WO2021001739A1 - Translational enhancers and related methods - Google Patents

Translational enhancers and related methods Download PDF

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Publication number
WO2021001739A1
WO2021001739A1 PCT/IB2020/056116 IB2020056116W WO2021001739A1 WO 2021001739 A1 WO2021001739 A1 WO 2021001739A1 IB 2020056116 W IB2020056116 W IB 2020056116W WO 2021001739 A1 WO2021001739 A1 WO 2021001739A1
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rna molecule
disclosure
rna
translational enhancer
methyl
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PCT/IB2020/056116
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French (fr)
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Justin T. Ernst
Paul A. Sprengeler
Craig Stumpf
Kevin R. Webster
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Effector Therapeutics, Inc.
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Publication of WO2021001739A1 publication Critical patent/WO2021001739A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • Protein replacement therapies involve supplementation of deficient or aberrant proteins and enzymes as well as modulation of cell behavior by expression of exogenous proteins. It is well known that nucleic acids can be used to modulate protein production in vivo. Because application of DNA or mRNA enables expression of virtually any desired protein inside host cells and tissues, expression of disease-relevant proteins to treat disease conditions can be achieved by intracellular delivery of plasmid DNA (pDNA) or messenger RNA (mRNA).
  • pDNA plasmid DNA
  • mRNA messenger RNA
  • mRNA-based protein replacement therapies offer several advantages over pDNA, including rapid and transient protein production, no risk of insertional mutagenesis, and greater efficacy of non-viral delivery by virtue of mRNA cytoplasmic activity.
  • m7G cap N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5’ to 5’ triphosphate linkage
  • the m7G cap N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5’ to 5’ triphosphate linkage
  • the m7G cap N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5’ to 5’ triphosphate linkage
  • Cap 1 structure 2’O methylation of +1 nucleotide (Cap 1 structure) is central to the non-self discrimination of innate immune response against foreign RNA (Daffis S., et al., 2010, Nature, 468:452-456; Devarkar S.C., et al., 2016, Proc. Natl. Acad. Sci. U.S.A., 113:596-601).
  • Cellular sensors RIG-I and MDA5 and effectors IFIT1 and IFIT5 of the Type I interferon (IFN) signaling pathway act by discriminating Cap 1 RNA from others.
  • Cap 1 structure abolished the interactions of RNA with RIG-I and MDA5 and hence did not activate the IFN signaling pathway (Zust, R., et al., 2011, Nat. Immunol., 12:137-143; Schuberth-Wagner C., et al., 2015, Immunity, 43:41-51).
  • the 5’ mRNA cap structure is essential for efficient gene expression from yeast to human. It plays a critical role in all aspects of the life cycle of an mRNA molecule.
  • the cap structure protects mRNAs from degradation by exonucleases and promotes transcription, polyadenylation, splicing, and nuclear export of mRNA and U-rich, capped snRNAs.
  • the cap structure is required for the optimal translation of the vast majority of cellular mRNAs, and it also plays a prominent role in the expression of eukaryotic mRNAs.
  • Cap-dependent translation initiation in eukaryotes is a highly regulated rate-limiting step, which involves recruitment and assembly of eukaryotic initiation factor 4F (eIF4F), a multiprotein complex on the 5 cap of the mRNA.
  • eIF4F consists of at least three proteins: the cap-binding protein eukaryotic initiation factor 4E (eIF4E), the ATP-dependent RNA helicase eukaryotic initiation factor 4A (eIF4A), and the scaffold protein eukaryotic initiation factor 4G (eIF4G).
  • eIF4E directly recognizes the cap structure of mRNAs, and is essential for cap- dependent translation initiation, while eIF4G interacts with the other eIF4F subunits as well as with the poly-A binding protein on the poly-A tail of the mRNA to create a close mRNA circle during translation initiation.
  • the present disclosure generally relates to novel translational enhancers, RNA molecules comprising the novel translational enhancers, and methods of using such RNA molecules for therapeutic uses.
  • the present disclosure provides a translational enhancer comprising an eIF4E ligand attached to at least one nucleotide.
  • the eIF4E ligand is attached to the at least one nucleotide via a linker.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to at least one nucleotide.
  • the eIF4E ligand is attached to the at least one nucleotide via a linker.
  • the translational enhancer is attached to the 5’ end of the RNA molecule. In some aspects, the translational enhancer functions as a 5’ cap structure.
  • the present disclosure provides a method of making a capped RNA molecule comprising: (a) reacting a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase and under conditions conducive to transcription by the RNA polymerase to generate at least one RNA molecule from the polynucleotide template; and (b) co-transcriptionally coupling to a 5’ end of the at least one RNA molecule a translational enhancer of the disclosure.
  • the present disclosure provides a method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject a therapeutically effective amount of an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to a dinucleotide.
  • FIG.1 shows co-transcriptional synthesis of 5’ capped Firefly Luciferase (FLuc) mRNA by in vitro transcription, and subsequent t FLuc protein expression from the mRNA.1G is absolutely required for transcription, and yield is compromised when the +2 nucleotide is not G.2 Gs are necessary for reasonable yields of in vitro transcripts and is recommended for most transcripts.3 Gs are present in the consensus T7 promoter.4Gs (not pictured) may result in increased 5’ transcript heterogeneity. DETAILED DESCRIPTION
  • the present disclosure relates to RNA-based protein replacement therapies that involve supplementation of deficient or aberrant proteins and enzymes.
  • the present disclosure provides a novel translational enhancer comprising an eIF4E ligand attached to a dinucleotide.
  • the present disclosure provides an RNA molecule comprising a translational enhancer of the disclosure.
  • the present disclosure provides a method of making an RNA molecule that comprises a translational enhancer of the disclosure.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term“about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms“a” and“an” as used herein refer to“one or more” of the enumerated components.
  • a protein domain, region, or module e.g., a binding domain, hinge region, linker module
  • a protein which may have one or more domains, regions, or modules
  • Amino refers to the -NH 2 substituent.
  • Aminocarbonyl refers to the–C(O)NH 2 substituent.
  • Carboxyl refers to the–CO 2 H substituent.
  • Cyanoalkylene refers to the -(alkylene)CoN substituent.
  • Haldroxyalkylene refers to the -(alkylene)OH subsituent.
  • Alkyl refers to a saturated, straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, having from one to twelve carbon atoms (C 1 -C 12 alkyl), from one to eight carbon atoms (C 1 -C 8 alkyl) or from one to six carbon atoms (C 1 -C 6 alkyl), and which is attached to the rest of the molecule by a single bond.
  • alkyl groups include methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.
  • “Lower alkyl” has the same meaning as alkyl defined above but having from one to four carbon atoms (C 1 -C 4 alkyl).
  • Alkenyl refers to an unsaturated alkyl group having at least one double bond and from two to twelve carbon atoms (C 2 -C 12 alkenyl), from two to eight carbon atoms (C 2 -C 8 alkenyl) or from two to six carbon atoms (C 2 -C 6 alkenyl), and which is attached to the rest of the molecule by a single bond, e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.
  • Alkynyl refers to an unsaturated alkyl group having at least one triple bond and from two to twelve carbon atoms (C 2 -C 12 alkynyl), from two to ten carbon atoms (C 2 -C 10 alkynyl) from two to eight carbon atoms (C 2 -C 8 alkynyl) or from two to six carbon atoms (C 2 -C 6 alkynyl), and which is attached to the rest of the molecule by a single bond, e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • Alkylene or“alkylene chain” refers to a straight or branched divalent hydrocarbon (alkyl) chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, respectively.
  • Alkylenes can have from one to twelve carbon atoms, e.g., methylene, ethylene, propylene, n-butylene, and the like.
  • the alkylene chain is attached to the rest of the molecule through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule can be through one carbon or any two carbons within the chain.“Optionally substituted alkylene” refers to alkylene or substituted alkylene.
  • Alkynylene refers to divalent alkyne. Examples of alkynylene include without limitation, ethynylene, propynylene.“Substituted alkynylene” refers to divalent substituted alkyne.
  • Alkoxy refers to a radical of the formula -OR a where R a is an alkyl having the indicated number of carbon atoms as defined above. Examples of alkoxy groups include without limitation–O-methyl (methoxy), -O-ethyl (ethoxy), -O-propyl (propoxy), -O-isopropyl (iso propoxy) and the like.
  • Alkylaminyl refers to a radical of the formula -NHR a or -NR a R a where each R a is, independently, an alkyl radical having the indicated number of carbon atoms as defined above.
  • Cycloalkylaminyl refers to a radical of the formula -NHR a or -NR a R a where R a is a cycloalkyl radical as defined herein.
  • Alkylcarbonylaminyl refers to a radical of the formula -NHC(O)R a or -NR a C(O)R a , where R a is an alkyl radical having the indicated number of carbon atoms as defined herein.
  • Cycloalkylcarbonylaminyl refers to a radical of the formula -NHC(O)R a , where R a is a cycloalkyl radical as defined herein.
  • Aryl refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring.
  • exemplary aryls are hydrocarbon ring system radical comprising hydrogen and 6 to 9 carbon atoms and at least one aromatic ring;
  • hydrocarbon ring system radical comprising hydrogen and 9 to 12 carbon atoms and at least one aromatic ring
  • hydrocarbon ring system radical comprising hydrogen and 12 to 15 carbon atoms and at least one aromatic ring
  • hydrocarbon ring system radical comprising hydrogen and 15 to 18 carbon atoms and at least one aromatic ring.
  • the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
  • Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.“Optionally substituted aryl” refers to an aryl group or a substituted aryl group.
  • Arylene denotes divalent aryl
  • “substituted arylene” refers to divalent substituted aryl
  • “Aralkyl” or“araalkylene” may be used interchangeably and refer to a radical of the formula -R b -R c where R b is an alkylene chain as defined herein and R c is one or more aryl radicals as defined herein, for example, benzyl, diphenylmethyl and the like.
  • Cycloalkyl refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, three to nine carbon atoms, three to eight carbon atoms, three to seven carbon atoms, three to six carbon atoms, three to five carbon atoms, a ring with four carbon atoms, or a ring with three carbon atoms.
  • the cycloalkyl ring may be saturated or unsaturated and attached to the rest of the molecule by a single bond.
  • Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl,
  • Cycloalkylalkylene or“cycloalkylalkyl” may be used interchangeably and refer to a radical of the formula -R b R e where R b is an alkylene chain as defined herein and R e is a cycloalkyl radical as defined herein.
  • R b is further substituted with a cycloalkyl group, such that the cycloalkylalkylene comprises two cycloalkyl moieties.
  • Cyclopropylalkylene and cyclobutylalkylene are exemplary cycloalkylalkylene groups, comprising at least one cyclopropyl or at least one cyclobutyl group, respectively.
  • fused refers to any ring structure described herein which is fused to an existing ring structure in the compounds or translational enhancers of the present disclosure.
  • fused ring is a heterocyclyl ring or a heteroaryl ring
  • any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with a nitrogen atom.
  • Halo or“halogen” refers to bromo (bromine), chloro (chlorine), fluoro (fluorine), or iodo (iodine).
  • Haloalkyl refers to an alkyl radical having the indicated number of carbon atoms, as defined herein, wherein one or more hydrogen atoms of the alkyl group are substituted with a halogen (halo radicals), as defined above.
  • the halogen atoms can be the same or different.
  • Exemplary haloalkyls are trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.
  • Heterocyclyl refers to a stable 3- to 18- membered saturated or unsaturated radical which consists of two to twelve carbon atoms and from one to six heteroatoms, for example, one to five heteroatoms, one to four heteroatoms, one to three heteroatoms, or one to two heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.
  • Exemplary heterocycles include without limitation stable 3-15 membered saturated or unsaturated radicals, stable 3-12 membered saturated or unsaturated radicals, stable 3-9 membered saturated or unsaturated radicals, stable 8-membered saturated or unsaturated radicals, stable 7-membered saturated or unsaturated radicals, stable 6-membered saturated or unsaturated radicals, or stable 5-membered saturated or unsaturated radicals.
  • the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated.
  • non-aromatic heterocyclyl radicals include, but are not limited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl,
  • Heterocyclyls include heteroaryls as defined herein, and examples of aromatic heterocyclyls are listed in the definition of heteroaryls below.
  • Heterocyclylalkyl or“heterocyclylalkylene” refers to a radical of the formula -R b R f where R b is an alkylene chain as defined herein and R f is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to the alkyl radical at the nitrogen atom.
  • Heteroaryl or“heteroarylene” refers to a 5- to 14-membered ring system radical comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring.
  • the heteroaryl radical may be a stable 5-12 membered ring, a stable 5-10 membered ring, a stable 5-9 membered ring, a stable 5-8 membered ring, a stable 5-7 membered ring, or a stable 6 membered ring that comprises at least 1 heteroatom, at least 2 heteroatoms, at least 3 heteroatoms, at least 4 heteroatoms, at least 5 heteroatoms or at least 6 heteroatoms.
  • Heteroaryls may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, 2 carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized.
  • the heteroatom may be a member of an aromatic or non-aromatic ring, provided at least one ring in the heteroaryl is aromatic.
  • Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,
  • benzodioxolyl benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimi
  • Heteroarylalkyl or“heteroarylalkylene” refers to a radical of the formula -R b R g where R b is an alkylene chain as defined above and R g is a heteroaryl radical as defined above.
  • Thioalkyl refers to a radical of the formula -SR a where R a is an alkyl radical as defined above containing one to twelve carbon atoms, at least 1-10 carbon atoms, at least 1-8 carbon atoms, at least 1-6 carbon atoms, or at least 1-4 carbon atoms.
  • Heterocyclylaminyl refers to a radical of the formula–NHR f where R f is a heterocyclyl radical as defined above.
  • “Sulfoxide” refers to a–S(O)- group in which the sulfur atom is covalently attached to two carbon atoms.
  • “Sulfone” refers to a–S(O) 2 - group in which a hexavalent sulfur is attached to each of the two oxygen atoms through double bonds and is further attached to two carbon atoms through single covalent bonds.
  • the compounds or translational enhancers provided in the present disclosure can exist in various isomeric forms, as well as in one or more tautomeric forms, including both single tautomers and mixtures of tautomers.
  • the term“isomer” is intended to encompass all isomeric forms of a compound of the present disclosure, including tautomeric forms of the compound.
  • Some compounds or translational enhancers described herein can have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms.
  • a compound provided in the present disclosure can be in the form of an optical isomer or a diastereomer. Accordingly, the invention encompasses compounds or translational enhancers provided in the present disclosure and their uses as described herein in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture.
  • Optical isomers of the compounds or translational enhancers provided in the present disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, or via chemical separation of stereoisomers through the employment of optically active resolving agents.
  • stereoisomer means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound.
  • a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound.
  • a stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound.
  • a typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
  • the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. In some cases, however, where more than one chiral center exists, the structures and names may be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know if the compounds or translational enhancers are prepared as single enantiomers from the methods used to prepare them.
  • a“pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound of the present disclosure.
  • Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexyl
  • the term“derivative” refers to a modification of a compound by chemical or biological means, with or without an enzyme, which modified compound is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound.
  • a“derivative” differs from an“analog” in that a parent compound may be the starting material to generate a“derivative,” whereas the parent compound may not necessarily be used as the starting material to generate an“analog.”
  • a derivative may have different chemical, biological or physical properties from the parent compound, such as being more hydrophilic or having altered reactivity as compared to the parent compound.
  • Derivatization may involve substitution of one or more moieties within the molecule (e.g., a change in functional group).
  • a hydrogen may be substituted with a halogen, such as fluorine or chlorine, or a hydroxyl group (-OH) may be replaced with a carboxylic acid moiety (-COOH).
  • exemplary derivatizations include glycosylation, alkylation, acylation, acetylation, ubiqutination, esterification, and amidation.
  • derivative also refers to all solvates, for example, hydrates or adducts (e.g., adducts with alcohols), active metabolites, and salts of a parent compound.
  • adducts e.g., adducts with alcohols
  • active metabolites e.g., adducts with alcohols
  • salts of a parent compound e.g., adducts with alcohols
  • the type of salt depends on the nature of the moieties within the compound.
  • acidic groups such as carboxylic acid groups
  • alkali metal salts or alkaline earth metal salts e.g., sodium salts, potassium salts, magnesium salts, calcium salts, and also salts with physiologically tolerable quaternary ammonium ions and acid addition salts with ammonia and physiologically tolerable organic amines such as, for example, triethylamine, ethanolamine or tris-(2- hydroxyethyl)amine).
  • Basic groups can form acid addition salts with, for example, inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or with organic carboxylic acids or sulfonic acids such as acetic acid, citric acid, lactic acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, methanesulfonic acid or p-toluenesulfonic acid.
  • Molecules that simultaneously contain a basic group and an acidic group for example, a carboxyl group in addition to basic nitrogen atoms, can be present as zwitterions. Salts can be obtained by customary methods known to those skilled in the art, for example, by combining a compound with an inorganic or organic acid or base in a solvent or diluent, or from other salts by cation exchange or anion exchange.
  • eIF4E also referred to as“eukaryotic translation initiation factor-4E,” refers to a translation initiation factor that, when part of an eIF4F pre-initiation complex also comprising eIF4A RNA helicase and eIF4G scaffold protein, binds to the 7- methyl-guanosine (m7GpppX) 5’-cap structure on eukaryotic mRNAs and directs ribosomes to the cap structure.
