: or a pharmaceutically acceptable salt thereof, wherein each R al is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R al attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two R a attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH; x is 0, 1, 2 or 3; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -
  • x may be selected to be 1, 2 or 3; x may be selected to be 1 or 2; or x may be selected to be 1.
  • R al may be selected to be methyl, fluoro or chloro; or R al may be selected to be methyl.
  • x may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • the small molecule has a structure according to Formula VI: or a pharmaceutically acceptable salt thereof, wherein
  • X 4 is selected from CH, CR d and N;
  • X 6 is selected from CH, CR d and N;
  • X 7 is selected from CH, CR d and N; wherein 0 or 1 of X 4 , X 6 or X 7 is N;
  • X a is selected from N and CH; each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom, or alternatively two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, and halo; or additionally or alternatively, two R a2 attached to the same carbon atom form an oxo group;
  • R b2 is selected from H and Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl; n is 1 or 2; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; and w is 0, 1 or 2.
  • z may be selected to be 1, 2 or 3; z may be selected to be 1 or 2; or z may be selected to be 1.
  • R a2 may be selected to be methyl, fluoro or chloro; or R a2 may be selected to be methyl.
  • z may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • n may be 1.
  • X a may be N.
  • R b2 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 62 may be H or methyl.
  • R b2 may be H.
  • each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom; or each R a is methyl.
  • two R a attached to the same ring carbon atom form a 3- to 5- membered carbocyclic ring; or two R a taken together with the carbon to which they are attached may form a spiro-cyclopropyl group.
  • the small molecule has a structure according to Formula
  • each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom, or alternatively two R a attached to the same ring carbon atom form a 3- to 5-membered carbocyclic ring; each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, and halo; or additionally or alternatively, two R a2 attached to the same carbon atom form an oxo group;
  • R b2 is selected from H and Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl; n is 1 or 2; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and v is 0, 1 or 2.
  • z may be selected to be 1, 2 or 3; z may be selected to be 1 or 2; or z may be selected to be 1.
  • R a2 may be selected to be methyl, fluoro or chloro; or R a2 may be selected to be methyl.
  • z may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • n may be 1.
  • R b2 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be C3 to Ce cycloalkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b2 may be -CH3, -CH2CH3, cyclopropyl, -CH2CH2OH, -CH2CH2F and the like.
  • R 62 may be H or methyl.
  • R b2 may be H.
  • each R a is independently selected from Ci to C3 alkyl both of which Ci to C3 alkyl groups are bonded to the same ring carbon atom; or each R a is methyl.
  • two R a attached to the same ring carbon atom form a 3- to 5- membered carbocyclic ring; or two R a taken together with the carbon to which they are attached may form a spiro-cyclopropyl group.
  • the small molecule has a structure according to Formula
  • each R a2 is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, and halo; or additionally or alternatively, two R a2 attached to the same carbon atom form an oxo group; z is 0, 1, or 2; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and
  • z may be selected to be 1 or 2; or z may be selected to be 1.
  • R a2 may be selected to be methyl, fluoro or chloro; or R a2 may be selected to be methyl.
  • z may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • the small molecule has a structure according to Formula IX: or a pharmaceutically acceptable salt thereof, wherein
  • X 4 is selected from CH, CR d and N;
  • X 6 is selected from CH, CR d and N;
  • X 7 is selected from CH, CR d and N; wherein 0 or 1 of X 4 , X 6 or X 7 is N;
  • X b is selected from O, NH, and NCH3;
  • each R a3 is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R a3 attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two Ra attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH;
  • R b3 is Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl; p is 1 or 2; y is 0, 1, 2 or 3; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; and w is 0, 1 or 2.
  • y may be selected to be 0 or 1.
  • R a3 may be selected from halo or methyl; or R a3 may be selected to be methyl.
  • y may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • p may be 2.
  • X b may be O.
  • X b may be NH.
  • R b3 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b3 may be -CH3, - CH2CH3, -CH2CH2OH, -CH2CH2F and the like.
  • R /,J may be methyl.
  • the small molecule has a structure according to Formula X: or a pharmaceutically acceptable salt thereof, wherein each R a3 is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R a3 attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two Ra attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH;
  • R b3 is Ci to C3 alkyl optionally substituted with halo, OH, or O-C1-3 alkyl; p is 1 or 2; y is 0, 1, 2 or 3; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and v is 0, 1 or 2.
  • y may be selected to be 0 or 1.
  • R a3 may be selected from halo or methyl; or R a3 may be selected to be methyl. Alternatively, y may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl. Alternatively, w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • R b3 may be Ci to C3 alkyl, which may be unsubstituted or substituted with halo, OH, or O-C1-3 alkyl.
  • R b3 may be -CH3, - CH2CH3, -CH2CH2OH, -CH2CH2F and the like.
  • R /,J may be methyl.
  • the small molecule has a structure according to Formula
  • each R a3 is independently selected from Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, halo, hydroxyl and amino; or additionally or alternatively, two R a3 attached to the same carbon atom form an oxo group, or a 3- to 5-membered carbocyclic ring; or two Ra attached to different carbon atoms form a 4- to 6- membered carbocyclic ring or a 4- to 6- membered heterocyclic ring having 1 or 2 heteroatoms selected from O and NH; y is 0, 1, 2 or 3; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, -CN, hydroxyl and amino; each R
  • y may be selected to be 0 or 1.
  • R a3 may be selected from halo or methyl; or R a3 may be selected to be methyl.
  • y may be 0.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • the small molecule has a structure according to Formula
  • X 4 is selected from CH, CR d and N;
  • X 6 is selected from CH, CR d and N;
  • X 7 is selected from CH, CR d and N; wherein 0 or 1 of X 4 , X 6 or X 7 is N;
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2;
  • R b5 is selected from H and Ci to C3 alkyl; r is 1 or 2;
  • each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino
  • each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; and w is 0, 1 or 2.
  • r and s may be independently selected from 1 or 2. In embodiments, one of r and s are selected to be 1 and the other is 2. In embodiments, both r and s are 2. In other embodiments, both r and s are 1.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2.
  • R b4 may be -NH2, -NH(CH3), - NH(CH 2 CH 3 ), -N(CH 3 ) 2 , or N(CH 2 CH 3 )2, or R b4 may be -NH 2 , or -NH(CH 3 ).
  • R b5 may be Ci to C3 alkyl, such as CH3, CH2CH3, CH2CH2CH3, CH(CH 3 ) 2 , and particularly CH3.
  • R b5 may be H.
  • the small molecule has a structure according to Formula
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2;
  • R b5 is selected from H and Ci to C3 alkyl; r is 1 or 2;
  • each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino
  • each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and v is 0, 1 or 2.
  • r and s may be independently selected from 1 or 2. In embodiments, one of r and s are selected to be 1 and the other is 2. In embodiments, both r and s are 2. In other embodiments, both r and s are 1.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2.
  • R b4 may be -NH2, -NH(CH3), - NH(CH 2 CH 3 ), -N(CH 3 ) 2 , or N(CH 2 CH 3 )2, or R b4 may be -NH 2 , or -NH(CH 3 ).
  • R b5 may be Ci to C3 alkyl, such as CH3, CH2CH3, CH2CH2CH3, CH(CH 3 ) 2 , and particularly CH3.
  • R b5 may be H.
  • the small molecule has a structure according to Formula XIV: or a pharmaceutically acceptable salt thereof, wherein
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2;
  • R b5 is selected from H and Ci to C3 alkyl; each R c is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; each R d is independently selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, -CHF2, - CN, hydroxyl and amino; alternatively, two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH; w is 0, 1 or 2; and v is 0, 1 or 2.
  • w may be selected from 0 or 1.
  • R c may be selected from halo or methyl; or R c may be selected from F, Cl or methyl.
  • w may be 0.
  • each R d may be selected from halo, Ci to C3 alkyl, -OCH3, -CF3, -CH2F, and -CHF2; or R d may be selected from CH3, CH2F, CHF2, CF3, F, Cl, Br, and OCH3., or R d may be selected from F, Cl, and Br.
  • v is 0.
  • two R d on adjacent ring positions may be taken together to form a 5- or 6-membered aromatic ring having from 0 to 2 heteroatoms selected from O, S, N and NH.
  • R b4 is selected from OH, -NH2, -NH(Ci to C3 alkyl), and -N(Ci to C3 alkyl)2.
  • R b4 may be -NH2, -NH(CH3), - NH(CH 2 CH 3 ), -N(CH 3 ) 2 , or N(CH 2 CH 3 )2, or R b4 may be -NH 2 , or -NH(CH 3 ).
  • R b5 may be Ci to C3 alkyl, such as CH3, CH2CH3, CH2CH2CH3, CH(CH 3 ) 2 .
  • R b5 may be H.
  • the small molecule has a structure according to the compounds in Table A (or a pharmaceutically acceptable salt thereof): Table A
  • the small molecule ligands of the Formula I-XIV and Table A may provide one or more beneficial properties, including improved penetration of the blood-brain barrier, improved partitioning into the tissue of the eye, low toxicity; and good bioavailability.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups and branched-chain alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., Ci- Ce for straight chain, C3-C6 for branched chain). Alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like.
  • substituted alkyl refers to an alkyl group which has from 1 to 4 substituents independently selected from halo, amino, amido, sulfonamido, OH, OCH3, nitro and CN.
  • cycloalkyl refers to saturated, carbocyclic groups having from 3 to 6 carbons in the ring. Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • bicyclyl refers to saturated carbocyclic groups having two joined ring systems, which may be fused or bridged. Bicyclic groups include bicycle[2.1. l]hexane, bicycle[2.2.1]heptane, decalin, and the like.
  • tricyclyl refers to saturated carbocyclic groups having three joined ring systems, which may be fused and/or bridged. Tricyclic groups include adamantane and the like.
  • Carbocyclic refers to ring system that comprise only carbon atoms as ring atoms (i.e., the ring system does not have a heteroatom as a ring atom). Carbocyclic ring systems may be unsaturated, but preferred carbocyclic rings are not aromatic.