  • m7GpppX 7- methyl-guanosine
  • isoform 1 is the canonical sequence
  • isoform 2 contains an alternate in-frame exon in the 3’-coding region compared to isoform 1
  • isoform 3 uses an alternate 5’-terminal exon, which results in a different 5’-UTR and use of an alternate translation start codon compared to isoform 1
  • isoform 4 differs in its 5’-UTR and contains an alternate exon in its 5’-coding region compared to isoform 1.
  • eIF4E refers to eIF4E isoform 1, isoform 2, isoform 3, isoform 4, or any combination thereof.
  • eIF4E refers to the canonical eIF4E isoform 1.
  • eIF4E refers to human eIF4E.
  • attachment directly refers to two molecules that are attached or linked or connected or bound or coupled or fused to one another in the absence of an intervening molecule.
  • promoter refers to a region of dsDNA template that directs and controls the initiation of transcription of a particular DNA sequence (e.g. gene). Promoters are located on the same strand and upstream on the DNA (towards the 5’ region of the sense strand). Promoters are typically immediately adjacent to (or partially overlap with) the DNA sequence to be transcribed. Nucleotide positions in the promoter are designated relative to the transcriptional start site, where transcription of DNA begins (position +1). The initiating oligonucleotide primer is complementary to initiation site of promoter sequence (which, in certain embodiments, is at positions +1 and +2 and, in the case of initiating tetramers, at positions +1, +2 and +3).
  • RNA transcript As used herein, the terms“transcription” or“transcription reaction” refers to methods known in the art for enzymatically making RNA that is complementary to DNA template, thereby producing the number of RNA copies of a DNA sequence.
  • the RNA molecule synthesized in transcription reaction called“RNA transcript”,“primary transcript” or “transcript”. Transcription of DNA template may be exponential, nonlinear or linear.
  • a DNA template may be a double stranded linear DNA, a partially double stranded linear DNA, circular double stranded DNA, DNA plasmid, PCR amplicon, a modified nucleic acid template which is compatible with RNA polymerase.
  • translation or“translation reaction” refer to the process or mechanism of synthesizing a protein from a messenger RNA (mRNA).
  • Translation reaction involving the compositions and methods provided herein employs the translational enhancers of the disclosure.
  • an immune response refers to the action of an immune cell, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement), that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • an immune response comprises an antigen-specific T cell response.
  • the phrase“inducing an attenuated immune response” refers to causing a decreased stimulation of one or more components of a subject’s innate immune system and/or one or more components of a subject’s adaptive immune system.
  • the assay for detecting cytokine levels (e.g., IL-2, IL-10, IFNg) to determine whether an immune response is induced, enhanced, or attenuated is the multiplex assay described by Dossus et al. (J. Immunol. Methods 350:125, 2009).
  • the assay for detecting T cell proliferation to determine whether an immune response induced or enhanced is the assay described by Liu et al. (Clin. Cancer Res.21:1639, 2015).
  • the assay for determining increased antigen responsiveness is the assay described by Tumeh et al. (Nature 515:568, 2014).
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to chemical entities that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical entities that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • A“conservative substitution” refers to amino acid substitutions that do not
  • Conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W).
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile.
  • Other conservative substitutions groups include: sulfur- containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
  • cell-free translation extract refers to an in vitro system used for translating protein from mRNAs.
  • a cell-free translation extract contains all cellular components required for protein expression.
  • an exogenous mRNA molecule added to the cell-free translation extract is translated to the protein encoded by the mRNA.
  • a transcribed mRNA is incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce a desired polypeptide or fragment thereof.
  • “protein” or“polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers.
  • Nucleic acid molecule or“polynucleotide” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring).
  • Purine bases include adenine, guanine, hypoxanthine, and xanthine
  • pyrimidine bases include uracil, thymine, and cytosine.
  • Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double stranded.
  • the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand).
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
  • the term“agent” refers to any molecule, either naturally occurring or synthetic, e.g., peptide, protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule (e.g., an organic molecule having a molecular weight of less than about 2500 daltons, e.g., less than 2000, less than 1000, or less than 500 daltons), circular peptide, peptidomimetic, antibody, polysaccharide, lipid, fatty acid, inhibitory RNA (e.g., siRNA or shRNA), polynucleotide, oligonucleotide, aptamer, drug compound, or other compound.
  • small organic molecule e.g., an organic molecule having a molecular weight of less than about 2500 daltons, e.g., less than 2000, less than 1000, or less
  • inhibitor refers to an alteration, interference, reduction, down regulation, blocking, suppression, abrogation or degradation, directly or indirectly, in the expression, amount or activity of a target gene, target protein, or signaling pathway relative to (1) a control, endogenous or reference target or pathway, or (2) the absence of a target or pathway, wherein the alteration, interference, reduction, down regulation, blocking, suppression, abrogation or degradation is statistically, biologically, or clinically significant.
  • inhibitors or“inhibitor” includes gene“knock out” and gene“knock down” methods, such as by chromosomal editing.
  • Treatment refers to medical management of a disease, disorder, or condition of a subject (i.e., patient), which may be therapeutic,
  • a treatment may improve or decrease the severity at least one symptom of a disease, delay worsening or progression of a disease, or delay or prevent onset of additional associated diseases.“Reducing the risk of developing a disease” refers to preventing or delaying onset of a disease or reoccurrence of one or more symptoms of the disease (e.g., cancer).
  • the immune system e.g., cancer
  • modulation provided by the translational enhancers of this disclosure aids or augments treatment regimens or aids or augments a host organism’s immune system.
  • A“patient” or“subject” includes an animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig.
  • the animal can be a mammal, such as a non-primate and a primate (e.g., monkey and human).
  • a primate e.g., monkey and human.
  • a subject is a human, such as a human infant, child, adolescent or adult.
  • a“mammal” includes primates, such as humans, monkeys and apes, and non- primates such as domestic animals, including laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife or the like.
  • Effective amount refers to that amount of a composition described herein which, when administered to a mammal (e.g., human), is sufficient to aid in treating a disease.
  • the amount of a composition that constitutes a“therapeutically effective amount” will vary depending on the cell preparations, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
  • a therapeutically effective dose refers to that ingredient or composition alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients, compositions or both that result in the therapeutic effect, whether
  • therapeutically effective amount refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
  • hyperproliferative disorder or“hyperproliferative disease” refers to excessive growth or proliferation as compared to a normal cell or an undiseased cell.
  • exemplary hyperproliferative disorders include dysplasia, neoplasia, non-contact inhibited or oncogenically transformed cells, tumors, cancers, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis, or the like).
  • a cancer being treated by the compositions and methods of this disclosure includes carcinoma (epithelial), sarcoma (connective tissue), lymphoma or leukemia (hematopoietic cells), germ cell tumor (pluripotent cells), blastoma (immature“precursor” cells or embryonic tissue), or any combination thereof.
  • carcinoma epidermal
  • sarcoma connective tissue
  • lymphoma or leukemia hematopoietic cells
  • germ cell tumor pluripotot cells
  • blastoma immunoma
  • these various forms of hyperproliferative disease are known in the art and have established criteria for diagnosis and classification (e.g., Hanahan and Weinberg, Cell 144:646, 2011; Hanahan and Weinberg Cell 100:57, 2000; Cavallo et al., Canc. Immunol.
  • nucleobase refers to a nitrogen-containing heterocyclic moiety, which is the parts of the nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand in a sequence specific manner.
  • the most common naturally-occurring nucleobases are: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).
  • modified nucleobase refers to a moiety that can replace a nucleobase.
  • the modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner.
  • a modified nucleobase can pair with at least one of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex.
  • modified nucleoside or“modified nucleotide” refers to a nucleoside or nucleotide that contains a modified nucleobase and/or other chemical modification disclosed herein, such as modified sugar, modified phosphorus atom bridges or modified internucleoside linkage.
  • nucleobases include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine optionally having their respective amino groups protected by, e.g., acyl protecting groups, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5- methylcytosine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6- diaminopurine, azacytosine, 2-thiouracil, 2-thiothymine, 2-aminopurine, N9-(2-amino-6- chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8- aza-guanine), N8-(8-aza-7-deazaadenine), pyrimidine analog
  • nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol.7, 313.
  • mRNA messenger RNA
  • mRNA refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo.
  • An mRNA has been transcribed from a DNA sequence by an RNA polymerase enzyme, and interacts with a ribosome to synthesize genetic information encoded by DNA.
  • mRNA are classified into two sub-classes: pre-mRNA and mature mRNA.
  • Precursor mRNA is mRNA that has been transcribed by RNA polymerase but has not undergone any post-transcriptional processing (e.g., 5‘capping, splicing, editing, and polyadenylation). Mature mRNA has been modified via post-transcriptional processing (e.g. , spliced to remove introns and polyadenylated) and is capable of interacting with ribosomes to perform protein synthesis.
  • An mRNA can be isolated from tissues or cells by a variety of methods. For example, a total RNA extraction can be performed on cells or a cell lysate and the resulting extracted total RNA can be purified (e.g. , on a column comprising oligo-dT beads) to obtain extracted mRNA.
  • mRNA can be synthesized in a cell-free environment, for example by in vitro transcription (IVT).
  • IVT in vitro transcription
  • an IVT template encodes a 5’ untranslated region, contains an open reading frame, and encodes a 3’ untranslated region and a poly A tail.
  • An“polynucleotide template” as used herein, may also include ribonucleic acid (RNA).
  • A“5’ untranslated region refers to a region of an mRNA that is directly upstream (i.e., 5’) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a protein or peptide.
  • A“3’ untranslated region refers to a region of an mRNA that is directly downstream (i.e., 3’) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a protein or peptide.
  • An“open reading frame” is a continuous stretch of RNA beginning with a start codon (e.g., ATG), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a protein or peptide.
  • start codon e.g., ATG
  • stop codon e.g., TAA, TAG or TGA
  • A“poly-A tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, consecutive adenosine monophosphates.
  • a poly-A tail may contain 10 to 300 adenosine monophosphates.
  • a poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
  • a poly-A tail contains 50 to 250 adenosine monophosphates.
  • the poly-A tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus, and translation.
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”).
  • organic base e.g., a purine or pyrimidine
  • nucleotide is defined as a nucleoside including a phosphate group.
  • the modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides).
  • the polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages.
  • the linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. Any combination of base/sugar or linker may be incorporated into the polynucleotides of the disclosure.
  • a“structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural
  • the polynucleotide “ATCG” may be chemically modified to“AT-5meC-G”.
  • the same polynucleotide may be structurally modified from“ATCG” to“ATCCCG”.
  • the dinucleotide“CC” has been inserted, resulting in a structural modification to the polynucleotide.
  • polynucleotides of the disclosure may include at least one chemical modification.
  • the polynucleotides described herein can include various substitutions and/or insertions from native or naturally occurring polynucleotides, e.g., in addition to the modification on the 5’ terminal mRNA cap moieties disclosed herein.
  • the terms“chemical modification” or, as appropriate,“chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribnucleosides and the internucleoside linkages in one or more of their position, partem, percent or population. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5’-terminal mRNA cap moieties.
  • the modifications may be various distinct modifications.
  • the regions may contain one, two, or more (optionally different) nucleoside or nucleotide
  • a modified polynucleotide introduced to a cell may exhibit reduced degradation in the cell as compared to an unmodified polynucleotide.
  • Modifications of the polynucleotides of the disclosure include, but are not limited to those listed in detail below.
  • the polynucleotide may comprise modifications which are naturally occurring, non-naturally occurring or the polynucleotide can comprise both naturally and non-natural occurring modifications.
  • the polynucleotides of the disclosure can include any modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone).
  • One or more atoms of a pyrimidine or purine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
  • Non-natural modified nucleotides may be introduced to polynucleotides during synthesis or post-synthesis of the chains to achieve desired functions or properties.
  • the modifications may be on internucleotide lineage, the purine or pyrimidine bases, or sugar.
  • the modification may be introduced at the terminal of a chain or anywhere else in the chain; with chemical synthesis or with a polymerase enzyme. Any of the regions of the polynucleotides may be chemically modified.
  • the present disclosure provides for polynucleotides comprised of unmodified or modified nucleosides and nucleotides and combinations thereof. As described herein
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”).
  • organic base e.g., a purine or pyrimidine
  • nucleobase also referred to herein as“nucleobase”.
  • nucleotide is defined as a nucleoside including a phosphate group.
  • the modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides).
  • the polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages.
  • the linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. Any combination of base/sugar or linker may be incorporated into the polynucleotides of the disclosure.
  • RNA polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • chemical modification that are useful in the compositions and methods of the present disclosure include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6- methyladenosine; 2- methylthio-N6-threonyl carbamoyladenosine; N6- glycinylcarbamoyladenosine; N6- isopentenyladenosine; N6-methyladenosine; N6- threonylcarbamoyladenosine; l, 2’-0- dimethyladenosine; 1 -methyladenosine; 2’-0- methyladenosine; 2’-0-ribosyladenosine
  • aminoalkylaminocarbonylethylenyl -pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminocarbonylethylenyl)- 4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)- pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; l -(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil; 1- Methy 1-3 -(3
  • Imidizopyridinyl Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2- amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl;
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • LNA locked nucleic acid
  • the term“LNA” or“locked nucleic acid” refers to a methylene bridge between the 2 ⁇ and 4’C of the nucleotide monomer and it also refers to a sugar analog, a nucleoside, a nucleotide monomer, or a nucleic acid, each of which contains such bridge.
  • “Stable compound” and“stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • “Isomerism” means compounds or translational enhancers that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed
  • enantiomers and stereoisomers that are non-superimposable mirror images of each other are termed“enantiomers” or sometimes optical isomers.
  • enantiomers A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a“racemic mixture.”
  • a carbon atom bonded to four nonidentical substituents is termed a“chiral center.”
  • “Chiral isomer” means a compound with at least one chiral center.
  • Compounds or translational enhancers with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.”
  • a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center.
  • tautomer refers to a proton shift from one atom of a molecule to another atom of the same molecule.
  • a tautomer is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds.
  • Tautomers exist as a mixture of a tautomeric set in solution. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.
  • the expressions“one or more of A, B, or C,”“one or more A, B, or C,” “one or more of A, B, and C,”“one or more A, B, and C” and the like are used interchangeably and all refer to a selection from the group consisting of A, B, and /or C, i.e., one or more As, one or more Bs, one or more Cs, or any combination thereof.
  • nucleotides can be altered on the internucleoside linkage (e.g., phosphate backbone).
  • phosphate backbone in the context of the polynucleotide backbone, the phrases“phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.
  • the alternative nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein.
  • Examples of alternative phosphate groups include, but are not limited to, phosphorothioate,
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged
  • translational enhancer refers to any compound or biological molecule that increases the rate, or amount, or both of polypeptide or protein production from an mRNA.
  • a translational enhancer of the disclosure provides an induced or boosted polypeptide or protein production in vitro or in the cell from an mRNA comprising the translational enhancer, preferably more efficiently than a natural mRNA or an mRNA not comprising the translational enhancer of the disclosure.
  • a translational enhancer may induce or boost polypeptide or protein production from mRNAs, e.g., by increasing stability/half- life/bioavailability/biodistribution of endogenous and/or exogenous mRNAs, increasing the translation efficiency of endogenous and/or exogenous mRNAs, inducing an attenuated immune response to exogenous mRNAs, and/or enhancing delivery and/or permeability of exogenous mRNA molecules to cells.
  • a translational enhancer of the disclosure comprises an eIF4E ligand attached to a dinucleotide.
  • the eIF4E ligand is attached directly to the dinucleotide.
  • the eIF4E ligand is attached to the dinucleotide via a linker.
  • the translational enhancers of the disclosure function as a 5’ cap analog or 5’ cap mimetic. In other embodiments, the translational enhancers of the disclosure are a 5’ cap analog or 5’ cap mimetic.
  • binding of the eIF4E ligand to eIF4E does not affect the interaction of eIF4E with any of the other components of the cellular translational machinery (e.g., other translation initiation factors).
  • binding of the eIF4E ligand to eIF4E enhances the interaction of eIF4E with one or more components of the cellular translational machinery.
  • linker refers to moieties that connects or attaches the eIF4E ligand and the dinucleotide moiety of a translational enhancer of the disclosure.
  • “5’ cap analog” or“5’ cap mimetic” refers to a compound that mimics the activity of a natural mRNA 5’ cap structure and binds to eIF4E.
  • RNA molecule of the disclosure and“RNA molecules of the disclosure” are used interchangeably to refer to an RNA molecule (e.g., mRNA molecule) comprising a translational enhancer of the disclosure or a stereoisomer, tautomer, or salt thereof.
  • RNA molecules of the disclosure can be used as therapeutic agents or are therapeutic mRNAs.
  • therapeutic mRNA refers to an mRNA that encodes a therapeutic protein.
  • Therapeutic protein refers to a protein that, when administered to a cell or a subject has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • Therapeutic proteins mediate a variety of effects in a host cell or a subject in order to treat a disease or ameliorate the signs and symptoms of a disease.
  • an RNA or a multimeric structure described herein can be
  • RNA is translated in vivo to produce a therapeutic peptide in the subject in need thereof. Accordingly, provided herein are
  • RNAs e.g., mRNAs
  • RNAs e.g., mRNAs
  • “treating” or“treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of an active ingredient of the disclosure to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder.