  • alkenyl refers to unsaturated aliphatic groups, including straight-chain alkenyl groups and branched-chain alkenyl groups, having at least one carbon-carbon double bond. In preferred embodiments, the alkenyl group has two to six carbon atoms (e.g., C2-C6 alkenyl).
  • halogen designates -F, -Cl, -Br or -I, and preferably -F, -Cl or -Br.
  • alkoxyl refers to an alkyl group, as defined above, that is attached through an oxygen atom.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • amine and “amino” refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
  • R and R' are each independently selected from H and C1-C3 alkyl.
  • amido refers to both unsubstituted and substituted amide substituents, e.g., a moiety that can be represented by the general formula:
  • R and R' are each independently selected from H and C1-C3 alkyl.
  • sulfonamide or “sulfonamido” refer to both unsubstituted and substituted sulfonamide substituents, e.g., a moiety that can be represented by the general formula:
  • R and R' are each independently selected from H and C1-C3 alkyl.
  • aryl as used herein includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine.
  • aryl heterocycles or "heteroaryl” groups.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic. Accordingly, aryl includes 8- to 10-membered fused bicyclic aromatic groups that may include from zero to five heteroatoms, in which one or both rings are aromatic, for example napthylene, quinolone, isoquinoline, benzo[b]thiophene, tetrahydronapthelene, and the like.
  • Each aryl group may be unsubstituted or may be substituted with 1 to 5 substituents selected from halogen, hydroxyl, amino, cyano, amido, sulfonamide, nitro, -SH, Ci-Ce alkyl, C2-C6 alkenyl, C3-C7 cycloalkyl, Ce-Cio bicyclyl, Ci-Ce haloalkyl, Ci-Ce perhaloalkyl, -O-(Ci-Ce alkyl), O-(C3-C?
  • cycloalkyl -O-(Ci-Ce haloalkyl), - O-(Ci-Ce perhaloalkyl), aryl, -O-aryl, -(Ci-Ce alkyl)-aryl, -O-(Ci-Ce alkyl)-aryl, -S-(Ci-Ce alkyl), -S-(C3-C? cycloalkyl), -S-(Ci-Ce haloalkyl), -S-(Ci-Ce perhaloalkyl), -S-aryl, -S-(Ci- Ce alkyl)-aryl, heteroaryl and hetercyclyl.
  • heterocycle of “heterocyclyl” refer to non-aromatic heterocycles having from 1 to 3 ring heteroatoms.
  • Preferred heterocycles are 5- and 6-membered heterocyclic groups having from 1 to 3 heteroatoms selected from the group consisting of O, N and S.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur. [0190] As used herein, the definition of each expression, e.g. alkyl, R 1 , R 2 , etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • the aptamer ligands disclosed herein may exist in particular geometric or stereoisomeric forms well as mixtures thereof.
  • Such geometric or stereoisomeric forms include, but not limited to, cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)- isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group.
  • the compounds according to Formulas I to XIV may contain an acidic or basic functional group, and accordingly may be present in a salt form.
  • the salt form is a pharmaceutically acceptable salt.
  • pharmaceutically-acceptable salts refers to the relatively non-toxic, inorganic and organic acid and base addition salts of the compounds disclosed herein.
  • the compounds according to Formulas I to XIV may contain one or more basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids.
  • These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound disclosed herein in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).
  • the pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from nontoxic organic or inorganic acids.
  • such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
  • the compounds according to Formulas I to XIV may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases.
  • These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Organic amines useful for the formation of base addition salts include ethylamine, di ethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, e.g., Berge et al., supra).
  • the aptamers provided herein bind to, or otherwise respond to the presence of, one or more compounds of Formula I - XIV provided herein, and/or bind to, or otherwise respond to, a metabolite analog or derivative of a compound of Formula I - XIV.
  • the specificity of the binding of an aptamer to its ligand can be defined in terms of the comparative dissociation constants (Ka) of the aptamer for its ligand as compared to the dissociation constant of the aptamer for unrelated molecules.
  • the ligand may be considered to be a molecule that binds to the aptamer with greater affinity than to unrelated material.
  • the Ka for the aptamer with respect to its ligand will be at least about 10- fold less than the Ka for the aptamer with unrelated molecules.
  • the Ka will be at least about 20-fold less, at least about 50-fold less, at least about 100-fold less, and at least about 200-fold less, at least about 500-fold less, at least about 1000-fold less, or at least about 10,000-fold less than the Ka for the aptamer with unrelated molecules.
  • the aptamer and small molecules contemplated by the disclosure are used for the regulation of gene expression.
  • Regulation of the expression of a target gene e.g., a therapeutic transgene
  • a target gene e.g., a therapeutic transgene
  • techniques that enable regulated expression of transgenes in response to the presence of a small molecule can enhance safety and efficacy by allowing for the regulation of the level of target gene expression and its timing.
  • the regulation of gene expression allows a systematic investigation of different experimental conditions.
  • the sequence encoding the aptamer is part of a gene regulation cassette that provides the ability to regulate the expression level of a target gene in response to the presence or absence of a small molecule described herein.
  • the gene regulation cassette further comprises a target gene.
  • target gene refers to a transgene that is expressed in response to the presence or absence of the small molecule ligands disclosed herein due to the small molecule binding to the aptamers disclosed herein.
  • the target gene comprises the coding sequence for a protein (e.g., a therapeutic protein), a miRNA, or a siRNA.
  • the target gene is heterologous to the aptamer used for the regulation of target gene expression, is heterologous to the polynucleotide cassette used for the regulation of target gene and/or is heterologous to a portion of the polynucleotide cassette used for the regulation of target gene.
  • the aptamers described herein can be part of a polynucleotide cassette that encodes the aptamer as part of a riboswitch.
  • the terms “gene regulation cassette”, “regulatory cassette”, or “polynucleotide cassette” are used interchangeably herein.
  • the presence of a small molecule disclosed herein that binds to an aptamer disclosed herein leads to an increase in expression of a target gene as compared to the expression of the target gene in absence of the small molecule.
  • the aptamer constitutes an “on” switch.
  • the expression of the target gene is increased by at least 3-fold, by at least 5-fold, by at least 10-fold, by at least 15-fold, by at least 20-fold, by at least 25-fold, by at least 30-fold, by at least 40-fold, by at least 50-fold, by at least 100-fold, by at least 1000-fold, or by at least 10,000-fold in presence of the small molecule that binds to an aptamer disclosed herein as compared to in absence of the small molecule.
  • the expression of the target gene is increased by between 2-fold and 10-fold, between 5-fold and 10-fold, between 5-fold and 15-fold, between 5-fold and 20- fold, between 5-fold and 25-fold, between 5-fold and 30-fold, between 10-fold and 20-fold, between 10-fold and 30-fold, between 10-fold and 40-fold, between 10-fold and 50-fold, between 10-fold and 100-fold, between 10-fold and 500-fold, between 10-fold and 1,000- fold, between 50-fold and 100-fold, between 50-fold and 500-fold, between 50-fold and 100- fold, between 50-fold and 1,000-fold, between 100-fold and 1,000-fold, or between 100-fold and 10,000-fold in presence of the small molecule that binds to an aptamer disclosed herein as compared to in absence of the small molecule.
  • the presence of a small molecule disclosed herein that binds to an aptamer disclosed herein leads to a decrease in expression of a target gene as compared to the expression of the target gene in the absence of the small molecule.
  • the aptamer constitutes an “off’ switch.
  • the expression of the target gene is decreased by at least 3 -fold, by at least 5-fold, by at least 10-fold, by at least 15-fold, by at least 20-fold, by at least 25-fold, by at least 30-fold, by at least 40-fold, by at least 50-fold, by at least 100-fold, by at least 1000-fold, or by at least 10,000-fold in presence of the small molecule that binds to an aptamer disclosed herein as compared to in absence of the small molecule.
  • the expression of the target gene is decreased by between 2- fold and 10-fold, between 5-fold and 10-fold, between 5-fold and 15-fold, between 5-fold and 20-fold, between 5-fold and 25-fold, between 5-fold and 30-fold, between 10-fold and 20- fold, between 10-fold and 30-fold, between 10-fold and 40-fold, between 10-fold and 50-fold, between 10-fold and 100-fold, between 10-fold and 500-fold, between 10-fold and 1,000- fold, between 50-fold and 100-fold, between 50-fold and 500-fold, between 50-fold and 100- fold, between 50-fold and 1,000-fold, between 100-fold and 1,000-fold, or between 100-fold and 10,000-fold in presence of the small molecule that binds to an aptamer disclosed herein as compared to in absence of the small molecule.
  • the aptamer is part of a riboswitch.
  • Riboswitches are regulatory segments of an RNA polynucleotide that regulate the stability of the RNA polynucleotide and/or regulate the production of a protein from the RNA polynucleotide in response to the presence or absence of aptamer-specific ligand molecules.
  • the riboswitch comprises a sensor region (e.g., the aptamer region) and an effector region that together are responsible for sensing the presence of a ligand (e.g., a small molecule) and causing an effect that leads to increased or decreased expression of the target gene.
  • a ligand e.g., a small molecule
  • the riboswitches described herein are recombinant, utilizing polynucleotides from two or more sources.
  • the sensor and effector regions are joined by a polynucleotide linker.
  • the polynucleotide linker forms a RNA stem or paired region (i.e., a region of the RNA polynucleotide that is double-stranded).
  • the paired region linking the aptamer to the effector region comprises all, or some of an aptamer stem (e.g., for example all, or some of the aptamer Pl stem.).
  • Riboswitches comprising aptamer sequences may be used, for example, to control the formation of rho-independent transcription termination hairpins leading to premature transcription termination. Riboswitches comprising aptamer sequences may also induce structural changes in the RNA, leading to sequestration for the ribosome binding site and inhibition of translation. Alternative riboswitch structures comprising the aptamer sequences disclosed herein can further affect the splicing of mRNA in response to the presence of the small molecule ligand.
  • the aptamers described herein are encoded as part of a gene regulation cassette for the regulation of a target gene by aptamer/ligand mediated alternative splicing of the resulting RNA (e.g., pre-mRNA).