  • the term“treat” can also include treatment of a cell in vitro or an animal model.
  • An active ingredient of the disclosure can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes.
  • “preventing,”“prevent,” or“protecting against” or“ameliorating” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.
  • A“pharmaceutical composition” is a formulation containing the active ingredient of the disclosure in a form suitable for administration to a subject.
  • the pharmaceutical composition is in bulk or in unit dosage form.
  • the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial.
  • the quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
  • active ingredient e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof
  • the dosage will also depend on the route of administration.
  • routes of administration A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.
  • Dosage forms for the topical or transdermal administration of an active ingredient of the disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
  • the phrase“pharmaceutically acceptable” refers to those compounds, translational enhancers, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • an“effective amount” of the polynucleotides (e.g., RNA or mRNA) disclosed herein is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the multimeric structures, and other determinants.
  • an effective amount of RNA provides an induced or boosted polypeptide or protein production in the cell, preferably more efficiently than a natural RNA or an RNA not comprising the translational enhancer of the disclosure.
  • Increased polypeptide production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the multimeric structures), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified polynucleotide), or altered polypeptide production in the host cell.
  • the disclosure provides a translational enhancer comprising an eIF4E ligand attached to at least one nucleotide.
  • the eIF4E ligand is attached directly to the at least one nucleotide.
  • the eIF4E ligand is attached to the at least one nucleotide via a linker.
  • the disclosure provides a translational enhancer comprising an eIF4E ligand attached to a dinucleotide.
  • the eIF4E ligand is attached directly to the dinucleotide.
  • the eIF4E ligand is attached to the dinucleotide via a linker.
  • the dinucleotide is selected from the group consisting of an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, and a GG dinucleotide. In certain embodiments, the dinucleotide is an AG dinucleotide. In other embodiments, the dinucleotide is a GG dinucleotide.
  • one or both of the adenosines in an AA dinucleotide is an N 6 - methyladenosine (m 6 A).
  • the dinucleotide is an m 6 A-m 6 A dinucleotide.
  • the dinucleotide is an A-m 6 A or an m 6 A-A dinucleotide.
  • one or both of the adenosines in an AA dinucleotide is an N 6 , 2’-O-dimethyladenosine (m 6 Am).
  • the dinucleotide is an m 6 Am-m 6 Am dinucleotide.
  • the dinucleotide is an A-m 6 Am or an m 6 Am-A dinucleotide. In other instances, the dinucleotide is an m 6 A-m 6 Am or an m 6 Am-m 6 A dinucleotide.
  • the adenosine in an AG or a GA dinucleotide is an m 6 A. In other embodiments, the adenosine in an AG or a GA nucleotide is an m 6 Am. In certain embodiments, the guanosine in the AG or GA dinucleotides may also be modified to a 2’-O- methylguanosine (Gm).
  • the dinucleotide is an m 6 A-G dinucleotide, an m 6 A-Gm dinucleotide, a G-m 6 A dinucleotide, a Gm-m 6 A dinucleotide, an m 6 Am-G dinucleotide, an m 6 Am-Gm dinucleotide, a G-m 6 Am dinucleotide, or a Gm-m 6 Am dinucleotide.
  • the dinucleotide includes, without limitation, an AU dinucleotide, an m 6 A-U dinucleotide, an m 6 Am-U dinucleotide, a UA dinucleotide, a U-m 6 A dinucleotide, a U-m 6 Am dinucleotide, an AC dinucleotide, an m 6 A-C dinucleotide, an m 6 Am-C dinucleotide, a CA dinucleotide, a C-m 6 A dinucleotide, a C-m 6 Am dinucleotide, a GU dinucleotide, a Gm-U dinucleotide, a UG dinucleotide, a U-Gm dinucleotide, a GC dinucleotide, a Gm-C dinucleotide, a CG dinucleotide
  • the cytosine or uracil in the above-disclosed dinucleotides may also be modified to a 2’-O-methylcytosine (Cm) or a 2’-O-methyluracil (Um).
  • the translational enhancer of the disclosure comprises an eIF4E ligand attached directly to the dinucleotide.
  • the translational enhancer of the disclosure comprises an eIF4E ligand attached to the dinucleotide via a linker.
  • the linker can be cleavable or non-cleavable.
  • the term “linker,” or“linker molecule,” or“linking group” means an organic moiety that connects two parts of a compound (e.g., the eIF4E ligand and the dinucleotide of a translational enhancer of the disclosure).
  • the term“linker” refers to moieties that connect the eIF4E ligand and the dinucleotide moiety of a translational enhancer of the disclosure.
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO 2 , SO 2 NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or
  • alkynyl unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhe
  • alkynylheteroarylalkyl alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
  • alkylheterocyclylalkyl alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,
  • alkenylheterocyclylalkyl alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
  • alkynylheterocyclylalkyl alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO 2 , N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • the linker is between 1-24 atoms, e.g., 4-24 atoms, 6-18 atoms, 8-18 atoms, or 8- 16 atoms.
  • the linker is a phosphate linker.
  • the phosphate linker is a monophosphate linker.
  • the phosphate linker is a diphosphate linker.
  • the phosphate linker is a triphosphate linker.
  • the phosphate linker is a tetraphosphate linker.
  • the linker is a phosphonate-diphosphate linker.
  • linker is a surrogate linker.
  • the phosphonate-diphosphate linker is more stable than the monophosphate, diphosphate, triphosphate, and tetraphosphate linkers.
  • the structural formula of a translational enhancer of the disclosure represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like, it being understood that not all isomers may have the same level of activity.
  • a crystal polymorphism may be present for the translational enhancers of the invention. It is noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.
  • the translational enhancer of the disclosure has an improved binding affinity for eIF4E as compared to, e.g., natural mRNA caps (m7GTP).
  • the translational enhancer of the disclosure has greater than about 1.2 fold, about 1.5 fold, about 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 300 fold, about 400 fold, about 500 fold, or about 1,000 fold higher binding affinity for eIF4E as compared to, e.g., natural mRNA caps (m7GTP).
  • the translational enhancer of the disclosure has greater than about 1,000 fold higher binding affinity for eIF4E as compared to, e.g., natural mRNA caps (m7GTP). In certain aspects, the translational enhancer of the disclosure is greater than about 1,000 fold more potent than, e.g., natural mRNA caps (m7GTP) in binding eIF4E. In some aspects the translational enhancer of the disclosure exhibits picomolar cell potency for binding eIF4E. In certain aspects, the translational enhancer of the disclosure has an improved binding affinity for eIF4E as compared to Anti-Reverse Cap Analog (ARCA).
  • ARCA Anti-Reverse Cap Analog
  • the translational enhancer of the disclosure has greater than about 1.2 fold, about 1.5 fold, about 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 300 fold, about 400 fold, about 500 fold, or about 1,000 fold higher binding affinity for eIF4E as compared to ARCA. In specific aspects, the translational enhancer of the disclosure has greater than about 1,000 fold higher binding affinity for eIF4E as compared to ARCA. [0145] In certain embodiments, binding of the translational enhancer to eIF4E does not affect the interaction of eIF4E with any of the other components of the cellular translational machinery (e.g., other translation initiation factors). In certain embodiments, binding of the translational enhancer to eIF4E enhances the interaction of eIF4E with one or more components of the cellular translational machinery.
  • k off is the off-rate, calculated from the dissociation phase
  • k on is the on- rate, calculated from the association phase
  • K d or K D is the binding affinity, which is the ratio of k off / k on
  • the residence time, t is the inverse of k off .
  • the translational enhancers of the disclosure with an improved eIF4E binding affinity have a K d or K D of no more than 10 mM, e.g., using surface plasmon resonance (SPR).
  • K d of the compound is no more than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.7, 0.5, 0.3, or 0.1 mM.
  • the compound has an eIF4E K d of no more than 10 mM (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.9, 0.7, 0.5, 0.3, or 0.1 mM) and a t of about 2 seconds or longer (e.g., 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 50 seconds, 75 seconds, 80 seconds, 90 seconds, 100 seconds, or longer).
  • the translational enhancers of the disclosure with an improved eIF4E binding affinity have a K d in the picomolar range.
  • the translational enhancers of the invention are capable of being co-transcriptionally attached to an RNA molecule. In certain embodiments, the translational enhancers of the invention are capable of being chemically attached to an RNA molecule. In other embodiments, the translational enhancers of the invention are capable of being enzymatically attached to an RNA molecule. In certain embodiments, the translational enhancers of the invention are co-transcriptionally, chemically, or enzymatically attached to the 5’ end of an RNA molecule. In certain aspects, the translational enhancers of the invention act as 5’ RNA cap structures. In certain embodiments, the translational enhancers of the invention are co-transcriptionally, chemically, or enzymatically attached to the 5’ end of RNA molecules to provide 5’ capped RNAs.
  • the translational enhancers of the disclosure provide capping reagents for in vitro or in vivo transcription of 5’ capped RNA resulting in a Cap 1, Cap 2, Cap 3, or Cap 4 structure. In certain aspects, the translational enhancers of the disclosure provide capping reagents for in vitro transcription of 5’ capped RNA resulting in a Cap 1 structure. Thus, in certain aspects, the translational enhancers of the disclosure provide significant advantages over current methods and compositions involving use of various initiating nucleosides, nucleotides and oligonucleotides or use of polyphosphate dinucleotide derivatives containing Cap 0 structure, such as mCAP and ARCA.
  • the translational enhancers of the disclosure are compatible with existing transcription systems and reagents and no additional enzymes or reagents are required for the generation of the Cap 1 RNAs.
  • the use of the translational enhancers of the disclosure makes several non- enzymatic and enzymatic steps (such as capping and 2’-O-methylation) unnecessary thus reducing complexity of the process and a cost of RNA synthesis.
  • the invention is directed to a translational enhancer having a structure:
  • X 1 is CR 2 , -C-L 1 -Y or N;
  • X 2 , X 5 and X 6 are independently CR 2 or N,
  • X 3 is C, or X 3 is C or N when X 4 is a bond;
  • X 4 is a bond, CR 2 or N
  • Q is H or–L 1 -Y;
  • Ring B is a six-membered aryl, heteroaryl or heterocyclyl
  • R 1 is H, OH, halo, CN, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, (C 3 -C 6 )cycloalkyl or NR 5 R 5 ;
  • R 2 is independently H, halo, CN, NO, NO 2 , CoH, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, CH 2 SR 5 , OR 5 , NHR 5 , NR 5 R 5 , [(C 1 -C 8 )alkylene]heterocyclyl, [(C 1 -C 8 )alkylene]heteroaryl, [(C 1 -C 8 )alkylene]NHR 5 , [(C 1 -C 8 )alkylene]NR 5 R 5 , [(C 1 -C 8 )alkylyne]NR 5 R 5 , C(O)R 5 , C(O)
  • R 4 is H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, SR 7 or Z, wherein Z is
  • Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl
  • R 5 is independently H, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 3 -C 5 )cycloalkyl, CO 2 H, [(C 1 - C 3 )alkylene]heteroaryl, [(C 1 -C 3 )alkylene]aryl, [(C 1 -C 3 )alkylene]CO 2 H, heterocyclyl, aryl or heteroaryl, or wherein two R 5 substituents together with a nitrogen atom form a 4-, 5-, 6- or 7- membered heterocyclyl;
  • R 6 is independently H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, NHR 7 , NR 7 R 7 , CO 2 H, [(C 1 -C 3 )alkylene]CO 2 H, (C 3 -C 5 )cycloalkyl, SR 7 , NH(CO)R 7 or
  • R 7 is independently H, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 8 is H, OH, CO 2 H, CO 2 R 7 , CF 2 C(R 6 ) 2 OH, C(R 6 ) 2 OH, C(CF 3 ) 2 OH, SO 2 H, SO 3 H, CF 2 SO 2 C(R 6 ) 3 , CF 2 SO 2 N(H)R 5 , SO 2 N(H)R 5 , SO 2 N(H)C(O)R 6 , C(O)N(H)SO 2 R 5 ,
  • n 0, 1, 2 or 3;
  • p 0, 1, 2 or 3;
  • any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH 3 , SO 2 CH 3, SO 2 NH 2 ,
  • X is a linker and Y is a dinucleotide.
  • the present invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure encompassed by the genus of Formula I, or stereoisomers, tautomers or pharmaceutically acceptable salts thereof.
  • the invention is directed to a translational enhancer, wherein the [0153] translational enhancer comprises an eIF4E ligand having a structure according to Formula II:
  • X 2 and X 5 are independently CR 2 or N,
  • Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl
  • R 1 is H, OH, halo, CN, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, (C 3 -C 6 )cycloalkyl or NR 5 R 5 ;
  • R 2 is independently H, halo, CN, NO, NO 2 , CoH, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, CH 2 SR 5 , OR 5 , NHR 5 , NR 5 R 5 , [(C 1 -C 8 )alkylene]heterocyclyl, [(C 1 -C 8 )alkylene]heteroaryl, [(C 1 -C 8 )alkylene]NHR 5 , [(C 1 -C 8 )alkylene]NR 5 R 5 , [(C 1 -C 8 )alkylyne]NR 5 R 5 , C(O)R 5 , C(O)
  • R 5 is independently H, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 3 -C 5 )cycloalkyl, CO 2 H, [(C 1 - C 3 )alkylene]heteroaryl, [(C 1 -C 3 )alkylene]aryl, [(C 1 -C 3 )alkylene]CO 2 H, heterocyclyl, aryl or heteroaryl, or wherein two R 5 substituents together with a nitrogen atom form a 4-, 5-, 6-, or 7- membered heterocyclyl;
  • R 6 is independently H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, NHR 7 , NR 7 R 7 , CO 2 H, [(C 1 -C 3 )alkylene]CO 2 H, (C 3 -C 5 )cycloalkyl, SR 7 , NH(CO)R 7 or
  • R 7 is independently H, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 8 is H, OH, CO 2 H, CO 2 R 7 , CF 2 C(R 6 ) 2 OH, C(R 6 ) 2 OH, C(CF 3 ) 2 OH, SO 2 H, SO 3 H, CF 2 SO 2 C(R 6 ) 3 , CF 2 SO 2 N(H)R 5 , SO 2 N(H)R 5 , SO 2 N(H)C(O)R 6 , C(O)N(H)SO 2 R 5 ,
  • n 0, 1, 2 or 3;
  • n 0, 1, 2 or 3;
  • p 0, 1, 2 or 3;
  • any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH 3 , SO 2 CH 3, SO 2 NH 2 ,
  • the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula III:
  • Ring C is a heteroaryl
  • R 1 is H, OH, halo, CN, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, (C 3 -C 6 )cycloalkyl or NR 5 R 5 ;
  • R 2 is independently H, halo, CN, NO, NO 2 , CoH, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, CH 2 SR 5 , OR 5 , NHR 5 , NR 5 R 5 , [(C 1 -C 8 )alkylene]heterocyclyl, [(C 1 -C 8 )alkylene]heteroaryl, [(C 1 -C 8 )alkylene]NHR 5 , [(C 1 -C 8 )alkylene]NR 5 R 5 , [(C 1 -C 8 )alkylyne]NR 5 R 5 , C(O)R 5 , C(O)
  • R 5 is independently H, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 3 -C 5 )cycloalkyl or
  • R 6 is independently H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, NHR 7 , NR 7 R 7 , CO 2 H, [(C 1 -C 3 )alkylene]CO 2 H, (C 3 -C 5 )cycloalkyl, SR 7 , NH(CO)R 7 or
  • R 7 is independently H, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 8 is H, OH, CO 2 H, CO 2 R 7 , CF 2 C(R 6 ) 2 OH, C(R 6 ) 2 OH, C(CF 3 ) 2 OH, SO 2 H, SO 3 H, CF 2 SO 2 C(R 6 ) 3 , CF 2 SO 2 N(H)R 5 , SO 2 N(H)R 5 , SO 2 N(H)C(O)R 6 , C(O)N(H)SO 2 R 5 ,
  • R 9 is H, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, cycloalkyl or heterocyclyl;
  • n 0, 1, or 2;
  • n 0, 1, 2 or 3;
  • p 0, 1, 2 or 3;
  • any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH 3 , SO 2 CH 3, SO 2 NH 2 ,
  • the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula IV:
  • X 2 and X 5 are independently CR 2 or N,
  • X 3 is C, or X 3 is C or N when X 4 is a bond; X 4 is a bond, CR 2 or N,
  • Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl
  • R 1 is H, OH, halo, CN, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, (C 3 -C 6 )cycloalkyl or NR 5 R 5 ;
  • R 2 is independently H, halo, CN, NO, NO 2 , CoH, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, CH 2 SR 5 , OR 5 , NHR 5 , NR 5 R 5 , [(C 1 -C 8 )alkylene]heterocyclyl, [(C 1 -C 8 )alkylene]heteroaryl, [(C 1 -C 8 )alkylene]NHR 5 , [(C 1 -C 8 )alkylene]NR 5 R 5 , [(C 1 -C 8 )alkylyne]NR 5 R 5 , C(O)R 5 , C(O)
  • R 5 is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C5)cycloalkyl, CO2H, [(C1- C 3 )alkylene]heteroaryl, [(C 1 -C 3 )alkylene]aryl, [(C 1 -C 3 )alkylene]CO 2 H, heterocyclyl, aryl or heteroaryl,
  • R 6 is independently H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, NHR 7 , NR 7 R 7 , CO 2 H, [(C 1 -C 3 )alkylene]CO 2 H, (C 3 -C 5 )cycloalkyl, SR 7 , NH(CO)R 7 or
  • R 7 is independently H, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 8 is H, OH, CO2H, CO2R 7 , CF2C(R 6 )2OH, C(R 6 )2OH, C(CF3)2OH, SO2H, SO3H, CF 2 SO 2 C(R 6 ) 3 , CF 2 SO 2 N(H)R 5 , SO 2 N(H)R 5 , SO 2 N(H)C(O)R 6 , C(O)N(H)SO 2 R 5 ,
  • p 0, 1, 2 or 3;
  • any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH 3 , SO 2 CH 3, SO 2 NH 2 ,
  • ring A forms a 5-membered heteroaryl wherein X 1 and X 5 can in addition to C be N.
  • the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula V:
  • Ring B is a six-membered aryl, heteroaryl or heterocyclyl
  • R 1 is H, OH, halo, CN, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, (C 3 -C 6 )cycloalkyl or NR 5 R 5 ;
  • R 2 is independently H, halo, CN, NO, NO 2 , CoH, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, CH 2 SR 5 , OR 5 , NHR 5 , NR 5 R 5 , [(C 1 -C 8 )alkylene]heterocyclyl, [(C 1 -C 8 )alkylene]heteroaryl, [(C 1 -C 8 )alkylene]NHR 5 , [(C 1 -C 8 )alkylene]NR 5 R 5 , [(C 1 -C 8 )alkylyne]NR 5 R 5 , C(O)R 5 , C(O)
  • R 4 is H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, SR 7 or Z, wherein Z is
  • Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl
  • R 5 is independently H, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 3 -C 5 )cycloalkyl, CO 2 H, [(C 1 - C3)alkylene]heteroaryl, [(C1-C3)alkylene]aryl, [(C1-C3)alkylene]CO2H, heterocyclyl, aryl or heteroaryl,
  • R 6 is independently H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, NHR 7 , NR 7 R 7 , CO 2 H, [(C 1 -C 3 )alkylene]CO 2 H, (C 3 -C 5 )cycloalkyl, SR 7 , NH(CO)R 7 or
  • R 7 is independently H, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 8 is H, OH, CO 2 H, CO 2 R 7 , CF 2 C(R 6 ) 2 OH, C(R 6 ) 2 OH, C(CF 3 ) 2 OH, SO 2 H, SO 3 H, CF 2 SO 2 C(R 6 ) 3 , CF 2 SO 2 N(H)R 5 , SO 2 N(H)R 5 , SO 2 N(H)C(O)R 6 , C(O)N(H)SO 2 R 5 ,
  • C(O)haloalkyl C(O)N(H)OR 5 , C(O)N(R 5 )OH, C(O)N(H)R 5 , P(O)(OR 5 )OH, P(O)(O)N(H)R 5 , P(O)(C(R 6 ) 3 )C(R 6 ) 3 , B(OH) 2 , heterocyclyl or heteroaryl;
  • n 0, 1, 2 or 3;
  • p 0, 1, 2 or 3;
  • q 0, 1, 2, 3 or 4;
  • any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH 3 , SO 2 CH 3, SO 2 NH 2 ,
  • the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula VI:
  • Q is–L 1 -Y;
  • Ring B is a six-membered aryl, heteroaryl or heterocyclyl
  • R 1 is H, OH, halo, CN, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, (C 3 -C 6 )cycloalkyl or NR 5 R 5 ;
  • R 2 is independently H, halo, CN, NO, NO 2 , CoH, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, CH 2 SR 5 , OR 5 , NHR 5 , NR 5 R 5 , [(C 1 -C 8 )alkylene]heterocyclyl, [(C 1 -C 8 )alkylene]heteroaryl, [(C 1 -C 8 )alkylene]NHR 5 , [(C 1 -C 8 )alkylene]NR 5 R 5 , [(C 1 -C 8 )alkylyne]NR 5 R 5 , C(O)R 5 , C(O)
  • R 4 is H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, SR 7 or Z, wherein Z is
  • Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl
  • R 5 is independently H, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 3 -C 5 )cycloalkyl, CO 2 H, [(C 1 - C 3 )alkylene]heteroaryl, [(C 1 -C 3 )alkylene]aryl, [(C 1 -C 3 )alkylene]CO 2 H, heterocyclyl, aryl or heteroaryl, or wherein two R 5 substituents together with a nitrogen atom form a 4-, 5-, or 6- membered heterocyclyl;
  • R 6 is independently H, OH, halo, CN, (C 1 -C 3 )alkyl, (C 1 -C 3 )haloalkyl, (C 1 -C 3 )alkoxy, NHR 7 , NR 7 R 7 , CO 2 H, [(C 1 -C 3 )alkylene]CO 2 H, (C 3 -C 5 )cycloalkyl, SR 7 , NH(CO)R 7 or
  • R 7 is independently H, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 8 is H, OH, CO 2 H, CO 2 R 7 , CF 2 C(R 6 ) 2 OH, C(R 6 ) 2 OH, C(CF 3 ) 2 OH, SO 2 H, SO 3 H, CF 2 SO 2 C(R 6 ) 3 , CF 2 SO 2 N(H)R 5 , SO 2 N(H)R 5 , SO 2 N(H)C(O)R 6 , C(O)N(H)SO 2 R 5 ,
  • n 0, 1, 2 or 3;
  • p 0, 1, 2 or 3;
  • q 0, 1, 2, 3 or 4;
  • any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH 3 , SO 2 CH 3, SO 2 NH 2 ,
  • X 2 of Formulae I, II, and III is N.
  • X 2 of Formulae I, II, and IV is N.
  • X 3 of Formulae I and IV is C.
  • X 4 of Formulae I and IV is CR 2 or N.
  • X 5 of Formulae I and IV is CR 2 .
  • L 1 of Formulae I, II, III, IV, V and VI is–(CH 2 ) 2 -O–
  • L 1 is–(CH 2 ) 2 -O–.
  • L 2 of Formulae I, II, III, IV, V and VI is a bond.
  • Ring B of Formulae I, V and VI is aryl.
  • Ring C of Formulae I, II, III, IV, V and VI is heteroaryl.
  • Ring C of Formulae I, II, III, IV, V and VI is
  • Ring C of Formula III is
  • R 1 of Formulae I, II, III, IV, V and VI is H, (C 1 -C 8 )alkyl or (C 1 - C 8 )haloalkyl.
  • R 1 of Formula IV is NHR 5 or N[(C 1 -C 3 )alkyl](R 5 ).
  • R 2 of Formulae I, II, III, IV, V and VI is halo, CN, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl or OR 5 .
  • R 2 is halo, CN or (C 1 -C 8 )haloalkyl.
  • R 3 of Formulae I, II, III, IV, V and VI is halo, CN, (C 1 -C 3 )alkyl or (C 1 -C 3 )haloalkyl.
  • R 4 of Formulae I, V and VI is Z, wherein Z is [0174]
  • R 5 of Formulae I, II, III, V and VI is H, (C 1 -C 3 )alkyl or (C 1 - C 3 )haloalkyl.
  • R 5 of Formula IV is aryl.
  • R 6 of Formulae I, II, III, IV, V and VI is H, OH, halo, CN, (C 1 - C 3 )alkyl, (C 1 -C 3 )haloalkyl or (C 1 -C 3 )alkoxy.
  • R 7 of Formulae I, II, III, IV, V and VI is H, (C 1 -C 8 )alkyl or (C 1 - C 8 )haloalkyl.
  • R 8 of Formulae I, II, III, IV, V and VI is CO 2 H or C(O)N(H)SO 2 R 5 .
  • R 9 of Formula III is (C 1 -C 8 )alkyl or (C 1 -C 8 )haloalkyl.
  • R 9 of Formula III is cycloalkyl or heterocyclyl.
  • the optional substituents of alkyl, cycloalkyl, heterocyclyl, heteroaryl or aryl are OH, CN, halogen, (C 1 -C 8 )alkyl, O(C 1 -C 8 )alkyl, haloalkyl, alkylene- C(O)NH 2 or alkylene-C(O)-NH(Me).
  • the optional substituents of alkyl, cycloalkyl, heterocyclyl, heteroaryl or aryl are cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with OH, halogen, (C 1 -C 8 )alkyl, (C 1 -C 8 )haloalkyl, O(C 1 -C 8 )alkyl or O(C 1 -C 8 )haloalkyl.
  • the compounds according to Formulae I, II, III, IV, V and VI are selected from
  • the present disclosure provides a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula I, II, III, IV, V or VI.
  • the present disclosure provides a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand of Formula I, II, III, IV, V or VI attached to a dinucleotide.
  • the eIF4E ligand of Formula I, II, III, IV, V or VI is attached directly to the dinucleotide.
  • the eIF4E ligand of Formula I, II, III, IV, V or VI is attached to the dinucleotide via a linker.
  • the translational enhancer of the disclosure comprises an eIFE ligand, wherein the eIF4E ligand has a structure according to Formula I.
  • the translational enhancer of the disclosure comprises an eIF4E ligand attached to a dinucleotide, wherein the eIF4E ligand has a structure according to Formula I.
  • the eIF4E ligand of Formula I is attached directly to the dinucleotide.
  • the eIF4E ligand of Formula I is attached to the dinucleotide via a linker.
  • the translational enhancer has a structure:
  • eIF4E ligand has a structure according to Formula I, II, III, IV, V, or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide.
  • the translational enhancer has a structure:
  • the translational enhancer has a structure:
  • the translational enhancer has a structure:
  • the translational enhancers of the disclosure are used to couple to RNA sequences of interest.
  • the translational enhancers of the disclosure are coupled to the 5’ end of RNA sequences of interest.
  • coupling of the translational enhancers of the disclosure to the 5’ end of RNA sequences of interest generates 5’ capped RNA molecules.
  • the translational enhancer functions as a 5’ cap structure.
  • the translational enhancer functions as a 5’ cap analog or 5’ cap mimetic.
  • the translational enhancer is a 5’ cap analog or 5’ cap mimetic.
  • the 5’ capped RNA molecules are Cap 0, Cap 1, Cap 2, Cap 3, or Cap 4 RNA molecules.
  • the 5’ capped RNA molecules are Cap 1 RNA molecules.
  • the present disclosure provides an RNA molecule comprising a translational enhancer of the disclosure.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer consists of or consists essentially of an eIF4E ligand.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to at least one nucleotide.
  • the eIF4E ligand is attached directly to the at least one nucleotide. In other aspects, the eIF4E ligand is attached to the at least one nucleotide via a linker. In other embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to a dinucleotide. In certain embodiments, the eIF4E ligand is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand is attached to the dinucleotide via a linker.
  • a translational enhancer of the disclosure is attached to the 5’ end of an RNA molecule.
  • the translational enhancer is attached directly to the 5’ end of the RNA molecule.
  • the translational enhancer is attached to the 5’ end of theRNA molecule via the at least one nucleotide.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand of Formula I, II, III, IV, V or VI.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer consists of or consists essentially of an eIF4E ligand of Formula I, II, III, IV, V or VI.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand of Formula I, II, III, IV, V or VI attached to a dinucleotide.
  • the eIF4E ligand is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand is attached to the dinucleotide via a linker.
  • the translational enhancer is attached to the 5’ end of an RNA molecule. In specific aspects, the translational enhancer is attached to the 5’ end of an RNA molecule via the Y moiety.
  • the present disclosure provides an RNA molecule comprising a translational enhancer comprising an eIF4E ligand, wherein the eIF4E ligand has a structure according to Formula I.
  • the present disclosure provides an RNA molecule comprising a translational enhancer consisting of or consisting essentially of an eIF4E ligand, wherein the eIF4E ligand has a structure according to Formula I.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, comprising an eIF4E ligand attached to a dinucleotide, wherein the eIF4E ligand has a structure according to Formula I.
  • the eIF4E ligand is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand is attached to the dinucleotide via a linker.
  • the translational enhancer is attached to the 5’ end of an RNA molecule. In specific aspects, the translational enhancer is attached to the 5’ end of an RNA molecule via the Y moiety.
  • coupling of the RNA molecules of the disclosure to the translational enhancer does not affect the interaction of the translational enhancer with any of the other components of the cellular translational machinery (e.g., eIF4E). In other embodiments, coupling of the RNA molecules of the disclosure to the translational enhancer enhances the interaction of the translational enhancer with one or more components of the cellular translational machinery (e.g., eIF4E). In other embodiments, coupling of the RNA molecules of the disclosure to the translational enhancer enhances the interaction of the translational enhancer with one or more components of the cellular translational machinery (e.g., eIF4E). In other embodiments, coupling of the RNA molecules of the disclosure to the translational enhancer enhances the interaction of the translational enhancer with one or more components of the cellular
  • translational machinery e.g., eIF4E
  • the dinucleotide is selected from the group consisting of an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, and a GG dinucleotide.
  • the dinucleotide is an AG dinucleotide.
  • the dinucleotide is a GG dinucleotide.
  • dinucleotide is an N 6 -methyladenosine (m 6 A).
  • the dinucleotide is an m 6 A-m 6 A dinucleotide.
  • the dinucleotide is an A-m 6 A or an m 6 A-A
  • the dinucleotide is an m 6 Am- m 6 Am dinucleotide. In other aspects, the dinucleotide is an A-m 6 Am or an m 6 Am-A
  • the adenosine in an AG or a GA dinucleotide is an m 6 A. In other embodiments, the adenosine in an AG or a GA nucleotide is an m 6 Am. In certain embodiments, the guanosine in the AG or GA dinucleotides may also be modified to a 2’-O- methylguanosine (Gm).
  • the dinucleotide is an m 6 A-G dinucleotide, an m 6 A-Gm dinucleotide, a G-m 6 A dinucleotide, a Gm-m 6 A dinucleotide, an m 6 Am-G dinucleotide, an m 6 Am-Gm dinucleotide, a G-m 6 Am dinucleotide, or a Gm-m 6 Am dinucleotide.
  • the dinucleotide includes, without limitation, an AU
  • the cytosine or uracil in the above-disclosed dinucleotides may also be modified to a 2’-O-methylcytosine (Cm) or a 2’-O-methyluracil (Um).
  • the linker is a phosphate linker.
  • the phosphate linker is a monophosphate linker.
  • the phosphate linker is a diphosphate linker.
  • the phosphate linker is a triphosphate linker.
  • the phosphate linker is a tetraphosphate linker.
  • the linker is a phosphonate-diphosphate linker.
  • linker is a surrogate linker.
  • the phosphonate-diphosphate linker is more stable than the monophosphate
  • diphosphate diphosphate, triphosphate, and tetraphosphate linkers.
  • the linkers of the invention typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as a, C(O), C(O)NH, SO, SO 2 , SO 2 NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or
  • alkenyl unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,
  • alkylheteroarylalkenyl alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl,
  • alkynylheteroarylalkynyl alkylheterocyclylalkyl, alkylheterocyclylalkenyl,
  • alkylhererocyclylalkynyl alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,
  • alkenylheterocyclylalkynyl alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
  • alkynylheterocyclylalkynyl alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl,
  • the linker is between 1-24 atoms, e.g., 4-24 atoms, 6-18 atoms, 8-18 atoms, or 8-16 atoms.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula I.
  • the present disclosure provides an RNA molecule comprising a translational enhancer having a structure:
  • the eIF4E ligand has a structure according to Formula I, II, II, IV, V or VI, X is a linker; Y is a dinucleotide, and wherein the translational enhancer is attached to the 5’ end of the RNA molecule.
  • the dinucleotide is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide.
  • the translational enhancer is attached to the 5’ end of the RNA molecule via the Y moiety.
  • the translational enhancers of the invention are co- transcriptionally attached to the 5’ end of an RNA molecule.
  • a method of making an RNA molecule comprising a translational enhancer of the disclosure or a stereoisomer, tautomer, or salt thereof (“the RNA molecule of the disclosure”), comprising reacting a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, and a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into one or more RNA molecules, whereby at least one RNA molecule incorporates at its 5’ end the translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof to generate an RNA molecule of the invention.
  • the method further comprises purifying the RNA molecule of the
  • a method of making a capped RNA molecule comprising: (a) reacting a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase and under conditions conducive to transcription by the RNA polymerase to generate at least one RNA molecule from the polynucleotide template; and (b) co-transcriptionally coupling to a 5’ end of the at least one RNA molecule a translational enhancer of the disclosure.
  • a method of making a capped RNA molecule comprising: (a) reacting a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase and under conditions conducive to transcription by the RNA polymerase to generate at least one RNA molecule from the polynucleotide template; and (b) co-transcriptionally coupling to a 5’ end of the at least one RNA molecule a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof , according to the structure:
  • eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide, thereby generating a capped RNA molecule.
  • Y is an AG dinucleotide. In other embodiments, Y is a GG dinucleotide. In certain cases, the linker is a phosphate linker. In other aspects, the linker is a phosphonate-diphosphate linker. In certain aspects, the RNA molecule is a single-stranded RNA molecule. In other aspects, the single-stranded RNA molecule is an mRNA.
  • the polynucleotide template is a complementary DNA (cDNA).