  • the gene regulation cassette comprises a riboswitch comprising a sensor region (e.g., the aptamers described herein) and an effector region that together are responsible for sensing the presence of a small molecule ligand and altering splicing to an alternative exon.
  • Splicing refers to the process by which an intronic sequence is removed from the nascent pre-messenger RNA (pre-mRNA) and the exons are joined together to form the mRNA.
  • Splice sites are junctions between exons and introns, and are defined by different consensus sequences at the 5' and 3' ends of the intron (i.e., the splice donor and splice acceptor sites, respectively). Splicing is carried out by a large multi-component structure called the spliceosome, which is a collection of small nuclear ribonucleoproteins (snRNPs) and a diverse array of auxiliary proteins. By recognizing various cis regulatory sequences, the spliceosome defines exon/intron boundaries, removes intronic sequences, and splices together the exons into a final message (e.g., the mRNA). In the case of alternative splicing, certain exons can be included or excluded to vary the final coding message thereby changing the resulting expressed protein.
  • spliceosome is a collection of small nuclear ribonucleoproteins (snRNPs) and a diverse array of auxiliary proteins.
  • the regulation of target gene expression is achieved by using any of the DNA constructs disclosed in WO2016/126747, which is hereby incorporated by reference in its entirety.
  • the riboswitches and polynucleotide cassettes disclosed in WO2016/126747 comprise an aptamer encoding sequence described herein in place of the aptamer sequence disclosed in WO2016/126747.
  • the polynucleotide cassette comprises (a) a riboswitch and (b) an alternatively-spliced exon, flanked by a 5' intron and a 3' intron, wherein the riboswitch comprises (i) an effector region comprising a stem forming sequence that includes the 5' splice site sequence of the 3' intron (and sequence complementary thereto), and (ii) an aptamer disclosed herein.
  • the effector region is a stem forming region that forms the Pl stem of the aptamer (see, e.g., Figs, la and lb).
  • the effector stem is, or comprises, the Pl stem of the aptamers disclosed herein.
  • the effector stem comprises a first sequence (the 5' effector stem arm) that is linked to the 5' end of the aptamers disclosed herein and a second sequence (the 3' effector stem arm) that is linked to the 3' end of the aptamers disclosed herein, wherein the first or second sequence includes the 5' splice site sequence of the 3' intron and the other includes sequence complementary to the 5' splice site sequence of the 3' intron.
  • the effector region stem comprises the intronic 5' splice site (“5' ss”) sequence of the intron that is immediately 3' of the alternative exon, as well as the sequence complimentary to the 5' ss sequence of the 3' intron.
  • 5' ss intronic 5' splice site
  • 5' splice site sequences are well known in the art. There is some variability among different 5' splice site sequences, and this variability is also well understood in the art. For example, Shapiro and Senapathy (Shapiro MB, Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 1987 Sep 11;15(17):7155-74 or Zhang MQ. Statistical features of human exons and their flanking regions. Hum Mol Genet.
  • Exemplary splice site sequences include, but are not limited to: A G G
  • the effector region forms a stem and thus prevents splicing to the splice donor site at the 3' end of the alternative exon, which prevents incorporation of the alternative exon in the target gene mRNA causing increased target gene expression in the present of the ligand.
  • the effector region is in a context that provides access to the splice donor site at the 3' end of the alternative exon, leading to inclusion of the alternative exon in the target gene mRNA thereby suppressing target gene expression.
  • the polynucleotide cassette is placed in the target gene to regulate expression of the target gene in response to a ligand.
  • the alternatively- spliced exon comprises a stop codon that is in-frame with the target gene when the alternatively-spliced exon is spliced into the target gene mRNA.
  • the gene regulation cassette comprises the sequences of SEQ ID NOs: 2 and 3, wherein SEQ ID NO: 2 is located 5' of an aptamer encoding sequence disclosed herein and SEQ ID NO: 3 is located 3' of the aptamer encoding sequence disclosed herein as shown below (where -X- comprises the aptamer encoding sequence). Lower case letters indicate paired stem sequence linking the aptamer to the remainder of the riboswitch.
  • the alternative exon (underlined in SEQ ID NO: 2, below) is replaced with another alternative exon sequence.
  • the alternative exon is flanked by 5' and 3' intronic sequences.
  • the 5' and 3' intronic sequences that can be used in the gene regulation cassettes disclosed herein can be any sequence that can be spliced out of the target gene creating either the target gene mRNA or the target gene comprising the alternative exon in the mRNA, depending upon the presence or absence of a ligand that binds the aptamer.
  • the 5' and 3' intronic sequences each have the sequences necessary for splicing to occur, i.e., splice donor, splice acceptor and branch point sequences.
  • the 5' and 3' intronic sequences of the gene regulation cassette are derived from one or more naturally occurring introns or portions thereof.
  • the 5' and 3' intronic sequences are derived from a truncated human beta-globin intron 2 (IVS2A), from intron 2 of the human 03 -globin gene, from the SV40 mRNA intron (used in pCMV-LacZ vector from Clontech Laboratories, Inc.), from intron 6 of human triose phosphate isomerase (TPI) gene (Nott Ajit, et al. RNA. 2003, 9:6070617), from an intron from human factor IX (Sumiko Kurachi, et al. J. Bio. Chem.
  • the alternative exon and riboswitch are engineered to be in an endogenous intron of a target gene. That is, the intron (or a substantially similar intronic sequence) naturally occurs at that position of the target gene.
  • the intronic sequence immediately upstream of the alternative exon is referred to as the 5' intron or 5' intronic sequence
  • the intronic sequence immediately downstream of the alternative exon is referred to as the 3' intron or 3' intronic sequence.
  • the endogenous intron is modified to contain a splice acceptor sequence and splice donor sequence flanking the 5' and 3' ends of the alternative exon.
  • the 5' and/or 3' introns are exogenous to the target gene.
  • the splice donor and splice acceptor sites in the alternative splicing gene regulation cassette can be modified to be strengthened or weakened. That is, the splice sites can be modified to be closer to the consensus for a splice donor or acceptor by standard cloning methods, site directed mutagenesis, and the like. Splice sites that are more similar to the splice consensus tend to promote splicing and are thus strengthened. Splice sites that are less similar to the splice consensus tend to hinder splicing and are thus weakened.
  • the consensus for the splice donor of the most common class of introns (U2) is A/C A GIIG T A/G A G T (where II denotes the exon/intron boundary).
  • the consensus for the splice acceptor is C A GIIG (where II denotes the exon/intron boundary).
  • C A GIIG where II denotes the exon/intron boundary.
  • the frequency of particular nucleotides at the splice donor and acceptor sites are described in the art (see, e.g., Zhang, M. Q., Hum Mol Genet. 1988. 7(5):919-932).
  • the strength of 5' and 3' splice sites can be adjusted to modulate splicing of the alternative exon.
  • Additional modifications to 5' and 3' introns present in the alternative splicing gene regulation cassette that can be made to modulate splicing include modifying, deleting, and/or adding intronic splicing enhancer elements, intronic splicing suppressor elements and or splice sites, and/or modifying the branch site sequence.
  • the 5' intron has been modified to contain a stop codon that will be in frame with the target gene.
  • the 5' and 3' intronic sequences can also be modified to remove cryptic slice sites, which can be identified with publicly available software (see, e.g., Kapustin, Y. et al. Nucl. Acids Res. 2011. 1-8).
  • the lengths of the 5' and 3' intronic sequences can be adjusted in order to, for example, meet the size requirements for viral expression constructs.
  • the 5' and/or 3' intronic sequences are about 50 to about 300 nucleotides in length. In one embodiment, the 5' and/or 3' intronic sequences are about 125 to about 240 nucleotides in length.
  • the stem portion of the effector region should be of a sufficient length (and GC content) to substantially prevent alternative splicing of the alternative exon upon ligand binding the aptamer, while also allowing access to the splice site when the ligand is not present in sufficient quantities.
  • the stem portion of the effector region comprises a stem sequence in addition to the 5' splice site sequence of the 3' intron and its complementary sequence of the 5' splice site sequence. In embodiments, this additional stem sequence comprises a sequence from the aptamer stem.
  • the length and sequence of the stem portion can be modified using known techniques in order to identify stems that allow acceptable background expression of the target gene when no ligand is present and acceptable expression levels of the target gene when the ligand is present.
  • the effector region stem of the riboswitch is about 7 to about 20 base pairs in length. In one embodiment, the effector region stem is 8 to 11 base pairs in length.
  • the GC base pair content of the stem can be altered to modify the stability of the stem.
  • the alternative exon that is part of the alternative splicing gene regulation cassettes disclosed herein is a polynucleotide sequence capable of being transcribed to a pre-mRNA and alternatively spliced into the mRNA of the target gene.
  • the alternative exon contains at least one sequence that inhibits translation such that when the alternative exon is included in the target gene mRNA, expression of the target gene from that mRNA is prevented or reduced.
  • the alternative exon contains a stop codon (TGA, TAA, TAG) that is in frame with the target gene when the alternative exon is included in the target gene mRNA by splicing.
  • the alternative exon comprises, in addition to a stop codon, or as an alternative to a stop codon, another sequence that reduces or substantially prevents translation when the alternative exon is incorporated by splicing into the target gene mRNA including, e.g., a microRNA binding site, which leads to degradation of the mRNA.
  • the alternative exon comprises a miRNA binding sequence that results in degradation of the mRNA.
  • the alternative exon encodes a polypeptide sequence which reduces the stability of the protein containing this polypeptide sequence.
  • the alternative exon encodes a polypeptide sequence which directs the protein containing this polypeptide sequence for degradation.
  • the basal or background level of splicing of the alternative exon can be optimized by altering exon splice enhancer (ESE) sequences and exon splice suppressor (ESS) sequences and/or by introducing ESE or ESS sequences into the alternative exon.
  • ESE exon splice enhancer
  • ESS exon splice suppressor
  • Such changes to the sequence of the alternative exon can be accomplished using methods known in the art, including, but not limited to site directed mutagenesis.
  • oligonucleotides of a desired sequence e.g., comprising all or part of the alternative exon
  • Identification of ESS and ESE sequences can be accomplished by methods known in the art, including, for example using ESEfinder 3.0 (Cartegni, L. et al. ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acid Research, 2003, 31(13): 3568-3571) and/or other available resources.
  • ESEfinder 3.0 Cartegni, L. et al. ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acid Research, 2003, 31(13): 3568-3571
  • the alternative exon is a naturally-occurring exon.
  • the alternative exon is derived from all or part of a known exon.
  • “derived” refers to the alternative exon containing sequence that is substantially homologous to a naturally occurring exon, or a portion thereof, but may contain various mutations, such a mutations generated by altering exon splice enhancer (ESE) sequences and exon splice suppressor (ESS) sequences and/or by introducing ESE or ESS sequences into the alternative exon.
  • ESE exon splice enhancer
  • ESS exon splice suppressor
  • homology can be determined by a direct comparison of two polypeptide molecules by aligning their sequences and using readily available computer programs.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single- stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two polynucleotide or two polypeptide sequences are “substantially homologous” to each other when, after optimally aligned with appropriate insertions or deletions, at least about 80%, at least about 85%, at least about 90%, and at least about 95% of the nucleotides or amino acids, respectively, match over a defined length of the molecules, as determined using the methods above.
  • the alternative exon is exogenous to the target gene, although it may be derived from a sequence originating from the organism where the target gene will be expressed.
  • exogenous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • the alternatively-spliced exon is derived from exon 2 of the human dihydrofolate reductase gene (DHFR), mutant human Wilms tumor 1 exon 5, mouse calcium/calmodulin-dependent protein kinase II delta exon 16, or SIRT1 exon 6.
  • the alternatively-spliced exon is, or comprises, the modified DHFR exon 2 in SEQ ID NO: 5.
  • GAATGAATTCAGATATTTCCAGAGAATGAAAAAAAAAATCTTCAGTAGAAG is, or comprises, the modified DHFR exon 2 in SEQ ID NO: 6
  • GAATGAATTCAGATATTTCCAGAGAATGAAAAAAAATCTTCAGTAGAAG GAATGAATTCAGATATTTCCAGAGAATGAAAAAAAATCTTCAGTAGAAG.
  • the aptamer-mediated expression of the target gene is regulated by an aptamer-mediated modulation of small endonucleolytic ribozymes.
  • a ribozyme is an RNA enzyme that catalyzes a chemical reaction.
  • a ribozyme may be any small endonucleolytic ribozyme that will self-cleave in the target cell type including, but not limited to a hammerhead, hairpin, the hepatitis delta virus, the Varkud satellite, twister, twister sister, pistol or hatchet ribozyme.
  • a riboswitch and a gene expression cassette comprising the riboswitch that contains a ribozyme linked to an aptamer disclosed herein.
  • WO2017/136608 which is incorporated in its entirety by reference herein, describes such riboswitches that activate ribozyme self-cleavage in the presence of aptamer ligand (“off’ switch) or riboswitches that inhibit ribozyme self-cleavage in the presence of aptamer (“on” switch).
  • aptamer/ligand binding increases the ribonuclease function of the ribozyme, leading to cleavage of the target gene RNA that contains the polynucleotide cassette, thereby reducing target gene expression.
  • Examples of such an off switch include a polynucleotide cassette for the regulation of the expression of a target gene comprising a riboswitch that comprises a twister ribozyme linked by a stem to an aptamer, wherein the stem linking the twister ribozyme to the aptamer attaches to the ribozyme at the location of the P3 stem of the twister ribozyme and wherein the target gene is linked to the Pl stem of the twister ribozyme (see, e.g. Figs, la, lb, or 3a of WO2017/136608 and the associated text, incorporated herein by reference).
  • aptamer/ligand binding inhibits the ribonuclease function of the ribozyme, decreasing cleavage of the target gene RNA that contains the polynucleotide cassette, thereby increasing target gene expression in the presence of ligand.
  • Examples of an on switch include a riboswitch that comprises a twister ribozyme linked to an aptamer, wherein the aptamer is linked to the 3' or 5' end of the twister ribozyme Pl stem, wherein when the aptamer is linked to the 3' end of the twister ribozyme Pl stem, a portion of the 3' arm of the twister ribozyme Pl stem is alternatively the 5' arm of the aptamer Pl stem, and wherein when the aptamer is linked to the 5' end of the twister ribozyme Pl stem, a portion of the 5' arm of the twister ribozyme Pl stem is alternatively the 3' arm of the aptamer Pl stem (see, e.g., Figs. 6a-6b of WO2017/136608 and the associated text, incorporated herein by reference).
  • the expression of a target gene is regulated by aptamer-modulated polyadenylation.
  • the 3' end of almost all eukaryotic mRNAs comprises a poly(A) tail — a homopolymer of 20 to 250 adenosine residues. Because addition of the poly(A) tail to mRNA protects it from degradation, expression of a gene can be influenced by modulating the polyadenylation the corresponding mRNA.
  • the expression of the target gene is regulated through aptamer- modulated accessibility of polyadenylation sequences as described in and WO2018/156658, which is incorporated in its entirety by reference herein.
  • the riboswitch comprises an effector stem-loop and an aptamer described herein, wherein the effector stem-loop comprises a polyadenylation signal, and wherein the aptamer and effector stem-loop are linked by an alternatively shared stem arm comprising a sequence that is complementary to the unshared arm of the aptamer stem (e.g., the aptamer Pl stem) and to the unshared arm of the effector stem loop (see, e.g., Figs la, lb, 2a, and 5a of WO2018/156658 and the associated text, incorporated herein by reference).
  • the effector stem-loop is positioned 3' of the aptamer such that the alternatively shared stem arm comprises all or a portion of the 3' aptamer stem arm and all or a portion of the 5' arm of the effector stem. In one embodiment, the effector stem-loop is positioned 5' of the aptamer such that the alternatively shared stem arm comprises all or a portion of the 5' aptamer stem arm and all or a portion of the 3' arm of the effector stem.
  • the polyadenylation signal comprises AATAA or ATTAA. In one embodiment, the polyadenylation signal is AATAAA or ATTAAA. In embodiments, the polyadenylation signal is a downstream element (DSE).
  • the polyadenylation signal is an upstream sequence element (USE).
  • the polynucleotide cassette comprises two riboswitches, wherein the effector stem loop of the first riboswitch comprises all or part of the polyadenylation signal AATAAA or ATTAAA and the effector stem loop of the second riboswitch comprises all or part of the downstream element (DSE).
  • the two riboswitches each comprise aptamers that bind the same ligand.
  • the two riboswitches comprise different aptamers that bind different ligands.
  • the riboswitch comprises a sensing region (e.g., an aptamer described herein) and an effector region comprising a binding site for the small nuclear ribonucleoprotein (snRNP) Ul, which is part of the spliceosome.
  • snRNP small nuclear ribonucleoprotein
  • WO2017/136591 describes riboswitches wherein the effector region comprises a Ul snRNP binding site (and sequence complementary thereto), and is incorporated herein by reference in its entirety.
  • the effector region forms a stem and sequesters the Ul snRNP binding site from binding a Ul snRNP.
  • the effector region is in a context that provides access to the Ul snRNP binding site, allowing Ul snRNP to bind the mRNA and inhibit polyadenylation leading to degradation of the message.
  • the Ul snRNP binding site can be any polynucleotide sequence that is capable of binding the Ul snRNP, thereby recruiting the Ul snRNP to the 3' UTR of a target gene and suppressing polyadenylation of the target gene message.
  • the Ul snRNP binding site is CAGGTAAGTA, (CAGGUAAGUA, when in the mRNA).
  • the Ul snRNP binding site is a variation of this consensus sequence, including for example sequences that are shorter or have one or more nucleotides changed from the consensus sequence.
  • the Ul snRNP binding site contains the sequence CAGGTAAG.
  • the binding site is encoded by the sequence selected from CAGGTAAGTA, CAGGTAAGT, and CAGGTAAG.
  • the Ul snRNP binding site can be any 5' splice site sequence from a gene, e.g., the 5' splice site from human DHFR exon 2.
  • the expression of the target gene is regulated through aptamer- modulated ribonuclease cleavage.
  • Ribonucleases RNases
  • RNases recognize and cleave specific ribonuclease substrate sequences.
  • recombinant DNA constructs that, when incorporated into the DNA of a target gene, provide the ability to regulate expression of the target gene by aptamer/ligand mediated ribonuclease cleavage of the resulting RNA.
  • the aptamer encoding sequence described herein is part of a construct that contains or encodes a ribonuclease substrate sequence and a riboswitch comprising an effector region and the aptamer such that when the aptamer binds a ligand, target gene expression occurs (as described in W02018/161053, which is incorporated in its entirety by reference herein).
  • an RNase P substrate sequence is linked to a riboswitch wherein the riboswitch comprises an effector region and an aptamer described herein, wherein the effector region comprises a sequence complimentary to a portion of the RNase P substrate sequence. Binding of a suitable ligand to the aptamer induces structural changes in the aptamer and effector region, altering the accessibility of the ribonuclease substrate sequence for cleavage by the ribonuclease.
  • the aptamer sequence is located 5' to the RNase P substrate sequence and the effector region comprises all or part of the leader sequence and all or part of the 5' acceptor stem sequence of the RNase P substrate sequence. See, e.g., Figs, la, lb, and 3b of W02018/161053 and the associated text, incorporated herein by reference.
  • the acceptor stem of the RNase P substrate and the riboswitch effector region are separated by 0, 1, 2, 3, or 4 nucleotides.
  • the effector region stem includes, in addition to leader sequence (and its complement), 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 aptamer sequence of the polynucleotide cassette is located 3' to the RNase P substrate sequence and the effector region comprises sequence complimentary to the all or part of the 3' acceptor stem of the RNase P substrate sequence. See, e.g., Fig. 3a of W02018/161053 and the associated text, incorporated herein by reference.
  • the effector region sequence complimentary to the 3' acceptor stem of the RNase P substrate is 1 to 7 nucleotides.
  • the effector region stem includes 1 to 7 nucleotides of the acceptor stem and includes sequence that is complementary to this 1 to 7 nucleotides of the acceptor stem.