  • the polynucleotide template comprises any nucleotide base at its 5’ terminal.
  • the polynucleotide template comprises at least one guanine nucleobase at its 5’ terminal.
  • the polynucleotide template comprises two guanine nucleobases at its 5’ terminal.
  • the polynucleotide template comprises three guanine nucleobases at its 5’ terminal.
  • the presence of one or more guanine nucleobases results in efficient transcription of the polynucleotide template by the RNA polymerase to generate the RNA molecule.
  • the RNA polymerase includes, without limitation, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, eukaryotic RNA polymerase II, a thermostable RNA polymerase, and an RNA-dependent RNA polymerase.
  • the RNA polymerase is T7 RNA polymerase.
  • the translational enhancers of the invention are chemically attached to the 5’ end of an RNA molecule.
  • a method of making an RNA of the invention comprising chemically synthesizing at least one RNA molecule having attached at its 5’ end a translational enhancer of the disclosure.
  • a method of making an RNA molecule of the invention comprising: (a) providing the RNA molecule; and (b) chemically coupling to a 5’ end of the RNA molecule a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, thereby producing a 5’ capped RNA molecule.
  • the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand. In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer consists of or consists essentially of an eIF4E ligand. In certain embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to at least one nucleotide. In certain cases, the method further comprises purifying the RNA molecule of the invention from unreacted components of the reaction.
  • the RNA molecules of the invention, thus produced are used in downstream reactions without further purification.
  • the RNAs used in the chemical reaction are double stranded RNAs.
  • the RNAs used in the chemical reaction are single stranded RNAs including, without limitation, mRNAs.
  • the RNAs used in the chemical reaction are non-mammalian RNAs, including, without limitation, viral RNAs.
  • the RNAs used in the chemical reaction include, without limitation, short interfering RNAs (siRNAs), inhibitory RNAs (RNAi), micro RNAs (miRNAs), short hairpin-loop RNAs (shRNAs), small nuclear RNAs (snRNAs), single guide RNAs (sgRNAs), or a derivative thereof.
  • the RNAs used in the chemical reaction are mRNAs.
  • the translational enhancers of the invention are
  • RNA molecules of the invention comprising reacting a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, and a RNA molecule in the presence one or more capping enzymes, whereby at least one RNA molecule incorporates at its 5’ end the translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof to generate an RNA molecule of the invention.
  • the method further comprises purifying the RNA molecule of the invention from unreacted components of the reaction.
  • the RNA molecules of the invention, thus produced are used in downstream reactions without further purification.
  • the RNAs used in the enzymatic reaction are double stranded RNAs. In other embodiments, the RNAs used in the enzymatic reaction are single stranded RNAs including, without limitation, mRNAs. In other embodiments, the RNAs used in the enzymatic reaction are non-mammalian RNAs, including, without limitation, viral RNAs. In certain embodiments, the RNAs used in the enzymatic reaction include, without limitation, short interfering RNAs (siRNAs), inhibitory RNAs (RNAi), micro RNAs (miRNAs), short hairpin-loop RNAs (shRNAs), small nuclear RNAs (snRNAs), or a derivative thereof.
  • siRNAs short interfering RNAs
  • RNAi inhibitory RNAs
  • miRNAs micro RNAs
  • shRNAs short hairpin-loop RNAs
  • snRNAs small nuclear RNAs
  • the RNAs used in the enzymatic reaction are mRNAs.
  • the capping efficiency of the method is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 990%, or 100%.
  • the RNA molecule of the disclosure has enhanced resistance to degradation by one or more cellular exonucleases. In certain embodiments, the one or more cellular exonucleases are 5’-3’ exonucleases. In certain embodiments, the RNA molecule of the disclosure has enhanced resistance to degradation by one or more cellular exonucleases compared to an RNA molecule not comprising the translational enhancer. In certain aspects, the one or more cellular exonucleases are 5’-3’exonucleases. Thus, in certain aspects, the RNA molecule of the disclosure has enhanced resistance to degradation by one or more cellular 5’- 3’exonucleases compared to an RNA molecule not comprising the translational enhancer.
  • the RNA molecule of the disclosure has an increased half-life in a cellular environment compared to that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain aspects, the RNA molecule of the disclosure has a half-life in a cellular environment that is at least 1.2 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure.
  • the half-life of the RNA molecule of the invention in a cellular environment is at least 1.5, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising the translational enhancer of the disclosure.
  • the RNA molecule of the disclosure has half-life in a cellular environment that is at least 1.2 times of that of an RNA molecule not comprising the translational enhancer.
  • the RNA molecule of the disclosure when administered to a subject, has an increased half-life compared to that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain embodiments, when administered to a subject, the RNA molecule of the disclosure has a half- life in a cellular environment that is at least 1.2 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure.
  • the half-life of the RNA molecule of the invention is at least 1.5, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising the translational enhancer of the disclosure.
  • the RNA molecule of the disclosure when administered to a subject, has half-life that is at least 1.2 times of that of an RNA molecule not comprising the translational enhancer.
  • the RNA molecule of the disclosure has an enhanced biodsitribution when administered to a subject compared to the biodistribution of a natural RNA molecule or an RNA molecule not comprising the translational enhancer of the disclosure.
  • RNA molecules can be decapped in a process known as RNA decapping.
  • the process of mRNA decapping consists of hydrolysis of the 5’ cap structure on the RNA by enzymes dcp1 and dcp2, exposing a 5’ monophosphate.
  • this 5’ monophosphate is a substrate for the 5’ exonulease Xrn1 and the mRNA is quickly destroyed.
  • the RNA molecule of the disclosure is not recognized by one or more de-capping enzymes.
  • the RNA molecule of the disclosure is recognized by one or more de-capping enzymes to a lesser extent compared to an RNA molecule not comprising a translational enhancer of the disclosure. In some embodiments, the RNA molecule of the disclosure is resistant to activity of one or more de-capping enzymes. In other embodiments, the RNA molecule of the disclosure has enhanced resistance to activity of one or more de-capping enzymes compared to an RNA molecule not comprising a translational enhancer of the disclosure. In certain aspects, the one or more de-capping enzymes include, but are not limited to, dcp1 and dcp2. In certain embodiments, the RNA molecule of the disclosure is resistant to activity of one or more de-capping enzymes selected from the group consisting of dcp1 and dcp2.
  • the present disclosure provides an RNA molecule comprising a translational enhancer having an improved eIF4E binding affinity, enhanced resistance to degradation, or both, as compared to, e.g., natural mRNA molecules and RNA molecules not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure have improved binding affinity for eIF4E compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • RNA molecules of the disclosure have greater than about 1.2 fold, about 1.5 fold, about 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 300 fold, about 400 fold, about 500 fold, or about 1,000 fold higher binding affinity for eIF4E compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure have greater than about 1,000 fold higher binding affinity for eIF4E as compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure are greater than about 1,000 fold more potent in binding eIF4E than an RNA molecule not comprising a translational enhancer of the disclosure.
  • binding of the RNA molecules of the disclosure to eIF4E does not affect the interaction of eIF4E with any of the other components of the cellular translational machinery (e.g., other translation initiation factors). In certain embodiments, binding of the RNA molecules of the disclosure to eIF4E enhances the interaction of eIF4E with one or more components of the cellular translational machinery.
  • the RNA molecule of the disclosure is a double stranded RNA. In other embodiments, the RNA molecule of the disclosure is a single stranded RNA. In other embodiments, the RNA molecules of the disclosure include, without limitation, short interfering RNAs (siRNAs), inhibitory RNAs (RNAi), micro RNAs (miRNAs), short hairpin- loop RNAs (shRNAs), small nuclear RNAs (snRNAs), single guide RNAs (sgRNAs), or a derivative thereof.
  • siRNAs short interfering RNAs
  • RNAi inhibitory RNAs
  • miRNAs micro RNAs
  • shRNAs short hairpin- loop RNAs
  • snRNAs small nuclear RNAs
  • sgRNAs single guide RNAs
  • the RNA molecule of the disclosure is a single-stranded RNA molecule.
  • the single-stranded RNA molecule is a messenger RNA (mRNA) molecule.
  • mRNA messenger RNA
  • the RNA molecule of the disclosure is an mRNA molecule.
  • the mRNA molecule of the disclosure comprises (a) a first region of linked nucleosides encoding a polypeptide of interest; (b) a first terminal region located 5’ relative to said first region comprising a 5’ untranslated region (UTR); (c) a second terminal region located 3’ relative to said first region; and (d) a poly (A) tail region.
  • the RNA molecule of the disclosure comprises a 5’ UTR element, an open reading frame, a 3’ UTR element, a poly-A tail and/or a polyadenylation signal.
  • the open reading frame is codon optimized for expression in a non-mammalian subject. In other embodiments, the open reading frame is codon optimized for expression in a mammalian subject. In certain aspects, the open reading frame is codon optimized for expression in a human subject.
  • the RNA molecule of the disclosure comprises a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5’-terminus of the first region (e.g., a 5’-UTR), a second flanking region located at the 3’-terminus of the first region (e.g., a 3’-UTR), at least one 5’-cap region, and a 3’- stabilizing region.
  • an RNA molecule of the disclosure further comprises a poly-A region or a Kozak sequence (e.g., in the 5’-UTR).
  • an RNA molecule of the disclosure may contain one or more intronic nucleotide sequences capable of being excised from the RNA molecule.
  • an RNA molecule of the disclosure e.g., an mRNA
  • any one of the regions of the RNA molecule of the disclosure comprises at least one alternative nucleoside.
  • the 3’-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2’-0-methyl nucleoside and/or the coding region, 5’-UTR, 3’-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5-methoxyuridine), a 1 -substituted
  • pseudouridine e.g., 1-methyl-pseudouridine
  • a 5-substituted cytidine e.g., 5-methyl- cytidine
  • the RNA molecule of the disclosure includes from about 200 to about 3,000 nucleotides (e.g., from about 200 to about 500, from about 200 to about 1,000, from about 200 to about 1,500, from about 200 to about 3,000, from about 500 to about 1,000, from about 500 to about 1,500, from about 500 to about 2,000, from about 500 to about 3,000, from about 1,000 to about 1,500, from about 1,000 to about 2,000, from about 1,000 to about 3,000, from about 1,500 to about 3,000, or from about 2,000 to about 3,000 nucleotides).
  • nucleotides e.g., from about 200 to about 500, from about 200 to about 1,000, from about 200 to about 1,500, from about 200 to about 3,000, from about 500 to about 1,000, from about 500 to about 1,500, from about 500 to about 2,000, from about 500 to about 3,000, from about 1,000 to about 1,500, from about 1,000 to about 2,000, from about 1,000 to about 3,000, from about 1,500 to about 3,000, or from about 2,000 to about 3,000 nucle
  • the mRNA molecule of the disclosure is transcribed from a polynucleotide template having a sequence complementary to the RNA molecule.
  • the polynucleotide template is cloned into a vector downstream of a viable promoter.
  • the polynucleotide template is a complementary DNA (cDNA).
  • the polynucleotide template comprises any nucleotide base at its 5’ terminal.
  • the polynucleotide template comprises at least one guanine nucleobase at its 5’ terminal.
  • the polynucleotide template comprises two guanine nucleobases at its 5’ terminal.
  • the polynucleotide template comprises three guanine nucleobases at its 5’ terminal.
  • the presence of one or more guanine nucleobases results in efficient transcription of the polynucleotide template by the RNA polymerase to generate the RNA molecule.
  • the polynucleotide template is genomic DNA. In yet other embodiments, the polynucleotide template is an RNA. In certain embodiments, the
  • the polynucleotide template is a complementary DNA (cDNA).
  • the promoter is recognized by an RNA polymerase promoter.
  • the RNA polymerase binds to the promoter and transcribes the polynucleotide template in the presence of a translational enhancer of the disclosure to generate an RNA molecule of the disclosure.
  • the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, eukaryotic RNA polymerase II, an RNA polymerase from other sources including a thermostable RNA polymerase, and an RNA-dependent RNA polymerase.
  • the RNA polymerase is T7 RNA polymerase.
  • translation of an RNA molecule of the disclosure can be in vivo, ex vivo, in culture, or in vitro.
  • methods of inducing translation of an RNA molecule of the disclosure to produce a polypeptide in a cell-free extract are provided.
  • methods of inducing translation of an RNA molecule of the disclosure to produce a polypeptide in a cell population are provided.
  • the RNA molecule of the disclosure is an in vitro transcribed RNA molecule (IVT RNA).
  • IVT RNA in vitro transcribed RNA molecule
  • the RNA molecule of the disclosure is an mRNA molecule.
  • an RNA molecule is recombinantly expressed in cells, purified, and co-transcriptionally, chemically, or enzymatically coupled to a translational enhancer of the disclosure to generate an RNA molecule of the disclosure.
  • the cell is a bacterial cell.
  • the cell is an eukaryotic cell including, but limited to, a yeast cell, a non-mammalian cell, a mammalian cell, a human cell, a cancer cell, an eukaryotic cell including, but limited to, a yeast cell, a non-mammalian cell, a mammalian cell, a human cell, a cancer cell, an eukaryotic cell including, but limited to, a yeast cell, a non-mamm
  • an RNA molecule is purified from natural sources and chemically or enzymatically coupled to a translational enhancer of the disclosure to generate an RNA molecule of the disclosure.
  • an RNA molecule is chemically synthesized and chemically or enzymatically coupled to a translational enhancer of the disclosure to generate an RNA molecule of the disclosure.
  • IVT mRNA molecules disclosed herein may function as mRNA but are distinguished from natural mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics.
  • the IVT mRNA may be structurally modified or chemically modified.
  • the length of the RNA molecule of the disclosure (e.g., IVT mRNA) encoding a polypeptide of interest is generally greater than about 30 nucleotides (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).
  • nucleotides e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250,
  • the RNA molecule of the disclosure comprises from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000
  • the RNA molecules of the disclosure when introduced to a cell, exhibit reduced degradation as compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure. In other embodiments, when administered to a subject, the RNA molecules of the disclosure exhibit reduced degradation as compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure do not induce an innate immune response when administered to a subject. In other embodiments, the RNA molecules of the disclosure do not substantially induce an innate immune response when administered to a subject. In certain aspects, when administered to a subject, the immune response induced by the RNA molecules of the disclosure is attenuated compared to the immune response induced by a natural RNA or an RNA molecule not comprising the translational enhancer of the disclosure. In certain aspects, the immune response is an innate immune response.
  • the RNA molecules of the disclosure do not induce an immune response of a cell into which the RNA molecule is introduced. In other embodiments, the RNA molecules of the disclosure do not substantially induce an immune response of a cell into which the RNA molecule is introduced. In some embodiments induce an attenuated immune response of a cell into which the RNA molecule is introduced compared to the immune response induced by a natural RNA or an RNA molecule not comprising the translational enhancer of the disclosure. In certain aspects, the immune response is an innate immune response.
  • an induced innate immune response include 1) increased expression of pro- inflammatory cytokines, 2) activation of innate immune sensors (e.g., intracellular PRRs such as RIG-I, MDA5, etc.) termination or reduction in protein translation.
  • innate immune sensors e.g., intracellular PRRs such as RIG-I, MDA5, etc.
  • the RNA molecules of the disclosure do not activate one or more innate immune sensors when administered to a subject.
  • the RNA molecule of the disclosure activates one or more innate immune sensors to a lesser extent when administered to a subject compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecule of the disclosure is not recognized by one or more innate immune sensors when administered to a subject.
  • the RNA molecule of the disclosure is recognized to a lesser extent by one or more innate immune sensors when administered to a subject compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecule of the disclosure does not activate one or more innate immune sensors when administered to a subject.
  • the one or more innate immune sensors include, without limitation, RIG-I, MDA5, IFIT-1, IFIT-5, protein kinase R (PKR), Toll-like receptor 3 (TLR 3), TLR 7, or TLR 8.
  • the one or more innate immune sensors comprise RIG-I.
  • the RNA molecule of the disclosure does not activate one or more innate immune sensors when administered to a subject, wherein the one or more innate immune sensors are selected from the group consisting of RIG-I, MDA5, IFIT-1, protein kinase R (PKR), Toll-like receptor 3 (TLR 3), TLR 7, and TLR 8.
  • the one or more innate immune sensors are selected from the group consisting of RIG-I, MDA5, IFIT-1, protein kinase R (PKR), Toll-like receptor 3 (TLR 3), TLR 7, and TLR 8.
  • the mRNA molecules of the disclosure encode a protein.
  • the RNA molecule of the disclosure exhibits increased cell permeability compared to an RNA molecule not comprising the translational enhancer. In some embodiments, the RNA molecule of the disclosure is translated with greater efficiency compared to an RNA molecule not comprising the translational enhancer. In other embodiments, the RNA molecule of the disclosure is translated with less efficiency compared to an RNA molecule not comprising the translational enhancer. In some embodiments, the RNA molecule of the disclosure is translated with greater efficiency in vitro compared to an RNA molecule not comprising the translational enhancer.
  • the RNA molecule of the disclosure is translated with greater efficiency in vivo or ex vivo, compared to an RNA molecule not comprising the translational enhancer of the disclosure.