  • the riboswitch is located 3' of the RNase P substrate so the effector region stem and the acceptor stem of the RNase P substrate do not overlap. In embodiments, the effector region and the acceptor stem of the RNase P substrate are immediately adjacent (i.e., not overlapping). In other embodiments, the effector region and the acceptor stem of the RNase P substrate are separated by 1, 2, 3, 4, 5 or more nucleotides.
  • the aptamers and gene regulation cassettes disclosed herein can be used to regulate the expression of any target gene that can be expressed in a target cell, tissue or organism.
  • target gene refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and translated and/or expressed under appropriate conditions.
  • the target gene is endogenous to the target cell and the gene regulation cassette is positioned into the target gene (for example into an existing untranslated region or intron of the endogenous target gene).
  • target gene is a polynucleotide encoding a therapeutic polypeptide.
  • the target gene is exogenous to the cell in which the recombinant DNA construct is to be transcribed.
  • target gene is endogenous to the cell in which the recombinant DNA construct is to be transcribed.
  • the target gene may be a gene encoding a protein, or a sequence encoding a non-protein coding RNA.
  • the target gene may be, for example, a gene encoding a structural protein, an enzyme, a cell signaling protein, a mitochondrial protein, a zinc finger protein, a hormone, a transport protein, a growth factor, a cytokine, an intracellular protein, an extracellular protein, a transmembrane protein, a cytoplasmic protein, a nuclear protein, a receptor molecule, an RNA binding protein, a DNA binding protein, a transcription factor, translational machinery, a channel protein, a motor protein, a cell adhesion molecule, a mitochondrial protein, a metabolic enzyme, a kinase, a phosphatase, exchange factors, a chaperone protein, and modulators of any of these.
  • the target gene encodes erythropoietin (Epo), human growth hormone (hGH), transcription activator-like effector nucleases (TALEN), human insulin, CRISPR associated protein 9 (cas9), or an immunoglobulin (or portion thereof), including, e.g., a therapeutic antibody.
  • Epo erythropoietin
  • hGH human growth hormone
  • TALEN transcription activator-like effector nucleases
  • human insulin e.g., human insulin
  • cas9 CRISPR associated protein 9
  • an immunoglobulin or portion thereof
  • the target gene is Cas9 or CasRx and the expression construct further comprises a sequence encoding a guide RNA (gRNA), for example a gRNA targeting PCSK9, which can be used to regulate expression of the gRNA target.
  • gRNA guide RNA
  • the target gene is PTH.
  • the target gene is insulin (e.g., comprising sequence comprising the A chain, B chain and C peptide) for use in regulating insulin levels in response to a small molecule for treating diabetes.
  • the target gene is a therapeutic antibody including an anti-PCSK9 antibody, anti-VEGFR2 antibody (e.g., for ophthalmological applications), anti -amyloid APp3-42 antibody, anti-IL-17 antibody, anti-PDl antibody, and anti-HER2 antibody.
  • the heavy and light chains can be expressed from a single message separated by a protein cleave site (furan, etc.) or peptide self-leaving site (e.g., 2A peptide such as T2A or P2A).
  • the target gene encodes an antibody against the SARS-CoV-2 viral proteins or antigens (such as the spike protein)(e.g., casirivimab and/or imdevimab (Regeneron), or bamlanivimab and/or etesevimab (Eli Lilly)).
  • the target gene encodes all or a portion of a SARS-CoV-2 spike protein, where induction of expression produces mRNA and thus functions like an inducible mRNA vaccine (mRNA-1273, Modema or Comirnaty, Pfizer-BioNTech).
  • the aptamers and gene regulation cassettes disclosed herein are used to regulate the expression of a target gene in eukaryotic cells for example, mammalian cells and more particularly human cells.
  • the aptamers and gene regulation cassettes disclosed herein are used to regulate the expression of a target gene in the eye (including cornea and retina), central nervous system (including the brain), liver, kidney, pancreas, heart, airway, muscle, skin, lung, cartilage, testes, arteries, thymus, bone marrow, or in tumors.
  • recombinant vectors and their use for the introduction of a polynucleotide comprising a target gene and a gene regulation cassette, wherein the gene regulation cassette comprises an aptamer disclosed herein.
  • the recombinant DNA constructs include additional DNA elements including DNA segments that provide for the replication of the DNA in a host cell and expression of the target gene in target cells at appropriate levels.
  • expression control sequences promoters, enhancers, and the like are selected based on their ability to promote expression of the target gene in the target cell.
  • Vector means a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini-circle or virus (including virus derived sequences) that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.
  • the recombinant vector is a viral vector or a combination of multiple viral vectors.
  • Viral vectors for the expression of a target gene in a target cell, tissue, or organism are known in the art and include adenoviral (AV) vectors, adeno-associated virus (AAV) vectors, retroviral and lentiviral vectors, and Herpes simplex type 1 (HSV1) vectors.
  • AV adenoviral
  • AAV adeno-associated virus
  • HSV1 Herpes simplex type 1
  • Adenoviral vectors include, for example, those based on human adenovirus type 2 and human adenovirus type 5 that have been made replication defective through deletions in the El and E3 regions.
  • the transcriptional cassette can be inserted into the El region, yielding a recombinant ElZE3-deleted AV vector.
  • Adenoviral vectors also include helperdependent high-capacity adenoviral vectors (also known as high-capacity, “gutless” or “gutted” vectors), which do not contain viral coding sequences. These vectors, contain the cis-acting elements needed for viral DNA replication and packaging, mainly the inverted terminal repeat sequences (ITR) and the packaging signal (CY). These helper-dependent AV vector genomes have the potential to carry from a few hundred base pairs up to approximately 36 kb of foreign DNA.
  • ITR inverted terminal repeat sequences
  • CY packaging signal
  • Recombinant adeno-associated virus “rAAV” vectors include any vector derived from any adeno-associated virus serotype, including, without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7 and AAV-8, AAV-9, AAV-10, and the like.
  • rAAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the Rep and/or Cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are retained for the rescue, replication, packaging and potential chromosomal integration of the AAV genome.
  • the ITRs need not be the wild-type nucleotide sequences, and may be altered (e.g., by the insertion, deletion or substitution of nucleotides) so long as the sequences provide for functional rescue, replication and packaging.
  • Lentiviral-based systems can transduce nondividing as well as dividing cells making them useful for applications targeting, for examples, the nondividing cells of the CNS.
  • Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome providing the potential for very long-term gene expression.
  • Polynucleotides including plasmids, YACs, minichromosomes and minicircles, carrying the target gene containing the gene regulation cassette can also be introduced into a cell or organism by nonviral vector systems using, for example, cationic lipids, polymers, or both as carriers.
  • Conjugated poly-L-lysine (PLL) polymer and polyethylenimine (PEI) polymer systems can also be used to deliver the vector to cells.
  • Other methods for delivering the vector to cells includes hydrodynamic injection and electroporation and use of ultrasound, both for cell culture and for organisms.
  • this disclosure provides a method of modulating the expression of a target gene (e.g., a therapeutic gene) comprising (a) inserting the polynucleotide cassette comprising an aptamer disclosed herein into the target gene, (b) introducing the target gene comprising the polynucleotide cassette into a cell, and (c) exposing the cell to a small molecule ligand that specifically binds the aptamer in an amount effective to induce expression of the target gene.
  • expression of the target gene in target cells confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic outcome.
  • a gene regulation cassette comprising an aptamer disclosed herein is inserted into the protein coding sequence of the target gene (rather than in the 5' or 3' untranslated regions).
  • a single gene regulation cassette comprising an aptamer disclosed herein is inserted into the target gene.
  • 2, 3, 4, or more gene regulation cassettes are inserted in the target gene, wherein one or more gene regulation cassettes comprise an aptamer disclosed herein.
  • two gene regulation cassettes are inserted into the target gene, wherein one or both gene regulation cassettes comprise an aptamer disclosed herein.
  • multiple gene regulation cassettes When multiple gene regulation cassettes are inserted into a target gene, they each can contain the same aptamer such that a single ligand can be used to modulate target gene expression. In other embodiments, multiple gene regulation cassettes are inserted into a target gene, each can contain a different aptamer so that exposure to multiple different small molecule ligands modulates target gene expression.
  • a method of regulating the level of a therapeutic protein delivered by gene therapy is provided.
  • the therapeutic gene sequence containing a regulatory cassette comprising an aptamer disclosed herein is delivered to the target cells in the body, e.g., by a vector.
  • the cell specificity of the target gene expression may be controlled by a promoter and/or other elements within the vector and/or by the capsid of the viral vector. Delivery of the vector construct containing the target gene, and the transfection of the target tissues resulting in stable transfection of the regulated target gene, is the first step in producing the therapeutic protein.
  • the target gene is not expressed at significant levels, i.e., it is in the “off state” in the absence of the specific ligand that binds to the aptamer contained within in the regulatory cassette riboswitch. Only when the aptamer specific ligand is administered is the target gene expression activated.
  • the delivery of the vector construct containing the target gene and the delivery of the activating ligand generally are separated in time.
  • the delivery of the activating ligand will control when the target gene is expressed, as well as the level of protein expression.
  • the ligand may be delivered by a number of routes including, but not limited to, intravitreal, intraocular, inhalation, subcutaneous, intramuscular, intradermal, intralesion, topical, intraperitoneal, intravenous (IV), intra-arterial, perivascular, intracerebral, intracerebroventricular, oral, sublingual, sublabial, buccal, nasal, intrathoracic, intracardiac, intrathecal, epidural, intraosseous, or intraarticular.
  • the timing of delivery of the ligand will depend on the requirement for activation of the target gene. For example, if the therapeutic protein encoded by the target gene is required constantly, an oral small molecule ligand may be delivered daily, or multiple times a day, to ensure continual activation of the target gene, and thus continual expression of the therapeutic protein. If the target gene has a long acting effect, the inducing ligand may be dosed less frequently, for example, once a week, every other week, once a month.
  • This aptamers described herein in the context of a gene regulation cassette comprising a riboswitch allow the expression of a therapeutic transgene to be controlled temporally, in a manner determined by the temporal dosing of the ligand specific to the aptamer.
  • the expression of the therapeutic transgene only on ligand administration increases the safety of a gene therapy treatment by allowing the target gene to be off in the absence of the ligand.
  • each therapeutic gene containing a regulatory cassette will have a specific aptamer within the cassette that will be activated by a specific small molecule. This means that each therapeutic gene can be activated only by the ligand specific to the aptamer housed within it. In these embodiments, each ligand will only activate one therapeutic gene. This allows for the possibility that several different “target genes” may be delivered to one individual and each will be activated on delivery of the specific ligand for the aptamer contained within the regulatory cassette housed in each target gene.
  • the aptamers disclosed herein in the context of a riboswitch allow any therapeutic protein whose gene can be delivered to the body (such as erythropoietin (EPO) or a therapeutic antibody) to be produced by the body when the activating ligand is delivered.
  • This method of therapeutic protein delivery may replace the manufacture of such therapeutic proteins outside of the body which are then injected or infused, e.g., antibodies used in cancer or to block inflammatory or autoimmune disease.
  • the body containing the regulated target gene becomes the biologies manufacturing factory, which is switched on when the genespecific ligand is administered.
  • the target protein may be a nuclease that can target and edit a particular DNA sequence.
  • nucleases include CasRx, Cas9, zinc finger containing nucleases, or TALENs.
  • the nuclease protein may be required for only a short period of time that is sufficient to edit the target endogenous genes. However, if an unregulated nuclease gene is delivered to the body, this protein may be present for the rest of the life of the cell. In the case of nucleases, there is an increasing risk of off-target editing the longer the nuclease is present. Regulation of expression of such proteins has a significant safety advantage.
  • vector containing the nuclease target gene containing a regulatory cassette could be delivered to the appropriate cells in the body.
  • the target gene is in the “off’ state in the absence of the cassette-specific ligand, so no nuclease is produced. Only when the activating ligand is administered, is the nuclease produced. When sufficient time has elapsed allowing sufficient editing to occur, the ligand will be withdrawn and not administered again. Thus the nuclease gene is thereafter in the “off’ state and no further nuclease is produced and editing stops.
  • This approach may be used to correct genetic conditions, including a number of inherited retinopathies such as LCA10 caused by mutations in CEP290 and Stargardts disease caused by mutations in ABCA4.
  • a regulated target gene encoding a therapeutic protein which is activated only on specific ligand administration may be used to regulate therapeutic genes to treat many different types of diseases, e.g., cancer with therapeutic antibodies, immune disorders with immune modulatory proteins or antibodies, metabolic diseases, rare diseases such as PNH with anti-C5 antibodies or antibody fragments as the regulated gene, or ocular angiogenesis with therapeutic antibodies, and dry AMD with immune modulatory proteins.
  • diseases e.g., cancer with therapeutic antibodies, immune disorders with immune modulatory proteins or antibodies, metabolic diseases, rare diseases such as PNH with anti-C5 antibodies or antibody fragments as the regulated gene, or ocular angiogenesis with therapeutic antibodies, and dry AMD with immune modulatory proteins.
  • a wide variety of specific target genes allowing for the treatment of a wide variety of specific diseases and conditions, are suitable for use as a target gene whose expression can be regulated using an aptamer/ligand described herein.
  • insulin or an insulin analog may be used as the target gene to treat type I diabetes, type II diabetes, or metabolic syndrome
  • human growth hormone may be used as the target gene to treat children with growth disorders or growth hormone- deficient adults
  • erythropoietin preferably human erythropoietin
  • Additional target genes compatibles with the aptamers and gene expression cassettes disclosed herein include, but are not limited to, cyclic nucleotide-gated cation channel alpha-3 (CNGA3) and cyclic nucleotide-gated cation channel beta-3 (CNGB3) for the treatment of achromatopsia, retinoid isomerohydrolase (RPE65) for the treatment of retinitis pigmentosa or Leber’s congential amaurosis, X-linked retinitis pigmentosa GTPase regulator (RPGR) for the treatment of X-linked retinitis pigmentosa, glutamic acid decarboxylase (GAD) including for the treatment of Parkinson's disease, regulator of nonsense transcripts 1 (UPF1) for the treatment amyotrophic lateral sclerosis, and aquaporin for the treatment of radiation-induced xerostomia and Sjogren’s syndrome.
  • CNGA3 cyclic nucleotide-gate
  • Additional target genes include ArchT (archaerhodopsin from Halorubrum strain TP009), Jaws (cruxhalorhodopsin derived from Haloarcula (Halobacterium) salinarum (strain Shark)), iClC2 (a variant of a C1C2 chimaera between channel rhodopsins ChRl and ChR2 from Chlamydomonas reinhardlii). or Rgs9-anchor protein (R9AP), a critical component of GTPase complex that mediates the deactivation of phototransduction cascade.
  • ArchT archaerhodopsin from Halorubrum strain TP009
  • Jaws crudexhalorhodopsin derived from Haloarcula (Halobacterium) salinarum (strain Shark)
  • iClC2 a variant of a C1C2 chimaera between channel rhodopsins ChRl and ChR2
  • the expression constructs comprising an aptamer disclosed herein may be especially suitable for treating diseases caused by single gene defects such as cystic fibrosis, hemophilia, muscular dystrophy, thalassemia, or sickle cell anemia.
  • diseases caused by single gene defects such as cystic fibrosis, hemophilia, muscular dystrophy, thalassemia, or sickle cell anemia.
  • human P-, y-, 5-, or ( ⁇ -globin may be used as the target gene to treat P-thalassemia or sickle cell anemia
  • human Factor VIII or Factor IX may be used as the target gene to treat hemophilia A or hemophilia B.
  • the expression constructs/small molecules disclosed herein may be used to treat, prevent, or lessen the severity of a viral disease.
  • the disclosure provides a method for treating, preventing, or lessening the severity of COVID-19 by expressing antibodies against the SARS-CoV-2 viral proteins or antigens (e.g., spike protein) in response to administration of a small molecule ligand.
  • the disclosure provides a method for preventing (or lessening the severity of) infection by SARS- CoV-2 by expressing the spike protein (or multiple serotype spike proteins) or portions thereof, using the gene regulation cassettes described herein and administering ligand.
  • the target gene is an antibody against the SARS-CoV-2 viral proteins or antigens (such as the spike protein).
  • the target gene encodes all or a portion of one or more SARS-CoV-2 spike proteins, where induction of expression produces mRNA and thus functions like an inducible mRNA vaccine.
  • the expression construct is part of an AAV viral genome and the AAV vector comprising the expression construct is administered to, e.g., the muscle of a subject followed by administration of the ligand.
  • the disclosure provides a method for restoring hemocrit and a method of treating anemia by expression of Epo from a gene regulation construct described herein, wherein a vector comprising an Epo gene regulation construct is administered to the subject in need thereof followed by administration of a small molecule ligand described herein.
  • the anemia is due to chronic kidney disease in the subject.
  • the disclosure provides a method for restoring hemocrit and a method of treating chronic kidney disease by expression of Epo from a gene regulation construct described herein, wherein a vector comprising an Epo gene regulation construct is administered to the subject in need thereof followed by administration of a small molecule ligand described herein.
  • compositions suitable for administration to a patient are generally combined with one or more pharmaceutically acceptable carriers to form pharmaceutical compositions suitable for administration to a patient.
  • Pharmaceutically acceptable carriers include solvents, binders, diluents, disintegrants, lubricants, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, generally used in the pharmaceutical arts.
  • Pharmaceutical compositions may be in the form of tablets, pills, capsules, troches, eye drops, and the like, and are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, intranasal, subcutaneous, oral, inhalation, transdermal (topical), transmucosal, and ocular.
  • compositions comprising compounds of I-XVI are administered to a patient in a dosing schedule such that an amount of the compound sufficient to desirably regulate the target gene is delivered to the patient.
  • the dosage form is a tablet, pill, or the like
  • the pharmaceutical composition comprises from 0.1 mg to 10 g of the compound; from 0.5 mg to 5 g of the compound; from 1 mg to 1 g of the compound; from 2 mg to 750 mg of the compound; from 5 mg to 500 mg of the compound; from 10 mg to 250 mg of the compound; or from 150 mg to 300 mg of the compound.
  • the pharmaceutical compositions may be dosed once per day or multiple times per day (e.g., 2, 3, 4, 5, or more times per day). Alternatively, pharmaceutical compositions may be dosed less often than once per day, e.g., once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or once a month or once every few months. In some embodiments, the pharmaceutical compositions may be administered to a patient only a small number of times, e.g., once, twice, three times, etc.
  • a method of treating a patient in need of increased expression of a therapeutic protein encoded by a target gene comprising administering to the patient a pharmaceutical composition comprising a ligand, which an aptamer disclosed herein binds to or otherwise responds to, wherein the patient previously had been administered a recombinant DNA comprising the target gene, and where the target gene contains a gene regulation cassette disclosed herein that provides the ability to regulate expression of the target gene by the ligand of the aptamer.
  • a pharmaceutical composition comprising a ligand, which an aptamer disclosed herein binds to or otherwise responds to, for use in a method of treating a patient in need of increased expression of a therapeutic protein encoded by a target gene, wherein the patient previously had been administered a recombinant DNA comprising the target gene, and where the target gene contains a gene regulation cassette disclosed herein that provides the ability to regulate expression of the target gene by the ligand of the aptamer.
  • aptamers can be incorporated in biosensors, microfluidic devices and other detection platforms.
  • the aptamer is conjugated to a polyalkylene glycol moiety, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), polyoxyethylated glycerol (POG) and other polyoxyethylated polyols, polyvinyl alcohol (PVA) and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • POG polyoxyethylated glycerol
  • PVA polyvinyl alcohol
  • the aptamer is conjugated to a detectable moiety, including, but not limited to, fluorescent moieties or labels, imaging agents, radioisotopic moieties, radiopaque moieties, and the like, e.g. detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, nanoparticles (including, but not limited to gold, magnetic, and superparamagnetic nanoparticles), quantum dots, radiolabels.