  • the RNA molecules of the disclosure are translated with greater efficiency in vitro in a cell-free translation extract compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure are translated with greater efficiency in a cellular environment compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure are translated with greater efficiency when administered to a subject compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure are preferentially translated in vitro in a cell-free translation extract over an RNA molecule not comprising a translational enhancer of the disclosure. In other aspects, the RNA molecules of the disclosure are preferentially translated in a cellular environment over an RNA molecule not comprising a translational enhancer of the disclosure. In certain instances, the RNA molecules of the disclosure are more stable in a cell-free extract compared to an RNA molecule not comprising a translational enhancer of the disclosure. In other instances, the RNA molecules of the disclosure are more stable in a cellular environment compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure are translated to produce a protein.
  • the protein is a functional protein.
  • the protein is a therapeutic protein.
  • the mRNA molecules of the disclosure encode a therapeutic protein.
  • therapeutic protein refers to a protein that, when administered to a cell or a subject has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • the protein is an enzyme.
  • the protein is a structural protein.
  • the protein is a regulatory protein.
  • the shortest length of an RNA molecule of the invention is generally the length of the RNA sequence that is sufficient to encode for a dipeptide. In other embodiments, the length of the RNA molecule of the invention is sufficient to encode for a tripeptide. In some embodiments, the length of the RNA molecule of the invention is sufficient to encode for a tetrapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a pentapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a hexapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a heptapeptide.
  • the length of the polynucleotide sequence is sufficient to encode for an octapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a nonapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a decapeptide. Examples of dipeptides that the alternative polynucleotide sequences can encode for include, but are not limited to, carnosine and anserine.
  • the cell in which the RNA molecules of the disclosure are translated is a bacterial cell.
  • the cell is an eukaryotic cell including, but limited to, a yeast cell, a non-mammalian cell, a mammalian cell, a human cell, a cancer cell, an immortalized cell line, or an insect cell.
  • the RNA molecules of the disclosure are greater than about 30 nucleotides in length. In other cases, the RNA molecule of the disclosure is greater than about 35, greater than about 50 nucleotides, greater than about 100 nucleotides, greater than about 200 nucleotides, greater than about 300 nucleotides, greater than about 400 nucleotides, greater than about 500 nucleotides, greater than about 600 nucleotides, greater than about 700 nucleotides, greater than about 800 nucleotides, greater than about 900 nucleotides, greater than about 1,000 nucleotides, greater than about 2,000 nucleotides, greater than about 3,000 nucleotides, greater than about 4,000 nucleotides, or greater than about 5,000 nucleotides in length.
  • a polynucleotide is at least about 30 nucleotides in length. In other embodiments, the length is at least about 35 nucleotides. In other embodiments, the length is at least about 40 nucleotides. In other cases, the length is at least about 45 nucleotides. In certain aspects, the length is at least about 50 nucleotides. In other embodiments, the length is at least about 55 nucleotides. In other aspects the length is at least 60 nucleotides. In other embodiments, the length is at least about 80 nucleotides. In other embodiments, the length is at least about 90 nucleotides. In other embodiments, the length is at least about 100 nucleotides.
  • the length is at least about 120 nucleotides. In other embodiments, the length is at least about 140 nucleotides. In other embodiments, the length is at least about 160 nucleotides. In other embodiments, the length is at least about 180 nucleotides. In other embodiments, the length is at least about 200 nucleotides. In other embodiments, the length is at least about 250 nucleotides. In other embodiments, the length is at least about 300 nucleotides. In other embodiments, the length is at least about 350 nucleotides. In other embodiments, the length is at least 400 nucleotides. In other embodiments, the length is at least about 450 nucleotides.
  • the length is at least about 500 nucleotides. In other embodiments, the length is at least about 600 nucleotides. In other embodiments, the length is at least about 700 nucleotides. In other embodiments, the length is at least about 800 nucleotides. In other embodiments, the length is at least about 900 nucleotides. In other embodiments, the length is at least about 1000 nucleotides. In other embodiments, the length is at least about 1100 nucleotides. In other embodiments, the length is at least about 1200 nucleotides. In other embodiments, the length is at least about 1300 nucleotides. In other embodiments, the length is at least about 1400 nucleotides.
  • the length is at least about 1500 nucleotides. In other embodiments, the length is at least about 1600 nucleotides. In other embodiments, the length is at least about 1800 nucleotides. In other embodiments, the length is at least about 2000 nucleotides. In other embodiments, the length is at least about 2500 nucleotides. In other embodiments, the length is at least about 3000 nucleotides. In other embodiments, the length is at least about 4000 nucleotides. In other embodiments, the length is at least about 5000 nucleotides.
  • the RNA molecules of the disclosure comprise one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).
  • all or substantially of the nucleotides comprising (a) the 5’-UTR, (b) the open reading frame (ORF), (c) the 3’-UTR, (d) the poly A tail, and any combination of (a, b, c or d above) comprise naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U
  • the RNA molecule of the disclosure except for the 5’ end cap thereof, is an unmodified RNA molecule which has the same sequence and structure as that of a natural RNA molecule.
  • the RNA molecule of the disclosure in addition to the modifications on the 5’ end cap disclosed herein, may include at least one chemical modification as described herein.
  • the RNA molecules of the disclosure comprises one or more modifications.
  • the one or more modifications comprise one or more phosphorothioate backbone linkages.
  • the one or more modifications comprise one or more modified nucleobases.
  • the RNA molecules of the disclosure comprise one or more alternative components (e.g., in a 3’-stabilizing region), which impart useful properties including increased stability and/or the lack of a substantial induction of the innate immune response of a cell into which the RNA molecule is introduced.
  • a modified (e.g., altered or alternative) RNA molecule of the disclosure exhibits reduced degradation in a cell into which the RNA molecule is introduced, relative to a corresponding natural RNA molecule or an unaltered RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure enhance the efficiency of protein production, intracellular retention of the RNA molecules, and/or viability of contacted cells, as well as possess reduced immunogenicity.
  • the RNA molecules of the disclosure may be naturally or non-naturally occurring.
  • the RNA molecules of the disclosure include one or more modified (e.g., altered or alternative) nucleobases, nucleosides, nucleotides, or combinations thereof.
  • the RNA molecules of the disclosure include any suitable modification or alteration, such as to the nucleobase, the sugar, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone).
  • alterations are present in each of the nucleobase, the sugar, and the internucleoside linkage of the RNA molecules of the disclosure.
  • alterations according to the present disclosure may be alterations of RNAs, e.g., the substitution of the 2’-OH of the ribofuranosyl ring to 2’-H.
  • the RNA molecules of the disclosure are not uniformly altered along the entire length of the molecule.
  • one or more or all types of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotides X in an RNA molecule of the disclosure are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • RNA molecules of the disclosure include different sugar alterations and/or internucleoside linkages (e.g., backbone structures) exist at various positions in the RNA molecules of the disclosure.
  • nucleotide analogs or other alteration(s) may be located at any position(s) of a RNA molecule such that the function of the RNA molecule is not substantially decreased.
  • an alteration may also be a 5’- or 3’- terminal alteration.
  • the RNA molecules of the disclosure include an alteration at the 3’-terminus.
  • the RNA molecules of the disclosure contain from about 1 % to about 100% alternative nucleotides (either in relation to overall RNA content, or in relation to one or more types of RNA, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1 % to 20%, from 1 % to 25%, from 1 % to 50%, from 1% to 60%, from 1% to 70%, from 1 % to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 50% to 100%
  • the RNA molecules of the disclosure contain at a minimum one and at maximum 100% alternative nucleotides, or any intervening percentage, such as at least about 5% altemative nucleotides, at least about 10% alternative nucleotides, at least about 25% alternative nucleotides, at least about 50% altemative nucleotides, at least about 80% altemative nucleotides, or at least about 90% altemative nucleotides.
  • the RNA molecules of the disclosure contain an alternative pyrimidine such as an alternative uracil or cytosine.
  • At least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 80%, at least about 90% or about 100% of the uracil in the polynucleotide is replaced with an alternative uracil (e.g., a 5-substituted uracil).
  • the alternative uracil is replaced by a nucleobase having a single unique structure, or is replaced by a plurality of nucleobases having different structures (e.g., 2, 3, 4 or more unique structures).
  • cytosine in the RNA molecules of the disclosure is replaced with an alternative cytosine (e.g., a 5-substituted cytosine).
  • the alternative cytosine is replaced by a nucleobase having a single unique structure, or is replaced by a plurality of nucleobases having different structures (e.g., 2, 3, 4 or more unique structures).
  • the RNA molecules of the disclosure comprise one or more alternative nucleosides and nucleotides.
  • the alternative nucleosides and nucleotides include an alternative nucleobase.
  • a nucleobase of a nucleic acid is an organic base such as a purine or pyrimidine or a derivative thereof.
  • a nucleobase may be a canonical base (e.g., adenine, guanine, uracil, thymine, and cytosine). In certain cases, these nucleobases are altered or wholly replaced to provide RNA molecules having enhanced properties, e.g., increased stability such as resistance to nucleases.
  • Non-canonical or modified bases may include, for example, one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and/or thio substitutions; one or more fused or open rings; oxidation; and/or reduction.
  • the RNA molecules of the disclosure comprise one or more alternative nucleotide base pairings.
  • alternative nucleotide base pairing encompasses not only the standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and/or alternative nucleotides including non-standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non- standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.
  • nucleotides are altered on the internucleoside linkage (e.g., phosphate backbone).
  • phosphate backbone in the context of the polynucleotide backbone, the phrases “phosphate” and“phosphodiester” are used interchangeably.
  • backbone phosphate groups are altered by replacing one or more of the oxygen atoms with a different substituent.
  • the alternative nucleotides include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein.
  • alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker is altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene- phosphonates).
  • the alternative nucleosides and nucleotides include the
  • non-bridging oxygens replacement of one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl, and/or methoxy.
  • BH3 borane moiety
  • sulfur (thio) methyl, ethyl, and/or methoxy.
  • two non-bridging oxygens at the same position e.g., the alpha (a), beta (b) or gamma (g) position
  • the replacement of one or more of the oxygen atoms at the phosphate moiety is provided to confer stability (such as against exonucleases and endonucleases) to RNA molecules of the disclosure through the unnatural phosphorothioate backbone linkages.
  • the phosphorothioate RNA molecules have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • Other inter nucleoside linkages known in the art that may be employed according to the present disclosure, including inter nucleoside linkages which do not contain a phosphorous atom, are also contemplated.
  • the RNA molecules of the disclosure contain an internal ribosome entry site (IRES).
  • IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
  • an RNA molecule of the disclosure containing more than one functional ribosome binding site encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multicistronic mRNA).
  • RNA molecules of the disclosure are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from
  • picornaviruses e.g., FMDV
  • CFFV pest viruses
  • PV polio viruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot- and-mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV murine leukemia virus
  • SIV simian immune deficiency viruses
  • CrPV cricket paralysis viruses
  • the RNA molecules of the invention comprise a stem loop structure such as, but not limited to, a histone stem loop.
  • the stem loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length.
  • the histone stem loop is located 3’-relative to the coding region (e.g., at the 3’-terminus of the coding region).
  • the stem loop may be located at the 3’-UTR of an RNA molecule of the disclosure.
  • the stem loop may be located at the 5’-UTR of an RNA molecule of the disclosure.
  • an RNA molecule of the disclosure includes more than one stem loop (e.g., two stem loops). Examples of such stem loop sequences are well known in the art.
  • the RNA molecules of the invention comprise a poly-A tail.
  • the poly-A region is generally at least about 30 nucleotides in length. In other embodiments, the poly-A region is at least about 35 nucleotides in length. In other embodiments, the length is at least about 40 nucleotides. In other embodiments, the length is at least about 45 nucleotides. In other embodiments, the length is at least about 55 nucleotides. In other embodiments, the length is at least about 60 nucleotides. In other embodiments, the length is at least about 70 nucleotides. In other embodiments, the length is at least about 80
  • the length is at least about 90 nucleotides. In other embodiments the length is at least about 100 nucleotides. In other embodiments, the length is at least about 120 nucleotides. In other embodiments, the length is at least about 140 nucleotides. In other embodiments, the length is at least about 160 nucleotides. In other embodiments, the length is at least about 180 nucleotides. In other embodiments, the length is at least about 200 nucleotides. In other embodiments, the length is at least about 250 nucleotides. In other embodiments, the length is at least about 300 nucleotides. In other embodiments, the length is at least 350 nucleotides.
  • the length is at least about 400 nucleotides. In other embodiments, the length is at least about 450 nucleotides. In other embodiments, the length is at least about 500 nucleotides. In other embodiments, the length is at least about 600 nucleotides. In other embodiments, the length is at least about 700 nucleotides. In other embodiments, the length is at least about 800 nucleotides. In other embodiments, the length is at least about 900 nucleotides. In other embodiments, the length is at least about 1000 nucleotides. In other embodiments, the length is at least about 1100 nucleotides. In other embodiments, the length is at least about 1200 nucleotides.
  • the length is at least about 1300 nucleotides. In other embodiments, the length is at least about 1400 nucleotides. In other embodiments, the length is at least about 1500 nucleotides. In other embodiments, the length is at least about 1600 nucleotides. In other embodiments, the length is at least about 1700 nucleotides. In other embodiments, the length is at least about 1800 nucleotides. In other embodiments, the length is at least about 1900 nucleotides. In other embodiments, the length is at least about 2000 nucleotides. In other embodiments, the length is at least about 2500 nucleotides. In other embodiments, the length is at least about 3000 nucleotides.
  • engineered binding sites and/or the conjugation of RNA molecules of the disclosure for poly-A binding protein may be used to enhance expression.
  • the engineered binding sites are sensor sequences which operate as binding sites for ligands of the local microenvironment of the RNA molecules.
  • the RNA molecules of the disclosure include at least one engineered binding site to alter the binding affinity of poly-A binding protein (PABP) and analogs thereof.
  • PABP poly-A binding protein
  • the incorporation of at least one engineered binding site increases the binding affinity of the PABP and analogs thereof.
  • a poly-A- region may be used to modulate translation initiation. While not wishing to be bound by theory, the poly-A region recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis.
  • RNA molecules of the disclosure may also be used to protect the RNA molecules of the disclosure against 3’-5’-exonuclease digestion, thereby further increasing the stability of the RNA molecule.
  • a method of improving the stability of an RNA molecule comprising: (a) providing the RNA molecule; and (b) coupling to a 5’ end of the RNA molecule a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, according to the structure:
  • eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide, thereby generating at least one capped RNA molecule that exhibits improved stability compared to an RNA molecule not comprising a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof.
  • the present invention also contemplates the production of RNA molecules of the disclosure for use as therapeutic agents in a pharmaceutical composition, or the introduction of the RNA molecules of the disclosure into cells that utilize those RNAs to produce proteins that may have a therapeutic effect on the host cells, or the introduction of the RNA molecules of the disclosure into cells of a subject to treat a medical condition.
  • a cell population is contacted with an effective amount of a composition containing an RNA molecule of the disclosure, and a translatable region encoding a polypeptide.
  • the cell population is contacted under conditions such that the polynucleotide is localized into one or more cells of the cell population and the polypeptide is translated in the cell from the RNA molecule.
  • an effective amount of the composition of an RNA of the disclosure is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA molecule (e.g., size, and extent of modified nucleosides), and other determinants.
  • an effective amount of the composition provides efficient protein production in the cell, preferably more efficient than a composition containing a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure.
  • increased efficiency may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the polynucleotide), increased protein translation from the RNA molecule, decreased degradation of the RNA molecule (as demonstrated, e.g., by increased duration of protein translation from the RNA molecule), reduced innate immune response of the host cell to the RNA molecule, or improve therapeutic utility of the RNA molecule.
  • the present disclosure is directed to methods of inducing in vivo translation of an RNA of the disclosure in a subject in need thereof.
  • an effective amount of a composition containing the RNA molecule of the disclosure and a translatable region encoding a polypeptide of interest is administered to the subject using the delivery methods described herein.
  • the RNA molecule of the disclosure also comprises at least one modified nucleoside.
  • the RNA molecule of the disclosure is provided in an amount and under other conditions such that the RNA molecule is localized into a cell or cells of the subject and the polypeptide of interest is translated in the cell from the RNA molecule.
  • the cell in which the RNA molecule is localized, or the tissue in which the cell is present is targeted with one or more than one rounds of administration of the RNA molecule of the disclosure.
  • the subject is a mammal. In other aspects, the subject is a human.
  • compositions comprising the RNA molecules of the disclosure are formulated for administration intramuscularly, transarterially, intraperitoneally, intravenously, intranasally, subcutaneously, endoscopically, transdermally, or intrathecally. In some embodiments, the compositions are formulated for extended release.
  • the subject to whom the RNA molecule of the disclosure is administered suffers from or is at risk of developing a disease, disorder, or deleterious condition.
  • methods of identifying, diagnosing, and classifying subjects on these bases include clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art.
  • the administered RNA molecule of the disclosure directs production of one or more polypeptides that provide a functional activity which is substantially absent in the cell in which the polypeptide is translated.
  • the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
  • the RNA molecules of the disclosure encode a protein.
  • the protein is a therapeutic protein.
  • the protein is an enzyme.
  • the protein is a structural protein.
  • the protein is a regulatory protein.
  • the protein acts as a vaccine when expressed in a subject.