  • exemplary fluorophores include fluorescent dyes (e.g. fluorescein, rhodamine, and the like) and other luminescent molecules (e.g. luminal).
  • a fluorophore may be environmentally-sensitive such that its fluorescence changes if it is located close to one or more residues in the modified protein that undergo structural changes upon binding a substrate (e.g. dansyl probes).
  • exemplary radiolabels include small molecules containing atoms with one or more low sensitivity nuclei ( 13 C, 15 N, 2 H, 125 I, 123 I, "Tc, 43 K, 52 Fe, 67 Ga, 68 Ga, n i In and the like). Other useful moieties are known in the art.
  • the aptamer is conjugated to a therapeutic moiety, including, but not limited to, an anti-inflammatory agent, anti-cancer agent, anti- neurodegenerative agent, anti-infective agent, or generally a therapeutic agent.
  • the method comprises the steps of:
  • step (iv) optionally repeating steps (ii) and (iii) on an aptamer identified in step (iii) rather than an aptamer selected in step (i).
  • the parent aptamer sequence may be a TPP aptamer, including known TPP aptamer sequence or may be a putative TPP aptamer identified by searching for homologous sequences in available databases.
  • the parent aptamer sequence may be an aptamer sequence disclosed herein, e.g., CTGGGGAGTCCTTCATGCGGGGCTGAGAGGATGGAAGCAATCGACCATCGACCC
  • the step of selecting a parent aptamer sequence can involve, for example, (i) identifying a putative TPP aptamer; (ii) inserting the aptamer into a riboswitch that modulates the expression of a target gene (for example a reporter gene); and (iii) exposing the riboswitch/target gene construct to a thiamine or TPP analog or derivative (e.g., the compounds described herein).
  • Putative TPP aptamers can be identified from an appropriate sequence database such as the Rfam database, which is a collection of RNA families, each represented by multiple sequence alignments, consensus secondary structures and covariance models (CMs).
  • the putative TPP aptamer is identified from the Rfam TPP riboswitch family RF00059. In embodiments, the putative TPP aptamer has a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97% at least 98% or at least 99% identical to thiC (GUAAUGUGUCGGAGUGCCUUAGGGAUUAUUCCCCUAAAGCUGAGACCGCAUU GCGGGAUCCGUUGAACCUGAUCAGGCUAAUACCUGCGAAGGGAACACAUUAC, SEQ ID NO: 7) or thiM (GUAAUGUCUCGGGGUGCCCUUCUGCGUGAAGGCUGAGAAAUACCCGUAUCAC CUGAUCUGGAUAAUGCCAGCGUAGGGAAGACAUUAC, SEQ ID NO: 8).
  • the putative TPP aptamer can be inserted into a riboswitch using techniques known to the ordinarily skilled artisan.
  • the responsiveness of the aptamer to the presence of TPP and one or more thiamine or TPP analogs or derivatives can be tested in cell culture and/or in a cell-free system.
  • the cell culture system is a eukaryotic cell culture including, e.g., a mammalian, a plant, or an insect cell culture.
  • one or more nucleotide positions of the sequence encoding the aptamer are randomized. Areas of the sequence that can be randomized include J2-4; L3a; P4/J4-5 to J5- 4; and L5.
  • the nucleotide positions for randomization can be selected based on the structure of the parent aptamer sequence.
  • the predicted secondary structure can be obtained using available programs such as RNAfold (http://rna.tbi.univie.ac.at/cgi- bin/RNAWebSuite/RNAfold.cgi) and/or by comparison to the crystal structure of a related aptamer (e.g., the E. coli thiM riboswitch in Edwards, TE & Ferre-D'Amare, AR, Structure. 2006 Sep; 14(9): 1459-68).
  • a related aptamer e.g., the E. coli thiM riboswitch in Edwards, TE & Ferre-D'Amare, AR, Structure. 2006 Sep; 14(9): 1459-68.
  • unpaired regions of the aptamer including loop (L) regions (e.g., L3 and/or L5) and joining (J) regions (e.g., J3-2 (joining paired regions P3 and P2), J2-4, and/or J4-5), can be identified, and one or more nucleotides in one or more unpaired regions can be randomized to generate a library of aptamers.
  • one or more nucleotides adjacent to one or more unpaired regions are randomized.
  • one or more nucleotides in a paired (P) region can be randomized.
  • one or more nucleotides in an unpaired or paired region can be added or deleted.
  • the mutagenized aptamer sequences can be provided as a library of aptamer sequences in the context of a riboswitch.
  • the aptamer library is provided in the context of a riboswitch as part of a gene expression cassette disclosed herein.
  • the aptamer encoding sequences containing one or more mutations can be tested for responsiveness to the presence of one or more compounds described herein.
  • Aptamers that are responsive to the desired compound can be further mutagenized by randomizing nucleotides.
  • the nucleotides at selected positions, for example unpaired regions, can be randomized and a library created as described above.
  • Reporter proteins encoded by the reporter genes used in the methods disclosed herein are proteins that can be assayed by detecting characteristics of the reporter protein, such as enzymatic activity or spectrophotometric characteristics, or indirectly, such as with antibody -based assays.
  • reporter gene products include, but are not limited to, puromycin resistance marker (pac), 3 -galactosidase, luciferase, orotidine 5'-phosphate decarboxylase (URA3), arginine permease CAN1, galactokinase (GALI), beta-galactosidase (LacZ), or chloramphenicol acetyl transferase (CAT).
  • detectable signals include cell surface markers, including, but not limited to CD4.
  • Reporter genes suitable for the use in the methods for identifying aptamers disclosed herein also include fluorescent proteins (e.g., green fluorescent protein (GFP) and its derivatives), or proteins fused to a fluorescent tag.
  • fluorescent proteins e.g., green fluorescent protein (GFP) and its derivatives
  • fluorescent tags and proteins include, but are not limited to, (3-F)Tyr-EGFP, A44-KR, aacuGFPl, aacuGFP2, aceGFP, aceGFP-G222E-Y220L, aceGFP-h, AcGFPl, AdRed, AdRed-C148S, aeurGFP, afraGFP, alajGFPl, alajGFP2, alajGFP3, amCyanl, amFP486, amFP495, amFP506, amFP515, amilFP484, amilFP490, amilFP497, amilFP504, amilFP512, amilFP513, amilFP593, amilFP597, anmlGFPl, anmlGFP2, anm2CP, anobCFPl, anobCFP2, anobGFP, apulFP483, AQ14, AQ143, Aquamarine, asCP562, asFP499, AsRed2,
  • Methods for screening an aptamer library disclosed herein may include measuring the activity of the reporter gene under the control of the aptamer and/or comparing the activity of the reporter gene in presence of the thiamine or TPP analog used for the screen as compared to the activity of the reporter gene in absence of the thiamine or TPP analog used for the screen.
  • kits or articles of manufacture for use in the methods described herein.
  • the kits comprise the compositions described herein (e.g., compositions for delivery of a vector comprising the target gene containing the gene regulation cassette) in suitable packaging.
  • suitable packaging for compositions (such as ocular compositions for injection) described herein are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed.
  • kits comprising the compositions described herein.
  • kits may further comprise instruction(s) on methods of using the composition, such as uses described herein.
  • the kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing the administration of the composition or performing any methods described herein.
  • the kit comprises an rAAV for the expression of a target gene comprising a gene regulation cassette containing an aptamer sequence described herein, a pharmaceutically acceptable carrier suitable for injection, and one or more of: a buffer, a diluent, a filter, a needle, a syringe, and a package insert with instructions for performing the injections.
  • the kit is suitable for intraocular injection, intramuscular injection, intravenous injection and the like.
  • Example 1 A Regulation of Target Gene Expression in Response to Small Molecule Ligands Disclosed Herein
  • Riboswitch construct a sequence of aptamers.
  • Aptamers were synthesized by Integrated DNA Technologies, Inc.
  • the synthesized aptamer sequence here referred to as aptamer sequence 12C6-1, with C at 5' end and a complementary G at 3' end flanking the aptamer sequence: CTGGGGAGTCCTTCATGCGGGGCTGAGAGGATGGAAGCAATCGACCATCGACCC ATTGCACCTGATCCGGATCATGCCGGCGCAGGGAG (SEQ ID NO: 1).
  • SEQ ID NO: 4 (12C6-1 alternate splicing gene regulation cassette) gtgagtctatgggacccttgatgttttctttccccttttttctatggttaagttcatgtcataggaaggggagaagt aacagggtacacatattgaccaaatcagggtaattttgcatttgtaatttttaaaaatgctttctttttaatata cttttttgtttatcttttctaatacttttccctaatctcttttttcagggcaataatgatacaatgtatcatgccgagt aacgctgtfcfctoacttgtoggaatgaattcagatattttccagagaatgaaaaaaaaatctttcagtag
  • HEK 293 cells were plated in a 96-well flat bottom plate the day before transfection. Plasmid DNA (500 ng) was added to a tube or a 96-well U-bottom plate. Separately, TransIT-293 reagent (Minis; 1.4 pL) was added to 50 pL Optimum I media (Life Technologies) and allowed to sit for 5 minutes at room temperature (RT). Then, 50 pL of this diluted transfection reagent was added to the DNA, mixed, and incubated at RT for 20 min. Finally, 7 pL of this solution was added to a well of cells in the 96-well plate. Four hours after transfection, medium containing transfection solution was replaced by medium with either TPP or fursultiamine as aptamer ligands.
  • luciferase activity was expressed as mean arbitrary light units (ALU) ⁇ S.D., and fold induction was calculated as the quotient of the luciferase activity obtained from cells with the compound treatment divided by the luciferase activity obtained from cells without compound treatment.
  • ALU mean arbitrary light units
  • Comp. A may be prepared as disclosed in PCT/US2020/045022.
  • Nominal (low resolution) mass spectra were acquired on either a Waters LCT or an Applied Biosystems API 3000 mass spectrometer. All other LC-MS experiments were done on an Agilent 1100 HPLC coupled with an Agilent single quadrupole mass spectrometer. Compound purity was determined by a LC-MS with 230 nM and 254 nM wavelengths. All final compounds reported here have purity > 95%.
  • Step 4 tert-Butyl 3,3-dimethyl-4-(3-((quinoxalin-6-ylmethyl)amino)pyridin-4- yl)piperazine- 1 -carboxylate
  • Step 2 4-(4-Methylpiperazin-l-yl)-A-(quinoxalin-6-ylmethyl)pyri din-3 -amine
  • reaction mixture was filtered and the filtrate was purified by prep-HPLC (column: Waters Xbridge C18 150 x 50 mm x 10 pm; mobile phase: A- water (NH4HCO3) and B- CH3CN; B%: 15% - 45%, 10 min) to give the title compound (198 mg, 26.7%) as a yellow solid.
  • Step 4 tert-Butyl 7-(3-((quinoxalin-6-ylmethyl)amino)pyridin-4-yl)-4,7- di azaspiro [2.5 ] octane-4 -carb oxy 1 ate
  • Step 5 A-(Quinoxalin-6-ylmethyl)-4-(4,7-diazaspiro[2.5]octan-7-yl)pyridin-3- amine
  • Step 1 tert-Butyl 2,2-dimethyl-4-(3-((quinoxalin-6-ylmethyl)amino)pyridin-4- yl)piperazine- 1 -carboxylate
  • Step 2 /crZ-Butyl 7-(3-aminopyridazin-4-yl)-4,7-diazaspiro[2.5]octane-4- carb oxy late
  • Step 4 tert-Butyl 7-(3-((quinoxalin-6-ylmethyl)amino)pyridazin-4-yl)-4,7- di azaspiro [2.5 ] octane-4 -carb oxy 1 ate
  • Step 2 ZczV-Butyl 7-(3-bromo-5-formylpyridin-4-yl)-4,7-diazaspiro[2.5]octane-4- carb oxy late
  • Step 4 tert-Butyl 7-(3-(difluoromethyl)-5-((diphenylmethylene)amino)pyridin-4- yl)-4,7-diazaspiro[2.5]octane-4-carboxylate
  • Step 8 tert-Butyl 7-(3-(difluoromethyl)-5-((quinoxalin-6-ylmethyl)amino)pyridin- 4-yl)-4, 7 -diazaspiro[2.5 ] octane-4-carboxylate
  • Step 9 5-(Difluoromethyl)-A-(quinoxalin-6-ylmethyl)-4-(4,7-diazaspiro[2.5]octan- 7-yl)pyridin-3-amine
  • Step 1 tert-Butyl 7-(3-bromo-5-(trifluoromethyl)pyridin-4-yl)-4,7- di azaspiro [2.5 ] octane-4 -carb oxy 1 ate
  • Step 2 tert-Butyl 7-(3-((diphenylmethylene)amino)-5-(trifluoromethyl)pyridin-4- yl)-4,7-diazaspiro[2.5]octane-4-carboxylate
  • Step 6 tert-Butyl 7-(3-((quinoxalin-6-ylmethyl)amino)-5-(trifluoromethyl)pyridin- 4-yl)-4,7-diazaspiro[2.5]octane-4-carboxylate
  • Step 4 4-(4-Methylpiperazin-l-yl)-A-(quinoxalin-6-ylmethyl)pyridazin-3 -amine
  • Step 5 5-(Difluoromethyl)-4-(4-methylpiperazin-l-yl)-7V-(quinoxalin-6- ylmethyl)pyri din-3 -amine
  • Step 5 To a solution of f£J-7V-(5-(difluoromethyl)-4-(4-methylpiperazin-l-yl)pyri din-3 - yl)-l-(quinoxalin-6-yl)methanimine (552 mg, 1.44 mmol, 1.00 eq in MeOH (5.00 mL) was added NaBHsCN (90.7 mg, 1.44 mmol, 1.00 eq) and AcOH (86.6 mg, 1.44 mmol, 82.6 pL, 1.00 eq .
  • Step 5 (A)-4-((l-methylpyrrolidin-3-yl)oxy)-A-(quinoxalin-6-ylmethyl)pyri din-3 - amine
  • Step 1 tert-Butyl (A,E)-(l-(3-fluoro-5-((quinoxalin-6-ylmethylene)amino)pyridin- 4-yl)pyrrolidin-3-yl)carbamate
  • Step 3 fAJ-4-(3-Aminopyrrolidin-l-yl)-5-fluoro-A-(quinoxalin-6- ylmethyl)pyri din-3 -amine
  • reaction mixture was concentrated to give a residue which was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm* 10um;mobile phase: [water(HCl)-ACN];gradient: l%-15% B over 10 min) to the title compound (143 mg, 50.6%) as a yellow solid.
  • Step 5 (7?J-4-(3-Aminopyrrolidin-l-yl)-5-chloro-A-(quinoxalin-6- ylmethyl)pyri din-3 -amine
  • Step 1 tert-Butyl (S,E)-(l-(3-fluoro-5-((quinoxalin-6-ylmethylene)amino)pyridin-
  • the reaction mixture was concentrated under reduced pressure to remove methanol, diluted with H2O (20.0 mL), and extracted with ethyl acetate (20.0 mL x 2). The combined organic layers were washed with brine (50.0 mL), dried over ISfeSCU, filtered and concentrated under reduced pressure to give a residue.
  • the residue was purified by prep-HPLC (column: Waters Xbridge 150*25mm* 5um; mobile phase: [water (ammonia hydroxide v/v)-ACN];gradient:25%-55% B over 10 min) to afford the title compound (260 mg, 34.9%) as a yellow solid.
  • the crude product was purified by reversed-phase HPLC (column: Phenomenex luna Cl 8 150 x 25mm x 10um; mobile phase: [water(HCl)-ACN]; gradient: l%-20% B over 10 min) and lyophilized to give the title compound (120 mg, 30.8%) as a yellow solid.
  • Step 4 (7?J-4-(3-(Methylamino)pyrrolidin-l-yl)-A-(quinoxalin-6-ylmethyl)pyridin- 3 -amine
  • Step 5 tert-Butyl 5)-(l-(3-fluoro-5-((quinoxalin-6-ylmethyl)amino)pyridin-4-yl)- 3 -methylpyrrolidin-3 -yl)carbamate
  • Step 2 (7?J-3-((Diphenylmethylene)amino)-5-fluoro-A-(l-methylpyrrolidin-3- yl)pyridin-4-amine
  • Step 5 (7? -5-Fluoro-N4-(l-methylpyrrolidin-3-yl)-N3-(quinoxalin-6- ylmethyl)pyridine-3,4-di amine
  • Step 1 (S,E)-3-(((8-Fluoroquinoxalin-6-yl)methylene)amino)-A-(l- methylpyrrolidin-3-yl)pyridin-4-amine
  • Step 5 N4-(l-Methylazetidin-3-yl)-N3-(quinoxalin-6-ylmethyl)pyridine-3,4- diamine
  • N4-(l- methylazeti din-3 -yl)pyridine-3,4-diamine (0.25 g, 1.40 mmol, 1.00 eq) in THF (10 mL) was added Ti(i-PrO)4 (797 mg, 2.81 mmol, 828 pL, 2.00 eq).
  • Example 66 The title compound was synthesized following a similar procedure described for the preparation of Example 66 (Comp. 65) with appropriate starting materials and intermediates.
  • Step 4 tert-Butyl 4-(3-((quinoxalin-6-ylmethyl)amino)pyridin-4-yl)piperazine-l- carb oxy late
  • Step 6 4-(Piperazin-l-yl)-7V-(quinoxalin-6-ylmethyl)pyri din-3 -amine
  • the crude product was purified by reversed-phase HPLC (column: Phenomenex luna Cl 8 (250 x 70mm, 10 pm); mobile phase: [water (HCl)-ACN]; gradient: 1%- 15% B over 20 min) to provide the title comopund (100 mg, 22.2%) as a brown solid.
  • the crude product was purified by reversed-phase HPLC(column: Waters X bridge 150 * 25mm x 5um; mobile phase: [water (ammonia hydroxide v/v)-ACN]; gradient:3%-33% B over 10 min) and lyophilized to give the title compound (150 mg, 52.8%) as a yellow solid.
  • the crude product was purified by reversed-phase HPLC (column: Waters X bridge 150 * 25 mm x 5 U m; mobile phase: [water (ammonia hydroxide v/v)-ACN]; gradient: 10%-40% B over 10 min) and lyophilized to give the title compound (150 mg, 55.2%) as a yellow solid.
  • CD-I mice were treated with the compounds via oral administration.
  • the oral administration was by gavage with a methyl cellulose/Tween 80 formulation.
  • Blood samples were collected at the 2 hour timepoint post-dose and were transferred into EDTA-K3 anti coagulant tubes.
  • the blood plasma samples were obtained by centrifugation at 4000g for 5 min and were kept at -75 ⁇ 15 °C until bioanalysis.
  • the compound concentrations in the plasma samples were assayed using LC-MS/MS. Quality control samples (prepared with CD- 1 mouse plasma) were included in each analysis to ensure assay performance.
  • tissue collection the animal was fully exsanguinated prior to the tissue collection. The following procedure was followed: open the cavitas thoracis, expose the heart, catheterize from left ventricular, make an incision at the right atrial appendage, and perfuse (10 mL saline) from the left ventricular. The fluid outflows from the right atrial appendage. The fresh tissues were then collected, put in an appropriate tube, quickly frozen in liquid nitrogen and kept at -75 ⁇ 15 °C until analysis. The tissue samples were homogenized at a ratio of 1 :4 with PBS (W/V, 1 :4) and the compound concentration was determined using LC-MS/MS. Quality control samples (prepared with CD-I mouse tissue homogenate) were included in each analysis to ensure assay performance.

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Abstract

La présente invention concerne de petites molécules de formule (I) qui se lient à des aptamères. L'invention concerne également des ribocommutateurs et des cassettes polynucléotidiques pour réguler l'expression d'un gène cible en réponse à la petite molécule, les cassettes polynucléotidiques comprenant les aptamères selon l'invention. Les petites molécules divulguées qui se lient aux aptamères selon l'invention sont des modulateurs de l'expression génique cible où le gène cible contient un riborégulateur comprenant un aptamère selon l'invention.
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