  • the administered RNA molecule of the disclosure directs production of one or more polypeptides that replace a polypeptide (or multiple polypeptides) that is substantially absent in the cell in which the one or more polypeptides are translated. In certain cases, such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof. In other embodiments, the administered RNA molecule of the disclosure directs production of one or more polypeptides to supplement the amount of polypeptide (or multiple polypeptides) that is present in the cell in which the one or more polypeptides are translated.
  • the administered RNA molecule of the disclosure directs production of one or more polypeptides, which function to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell.
  • antagonized biological moieties include, without limitations, proteins (e.g., cancer), lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a polynucleotide, a carbohydrate, or a small molecule toxin.
  • the translated proteins are engineered for localization within the cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
  • RNA molecules of the disclosure of the disclosure are the capacity to reduce, evade, avoid or eliminate the innate immune response of a cell to an exogenous RNA.
  • the RNA molecules of the disclosure of the disclosure induces an attenuated immune response when administered to a subject compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • the RNA molecules of the disclosure of the disclosure induces an attenuated immune response in a cell or a population of cells compared to an RNA molecule not comprising a translational enhancer of the disclosure.
  • methods for performing the titration, reduction or elimination of the immune response in a cell or a population of cells are well known in the art.
  • RNA molecules of the disclosure are particularly advantageous in treating acute diseases such as sepsis, stroke, and myocardial infarction.
  • the lack of transcriptional regulation of the mRNA molecules of the disclosure is advantageous in that accurate titration of protein production is achievable. Multiple diseases are characterized by missing (or substantially diminished such that proper protein function does not occur) protein activity.
  • the present disclosure provides a method for treating such conditions or diseases in a subject by introducing the RNA molecules of the disclosure or cell-based therapeutics containing the RNA molecules of the disclosure, wherein the RNA molecules encode for a protein that replaces the protein activity missing from the target cells of the subject.
  • diseases characterized by dysfunctional or aberrant protein activity include, but not limited to, hyperproliferative diseases (e.g., cancer), genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, inflammatory diseases, viral infections, cardiovascular diseases, and metabolic diseases.
  • hyperproliferative diseases e.g., cancer
  • genetic diseases e.g., cystic fibrosis
  • autoimmune diseases e.g., cystic fibrosis
  • diabetes e.g., cystic fibrosis
  • neurodegenerative diseases e.g., inflammatory diseases, viral infections, cardiovascular diseases, and metabolic diseases.
  • the present disclosure provides a method for treating such conditions or diseases in a subject by introducing the RNA molecules of the disclosure or cell- based therapeutics containing the RNA molecules of the disclosure, wherein the RNA molecules encode for a protein that antagonizes or otherwise overcomes the aberrant protein activity present in the cell of a subject.
  • the RNA molecule of the disclosure is capable of treating, preventing, or ameliorating a disease when administered to a subject in need thereof.
  • the disease includes, but is not limited to, hyperproliferative disease, genetic disease, autoimmune disease, diabetes, neurodegenerative disease, inflammatory disease, viral infection, cardiovascular disease, and metabolic disease.
  • the disease is selected from the group consisting of
  • hyperproliferative disease inflammatory disease, viral infection, cardiovascular disease, genetic disease, autoimmune disease.
  • the genetic disease is cystic fibrosis.
  • the hyperproliferative disease is cancer.
  • the cancer includes, without limitation, solid tumor, melanoma, non-small cell lung cancer, renal cell carcinoma, renal cancer, a hematological cancer, prostate cancer, castration-resistant prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck cancer, thyroid cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, lung cancer, urothelial cancer, pancreatic cancer, glioblastoma, hepatocellular cancer, myeloma, multiple myeloma, leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, myelodysplastic syndrome, brain cancer, CNS cancer, malignant glioma, or any combination thereof.
  • a dysfunctional protein are the missense or nonsense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional or nonfunctional, respectively, protein variant of CFTR protein, which causes cystic fibrosis.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • RNA molecules of the disclosure having a translatable region that encodes a functional CFTR polypeptide, under conditions such that an effective amount of the CFTR polypeptide is produced in the cell.
  • a method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject a therapeutically effective amount of an mRNA molecule of the disclosure.
  • the disease includes, but is not limited to, hyperproliferative disease, inflammatory disease, viral infection, cardiovascular disease, genetic disease, and autoimmune disease.
  • a method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject an mRNA molecule comprising a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof:
  • the eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide, wherein the translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof is attached to the 5’ end of the mRNA molecule.
  • the mRNA molecule expresses a therapeutic protein when administered to the subject. In other aspects, expression of the therapeutic protein results in treating, preventing, or ameliorating the disease.
  • the subject is a non-mammalian subject. In other words, the subject is a non-mammalian subject.
  • the subject is a mammalian subject. In certain aspects, the subject is a human.
  • the target cells are epithelial cells, such as the lung, and methods of administration are determined in view of the target tissue; i.e., for lung delivery, the RNA molecules are formulated for administration by inhalation.
  • methods of the present disclosure enhance polynucleotide delivery into a cell population, in vivo, ex vivo, or in culture.
  • a cell culture containing a plurality of host cells e.g., eukaryotic cells such as yeast or mammalian cells
  • a composition that contains an RNA molecule of the disclosure is contacted with a composition that contains an RNA molecule of the disclosure.
  • composition also generally contains a transfection reagent or other compound that increases the efficiency of RNA uptake into the host cells.
  • the RNA molecules of the disclosure exhibit enhanced retention in the cell population, relative to a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain instances, the retention of the RNA of the disclosure is greater than the retention of the corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure.
  • the retention of the RNA of the disclosure is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greater than the retention of the natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In some cases, such retention advantage may be achieved by one round of transfection with the RNA of the disclosure, or may be obtained following repeated rounds of transfection.
  • the RNA of the disclosure is delivered to a target cell population with one or more additional polynucleotides. In certain cases, such delivery may be at the same time, or the RNA of the disclosure is delivered prior to delivery of the one or more additional polynucleotides.
  • the additional one or more polynucleotides may be RNA molecules of the disclosure or natural polynucleotides. It is understood that the initial presence of the RNA of the disclosure does not substantially induce an innate immune response of the cell population and, moreover, that the innate immune response will not be activated by the later presence of the natural polynucleotides. In this regard, in certain aspects, the RNA of the disclosure may not itself contain a translatable region, if the protein desired to be present in the target cell population is translated from the natural polynucleotides.
  • compositions comprising the RNA molecules of the disclosure, and, optionally, one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • pharmaceutical compositions comprising the RNA molecules of the disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • compositions optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances.
  • additional active substances e.g., therapeutically and/or prophylactically active substances.
  • pharmaceutical compositions of the present disclosure are sterile and/or pyrogen- free. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.
  • an RNA molecule of the disclosure comprising a translational enhancer of the disclosure is formulated as a pharmaceutical composition in an amount effective to treat a particular disease or condition of interest (e.g., cancer, cardiovascular disease, or autoimmune disease) upon administration of the pharmaceutical composition to a subject.
  • a particular disease or condition of interest e.g., cancer, cardiovascular disease, or autoimmune disease
  • the subject is a mammal.
  • the mammal is a human.
  • a“mammal” includes primates, such as humans, monkeys and apes, and non- primates such as domestic animals, including laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife or the like.
  • a pharmaceutical composition comprises an RNA molecule of the disclosure and a pharmaceutically acceptable carrier, diluent or excipient.
  • a “pharmaceutically acceptable carrier, diluent or excipient” includes any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • a pharmaceutical composition of the disclosure is prepared by combining or formulating an RNA molecule of the disclosure with an appropriate
  • a pharmaceutical composition of the disclosure is formulated into preparations in solid, semi-solid, liquid or gaseous forms, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • exemplary routes of administering such pharmaceutical compositions include oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • a pharmaceutical composition of the disclosure is formulated to be administered by routes selected from the group consisting of oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • pharmaceutical compositions of the disclosure are formulated to allow the active ingredients contained therein to be bioavailable upon administration to a subject or patient.
  • compositions that will be administered to a subject or patient take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container of an RNA molecule of the disclosure in aerosol form may hold a plurality of dosage units.
  • dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • a composition to be administered will, in any event, contain a therapeutically effective amount of an RNA molecule of the disclosure, or a pharmaceutically acceptable salt thereof, to aid in treatment of a disease or condition of interest in accordance with the teachings herein.
  • a pharmaceutical composition of an RNA molecule of the disclosure may be in the form of a solid or liquid.
  • the carrier(s) are particulate so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) are liquid, with a composition being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
  • a pharmaceutical composition of an RNA molecule of the disclosure is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • a pharmaceutical composition of an RNA molecule of the disclosure may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • one or more of the following may be additionally present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin
  • excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like
  • lubricants such as magnesium stearate or
  • the pharmaceutical composition when in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
  • a liquid carrier such as polyethylene glycol or oil.
  • the pharmaceutical composition is in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection.
  • preferred compositions contain, in addition to an RNA molecule of the disclosure, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions of RNA molecules of the disclosure may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride
  • fixed oils such as synthetic mono or digly
  • parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • physiological saline is a preferred adjuvant.
  • an injectable pharmaceutical composition is preferably sterile.
  • a liquid pharmaceutical composition of the disclosure intended for either parenteral or oral administration should contain an amount of an RNA molecule of the disclosure such that a suitable dosage will be obtained.
  • a pharmaceutical composition of an RNA molecule of the disclosure may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • thickening agents may be present in a pharmaceutical composition for topical administration.
  • a composition of an RNA molecule of the disclosure may be included with a transdermal patch or iontophoresis device.
  • the pharmaceutical composition of an RNA molecule of the disclosure is intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the RNA molecule.
  • a composition for rectal administration contains an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter or polyethylene glycol.
  • the pharmaceutical composition of an RNA molecule of the disclosure includes various materials that modify the physical form of a solid or liquid dosage unit.
  • the composition includes materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients are encased in a gelatin capsule.
  • the pharmaceutical composition of this disclosure in solid or liquid form include an agent that binds to an RNA molecule of the disclosure and thereby assist in the delivery of the RNA molecule.
  • suitable agents that act in this capacity include a protein or a liposome.
  • a pharmaceutical composition of an RNA molecule of the disclosure consist of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages.
  • delivery is accomplished by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients.
  • aerosols of the RNA molecules of the disclosure may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). In other
  • delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit.
  • activators e.g., a liquid crystal display
  • valves e.g., a valve
  • subcontainers e.g., a container that stores a kit.
  • a pharmaceutical composition of this disclosure may be prepared by methodology well-known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by combining an RNA molecule of the disclosure with a sterile solvent so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are molecules that non-covalently interact with a compound of this disclosure so as to facilitate dissolution or homogeneous suspension of the compound in an aqueous delivery system.
  • compositions of the disclosure comprise one or more additional therapeutically active substances.
  • a pharmaceutical composition of the disclosure comprise one or more additional therapeutically active substances.
  • a“combination” refers to a combination comprising an RNA molecule of the disclosure and one or more additional therapeutically active substances, each of which may be administered serially (sequentially), concurrently or simultaneously.
  • the phrase“active ingredient” refers to an RNA molecule of the disclosure, and salts thereof.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formulation or concurrently in separate formulations). The most effective doses may generally be determined using experimental models and/or clinical trials. Design and execution of pre- clinical and clinical studies for a therapeutic agent (including when administered for
  • prophylactic benefit described herein are well within the skill of a person skilled in the relevant art.
  • relative amounts of the active ingredient i.e., an RNA molecule of the disclosure
  • the pharmaceutically acceptable excipient i.e., any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the pharmaceutical composition may comprise between 0.1 % and 100% (w/w), e.g., between 0.1% and 99%, between 0.5 and 50%, between 1-30%, between 5-80%, or at least about 80% (w/w) of the active ingredient.
  • the RNA molecules of the disclosure are formulated using one or more excipients, for example, to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
  • excipients of the present disclosure can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with multimeric structures, hyaluronidase, nanoparticle mimics and
  • the RNA molecules of the disclosure are formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
  • pharmaceutical compositions of the nucleic acids or multimeric nucleic acid molecules include lipid
  • lipid nanoparticles are MC3-based lipid nanoparticles.
  • the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle ranges from about 1 RNA molecule to about 100 RNA molecules. In other embodiments, the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle ranges from about 50 to about 500 RNA molecules. In other aspects, the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle ranges from about 250 to about 1000 RNA molecules. In yet other embodiments, the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle is greater than 1000 RNA molecules.
  • the RNA molecules of the disclosure are formulated in a lipid- poly cation complex.
  • the formation of the lipid-poly cation complex may be accomplished by methods known in the art.
  • the poly cation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or
  • RNA molecules of the disclosure are formulated in a lipid-poly cation complex which further includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).
  • a non-cationic lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • the RNA molecules of the disclosure are formulated in a nanoparticle.
  • the nanoparticle comprises at least one lipid.
  • the lipid is selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC 3 -DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.
  • the lipid is a cationic lipid such as, but not limited to, DLin- DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.
  • nanoparticle compositions also includes one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components known in the art.
  • nanoparticle compositions includes any substance useful in pharmaceutical compositions.
  • nanoparticle compositions includes a lipid component and one or more additional components, such as an additional therapeutic agent.
  • the amount of a therapeutic agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic agent.
  • the amount of an RNA molecule of the disclosure useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA molecule.
  • the relative amounts of a therapeutic agent and other elements (e.g., lipids) in a nanoparticle composition may also vary.
  • the wt/wt ratio of the lipid component to a therapeutic agent in a nanoparticle composition may be from about 5 : 1 to about 60: 1 , such as 5 : 1, 6: 1, 7: 1 , 8: 1, 9: 1 , 10: 1, 11 : 1, 12: 1, 13: 1 , 14: 1 , 15: 1 , 16: 1 , 17: 1, 18: 1, 19: 1, 20: 1, 25 : 1, 30: 1, 35 : 1 , 40: 1 , 45: 1 , 50: 1 , and 60: 1.
  • the wt/wt ratio of the lipid component to a therapeutic agent may be from about 10: 1 to about 40:1. In specific aspects, the wt/wt ratio is about 20: 1.
  • the RNA molecules of the disclosure are designed to encode polypeptides of interest selected from any of several target categories including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.
  • target categories including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.
  • the RNA molecules of the disclosure encode a protein.
  • the protein is a therapeutic protein.
  • the protein is an enzyme.
  • the protein is a structural protein.
  • the protein is a regulatory protein.
  • therapeutic protein refers to a protein that, when administered to a cell or a subject has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • the term“therapeutically effective amount”, as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect.
  • the effect can be detected by any assay method known in the art.
  • the precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration.
  • therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
  • the disease or condition to be treated is cancer.
  • the disease or condition to be treated is a cell proliferative disorder.
  • a therapeutic agent is administered at a therapeutically effective amount or dose.
  • a therapeutically effective amount or dose will vary according to several factors, including the chosen route of administration, formulation of the composition, patient response, severity of the condition, the subject’s weight, and the judgment of the prescribing physician.
  • the dosage can be increased or decreased over time, as required by an individual subject.
  • a patient initially is given a low dose, which is then increased to an efficacious dosage tolerable to the subject.
  • a patient may be given a plurality of doses over a determined period of time and in particular time increments (such as daily, weekly, biweekly, monthly, quarterly, biannually, annually or the like). Determination of an effective amount or dosing regimen is well within the capability of those skilled in the art.
  • the route of administration of a composition in accordance with the disclosure can be oral, intraperitoneal, transdermal, subcutaneous, by intravenous or intramuscular injection, by inhalation, topical, intralesional, infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art.
  • compositions in accordance with the disclosure is
  • the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In some embodiments,
  • administrations e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.
  • the translational enhancers and RNA molecules of the disclosure can be characterized using a variety of assays known to those skilled in the art to determine whether they have biological activity.
  • the RNA molecules of the disclosure can be characterized by conventional assays, including but not limited to protein production assays (e.g., cell-free translation assays or cell based expression assays), degradation assays, cell culture assays (e.g., of neoplastic cells), animal models (e.g., rats, mice, rabbits, dogs, or pigs) and the like, to determine whether they have a predicted activity, e.g., binding activity and/or binding specificity, and stability.
  • protein production assays e.g., cell-free translation assays or cell based expression assays
  • degradation assays e.g., cell culture assays (e.g., of neoplastic cells)
  • animal models e.g., rats, mice, rabbits, dogs, or pigs
  • high-throughput screening can be used to speed up analysis using such assays. As a result, it can be possible to rapidly screen the molecules described herein for activity, using high-throughput screening.
  • General methodologies for performing high- throughput screening are well known in the art.
  • kits including, a translational enhancer of the disclosure for performing transcription are also contemplated.
  • kits comprise all transcription reagents for synthesis of common RNAs (e.g., FLuc mRNA). More specifically, in certain cases, a kit contains: a polynucleotide template having a sequence complementary to the RNA molecule, a translational enhancer of the disclosure, a container marked for transcription, instructions for performing RNA synthesis, and one or more reagents selected from the group consisting of one or more modified or unmodified initiating capped oligonucleotide primers, one or more unmodified NTPs, one or more modified NTPs (e.g., pseudouridine 5’-triphosphate), an RNA polymerase, other enzymes, a reaction buffer, and magnesium.
  • RNAs e.g., FLuc mRNA
  • kits for capping an RNA molecule comprising: (a) a polynucleotide template having a sequence complementary to the RNA molecule; (b) an RNA polymerase; and (c) a translational enhancer of the disclosure.
  • kits for capping an RNA molecule comprising: (a) a polynucleotide template having a sequence complementary to the RNA molecule; (b) an RNA polymerase; and (c) a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, according to the structure:
  • eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide.
  • Y is an AG dinucleotide. In other embodiments, Y is a GG dinucleotide. In certain cases, the linker is a phosphate linker. In other aspects, the linker is a phosphonate-diphosphate linker. In certain aspects, the RNA molecule is a single-stranded RNA molecule. In other aspects, the single-stranded RNA molecule is an mRNA.
  • the polynucleotide template is a complementary DNA (cDNA).
  • the polynucleotide template comprises any nucleotide base at its 5’ terminal.
  • the polynucleotide template comprises at least one guanine nucleobase at its 5’ terminal.
  • the polynucleotide template comprises two guanine nucleobases at its 5’ terminal.
  • the polynucleotide template comprises three guanine nucleobases at its 5’ terminal.
  • the presence of one or more guanine nucleobases results in efficient transcription of the polynucleotide template by the RNA polymerase to generate the RNA molecule.
  • the kit comprises nucleotides. In other embodiments, the kit further comprises a ribonuclease inhibitor. In other embodiments, the kit further comprises a buffer.
  • RNA polymerases known in the art for use in transcription reactions may be utilized with the compositions and methods of the present invention.
  • other enzymes including natural or mutated variants that may be utilized include, for example, SP6 and T3 RNA polymerases, eukaryotic RNA polymerase II, RNA polymerases from other sources including thermostable RNA polymerases, and RNA- dependent RNA polymerases.
  • the RNA polymerase includes, without limitation, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, eukaryotic RNA polymerase II, a thermostable RNA polymerase, and an RNA-dependent RNA polymerase.
  • the RNA polymerase is T7 RNA polymerase.
  • TLC thin layer chromatography
  • NMR spectra were obtained as CDCl 3 solutions (reported in ppm), using chloroform as the reference standard (7.27 ppm for the proton and 77.00 ppm for carbon), CD 3 OD solutions using 3.4 ppm and 4.8 ppm as reference standards for the protons and 49.3 ppm as a reference standard for carbon, DMSO-d 6 (2.49 ppm for proton), or internally tetramethylsilane (0.00 ppm) when appropriate. Other NMR solvents were used as needed. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m
  • protecting groups are used during the synthesis of intermediates, or if Formulae I, II, III, IV, V or VI compounds contain one or more protecting groups, then such protecting groups are removed by methods known in the chemical art.
  • reaction mixture was warmed to 0°C and stirred for 30 min It was again cooled to -78°C and a solution of 1,2-dibromo-5-fluoro-3-(trifluoromethyl)benzene (2 , 10.0 g, 31.15 mol) in dry tetrahydrofuran (70 mL) was added at -100°C and the reaction mixture was stirred for 45 min at -100°C. Carbon dioxide gas was bubbled through the reaction mass at this temperature for 15 min and it was gradually warmed to room temperature in 2 h. After completion, the reaction mixture was quenched with water and washed with diethyl ether.
  • the filtrate was concentrated in vacuo, and the residue (N-methyl-2-pyrrolidone solution) was loaded onto a 5g Phenomenex strata ion exchange column.
  • the cartridge was washed with water, acetonitrile, and MeOH (some product found in the combined filtrate/rinsing solution).
  • reaction mixture was cooled to 0 °C and a solution of 2,4,6- triisopropylbenzenesulfonyl chloride (10a) and N-methyl imidazole in dry acetonitrile (10 mL) was added to reaction mixture. The reaction mixture was allowed to react for 16 h.
  • reaction mixture was concentrated in vacuum and crude obtained was purified by flash column chromatography using silica gel (230-400 mesh) and 70-100% ethyl acetate in hexanes as eluents to afford (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)-4-methoxytetrahydrofuran-3-yl)oxy)(4-chlorophenoxy)phosphoryl)oxy)methyl)-5-(2- isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diyl bis(2- methylpropanoate) (11) as a brown solid
  • reaction mixture was then stirred at room temperature for 6 h. After completion, reaction mixture was cooled down and quenched by addition of saturated solution sodium bicarbonate and extracted with dichloromethane. Organic layer obtained was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude compound. Crude compound obtained was purified by flash column chromatography using silica gel (230-400 mesh) and 6 to 7 % methanol in dichloromethane as eluents. The desired fractions were concentrated under reduced pressure to afford
  • triphenyl phosphine (0.068 g, 0.26 mmol) is added and the mixture is stirred at room temperature for 8 h.
  • the product is precipitated from the reaction mixture with a solution of anhydrous sodium perchlorate in dry acetone.
  • reaction mixture was cooled to room temperature, diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure.
  • the crude residue obtained was purified by column chromatography using silica gel (100-200 mesh) and 0-60% ethyl acetate in hexanes to afford diethyl (7-(5-chloro-2-hydroxyphenyl)thieno[3,2-b]pyridin-3-yl)phosphonate (19) as a brownish sticky gum. Yield: 6.00 g, 51%; MS (ESI) m/z 398.10 [M+1]+.
  • reaction mixture was cooled down, poured onto ice cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to get crude product, which was purified by preparative HPLC to afford diethyl (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4- methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2- b]pyridin-3-yl)phosphonate (21) as a pale yellow solid. Yield: 0.024 g, 10 %; MS (ESI) m/z 775.07 [M+1]+.
  • reaction mixture is stirred under argon atmosphere at room temperature for 15 h.
  • the reaction mixture is cooled to 0 °C and sodium perchlorate (0.40 g) in 20 mL acetone is added and resulting solid is filtered and dried to afford (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)(1H-imidazol-1- yl)phosphinic acid (23).
  • Crude compound obtained is column purified by flash column chromatography using silica gel (230- 400 mesh) and 0 to 40 % ethyl acetate in hexanes as eluents to afford 7-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thieno[3,2-b]pyridine (2).
  • reaction mixture is cooled down, poured onto ice cold water and extracted with ethyl acetate.
  • the organic layer is dried over anhydrous sodium sulphate, filtered and concentrated to get crude product, which is purified by flash column chromatography using silica gel (230-400 mesh) and 60-90% ethyl acetate in hexanes as eluents to afford 3-(2-(4-chloro-2-(3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridin-7- yl)phenoxy)ethyl)-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (7).
  • reaction mixture is diluted with water and extracted with ethyl acetate.
  • organic layer is dried over anhydrous sodium sulphate, filtered and concentrated to get crude product, which is purified by flash column chromatography using silica gel (230-400 mesh) and 2-5 % methanol in dichloromethane as eluents to afford 3-(2-(4-chloro-2-(3-hydroxythieno[3,2-b]pyridin-7- yl)phenoxy)ethyl)-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (8).
  • reaction mixture is concentrated under reduced pressure and purified by flash chromatography using silica gel (230- 400 mesh) and 40-70% ethyl acetate in hexanes as eluents to afford 7-(5-chloro-2-(2-(5-cyano-2- methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl diethyl phosphate (9).
  • reaction mixture is stirred under argon atmosphere at room temperature for 15 h. After this time, the reaction mixture is cooled to 0 °C. Then, sodium perchlorate in acetone is added and the resulting solid is filtered and dried to afford 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4- oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl hydrogen (1H-imidazol-1-yl)phosphonate (11).
  • Mono- and bicistronic reporter mRNAs containing a 5’ CAP1 mimetic and/or a cap- independent translation element were generated using translational enhancer Compound 185F.
  • a DNA template consisting of a T7 RNA polymerase reporter upstream of the Renilla or Firefly luciferase (Fluc) open reading frame (ORF) was generated with and without a downstream, cap- independent cistron composed of the opposite luciferase ORF. The following components were added in the order specified:
  • the reaction was incubated at 37 o C for 2 hours to generate a 5’ CAP1 Fluc mRNA (FIG.1).
  • T7 RNA polymerase is recruited to the template DNA via the specific promoter sequence. Once bound, the polymerase will incorporate nucleotides that base pair with the template DNA. The sequence near the transcription initiation site can influence the efficiency of RNA production with a GG or AG dinucleotide yielding the best results.
  • the transcription reaction was carried out to completion.
  • the transcription reaction containing the 5’ CAP1 Fluc mRNA was directly added to a cell-free translation extract, incubated at 37 o C for an additional 30 minutes, leading to the synthesis of the Firefly luciferase protein from the mRNA. Luciferase protein expression was directly assayed by the measuring the light signal in the reaction mixture.
  • purified mRNAs containing either a 5’ ARCA or“compound 185F” cap and a cap-independent control reporter were delivered into HEK-293t cells by lipid mediated RNA transfection. Cells were cultured for 4-8 hours under normal conditions.

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Abstract

The present disclosure relates to novel translational enhancers comprising an eukaryotic initiation factor 4E (eIF4E) ligand attached to a dinucleotide. The present disclosure also relates to RNA molecules (e.g., mRNAs) comprising the novel translational enhancers, which imparts properties to the RNA molecules that are advantageous to therapeutic development, methods of using RNA molecules comprising such novel translational enhancers for therapeutic uses, as well as kits containing the translational enhancers.

Description

TRANSLATIONAL ENHANCERS AND RELATED METHODS BACKGROUND
[0001] Protein replacement therapies involve supplementation of deficient or aberrant proteins and enzymes as well as modulation of cell behavior by expression of exogenous proteins. It is well known that nucleic acids can be used to modulate protein production in vivo. Because application of DNA or mRNA enables expression of virtually any desired protein inside host cells and tissues, expression of disease-relevant proteins to treat disease conditions can be achieved by intracellular delivery of plasmid DNA (pDNA) or messenger RNA (mRNA).
mRNA-based protein replacement therapies, in particular, offer several advantages over pDNA, including rapid and transient protein production, no risk of insertional mutagenesis, and greater efficacy of non-viral delivery by virtue of mRNA cytoplasmic activity.
[0002] All naturally-occurring eukaryotic mRNA contains a cap structure - an N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5’ to 5’ triphosphate linkage (“the m7G cap”). During mRNA capping, Cap 0 (7MeG5’-ppp5’-N) is formed as an intermediate. Cap 0 formation is a co-transcriptional modification resulting from three enzymatic activities: RNA triphosphatase, guanylyltransferase and S-adenosyl-l-methionine (AdoMet) dependent (guanine-N7)-methyltransferase (N7MTase). Further methylation at the 2’O position of the ribose of the first nucleotide by an AdoMet-dependent (nucleoside-2’-O-)-methyltransferase (2’OMTase) leads to a Cap 1 structure (7MeG5’-ppp5’-N2¢OMe). See FIG.1. While the m7G Cap 0 structure is known to be required for efficient translation of mRNA, recent studies have revealed that 2’O methylation of +1 nucleotide (Cap 1 structure) is central to the non-self discrimination of innate immune response against foreign RNA (Daffis S., et al., 2010, Nature, 468:452-456; Devarkar S.C., et al., 2016, Proc. Natl. Acad. Sci. U.S.A., 113:596-601). Cellular sensors RIG-I and MDA5 and effectors IFIT1 and IFIT5 of the Type I interferon (IFN) signaling pathway act by discriminating Cap 1 RNA from others. Most significantly, the Cap 1 structure abolished the interactions of RNA with RIG-I and MDA5 and hence did not activate the IFN signaling pathway (Zust, R., et al., 2011, Nat. Immunol., 12:137-143; Schuberth-Wagner C., et al., 2015, Immunity, 43:41-51).
[0003] The 5’ mRNA cap structure is essential for efficient gene expression from yeast to human. It plays a critical role in all aspects of the life cycle of an mRNA molecule. The cap structure protects mRNAs from degradation by exonucleases and promotes transcription, polyadenylation, splicing, and nuclear export of mRNA and U-rich, capped snRNAs. In addition, the cap structure is required for the optimal translation of the vast majority of cellular mRNAs, and it also plays a prominent role in the expression of eukaryotic mRNAs.
[0004] Cap-dependent translation initiation in eukaryotes is a highly regulated rate-limiting step, which involves recruitment and assembly of eukaryotic initiation factor 4F (eIF4F), a multiprotein complex on the 5 cap of the mRNA. eIF4F consists of at least three proteins: the cap-binding protein eukaryotic initiation factor 4E (eIF4E), the ATP-dependent RNA helicase eukaryotic initiation factor 4A (eIF4A), and the scaffold protein eukaryotic initiation factor 4G (eIF4G). eIF4E directly recognizes the cap structure of mRNAs, and is essential for cap- dependent translation initiation, while eIF4G interacts with the other eIF4F subunits as well as with the poly-A binding protein on the poly-A tail of the mRNA to create a close mRNA circle during translation initiation.
[0005] Inside cells, there is a balance between the processes of translation and mRNA decay, and the steady state level of an mRNA is a function of its rate of synthesis and degradation. Messages which are being actively translated are bound by polysomes and the eukaryotic initiation factors eIF4E and eIF4G (in eukaryotes). This blocks access to the cap by the decapping enzymes and protects the mRNA molecule. In most eukaryotes, mRNAs are eventually usually decapped and subsequently degraded by a 5’-to-3’ exonuclease. Decapping is catalyzed by the enzymes dcp1 and dcp2 which compete with eIF4E to bind to the cap.
[0006] Despite the advantages provided by mRNA-based protein replacement therapies, therapeutic mRNA technology has been limited by a number of factors. Naked, single-stranded mRNAs are prone to nuclease degradation, are inefficiently translated in vivo, and are too large and negatively charged to passively cross the cell membrane, and must, therefore, be provided with additional means of cellular entry. Additionally, for maximal expression in cells or target organs, transfected mRNAs must avoid detection by pattern recognition receptors (PRRs) that evolved to sense pathogenic non-self RNAs. These include PRRs that recognize improperly capped RNAs and double stranded RNA. PRR activation leads to cytokine production, translational arrest and cell toxicity or death.
[0007] There is a need in the art for alternative, effective approaches and therapies for the treatment of protein and enzyme deficiencies using mRNAs, particularly strategies and therapies which overcome the challenges and limitations associated with the administration of mRNAs and the transfection of target cells. The present disclosure meets such needs, and further provides other related advantages. SUMMARY
[0008] The present disclosure generally relates to novel translational enhancers, RNA molecules comprising the novel translational enhancers, and methods of using such RNA molecules for therapeutic uses.
[0009] In certain embodiments, the present disclosure provides a translational enhancer comprising an eIF4E ligand attached to at least one nucleotide. In some embodiments, the eIF4E ligand is attached to the at least one nucleotide via a linker.
[0010] In certain embodiments the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to at least one nucleotide. In some embodiments, the eIF4E ligand is attached to the at least one nucleotide via a linker. In some embodiments, the translational enhancer is attached to the 5’ end of the RNA molecule. In some aspects, the translational enhancer functions as a 5’ cap structure.
[0011] In other embodiments the present disclosure provides a method of making a capped RNA molecule comprising: (a) reacting a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase and under conditions conducive to transcription by the RNA polymerase to generate at least one RNA molecule from the polynucleotide template; and (b) co-transcriptionally coupling to a 5’ end of the at least one RNA molecule a translational enhancer of the disclosure.
[0012] In other embodiments the present disclosure provides a method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject a therapeutically effective amount of an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to a dinucleotide.
[0013] The above embodiments and other aspects of this disclosure are readily apparent in the detailed description that follows. Various references are set forth herein which describe in more detail certain background information, procedures and/or compositions, and are each hereby incorporated by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG.1 shows co-transcriptional synthesis of 5’ capped Firefly Luciferase (FLuc) mRNA by in vitro transcription, and subsequent t FLuc protein expression from the mRNA.1G is absolutely required for transcription, and yield is compromised when the +2 nucleotide is not G.2 Gs are necessary for reasonable yields of in vitro transcripts and is recommended for most transcripts.3 Gs are present in the consensus T7 promoter.4Gs (not pictured) may result in increased 5’ transcript heterogeneity. DETAILED DESCRIPTION
[0015] The present disclosure relates to RNA-based protein replacement therapies that involve supplementation of deficient or aberrant proteins and enzymes. In certain embodiments, the present disclosure provides a novel translational enhancer comprising an eIF4E ligand attached to a dinucleotide. In other embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer of the disclosure. In other embodiments the present disclosure provides a method of making an RNA molecule that comprises a translational enhancer of the disclosure.
[0016] Reference throughout this specification to“one embodiment” or“an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases“in one embodiment” or“in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.
Definitions
[0017] Prior to setting forth this disclosure in more detail, it may be helpful to an
understanding thereof to provide definitions of certain terms to be used herein. Unless defined otherwise, all terms used herein have the same meaning as are commonly understood by one of skill in the art to which this invention belongs. All patents, patent applications and publications referred to throughout the disclosure herein are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail.
[0018] As used herein, and unless noted to the contrary, the following terms and phrases have the meaning noted below.
[0019] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term“about” means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms“a” and“an” as used herein refer to“one or more” of the enumerated components. The use of the alternative (e.g.,“or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms“include,”“have” and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
[0020] In addition, it should be understood that the individual compounds or translational enhancers, or groups of compounds or translational enhancers, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or translational enhancer, or group of compounds or translational enhancers was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure.
[0021] The term“consisting essentially of” limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic characteristics of a claimed invention. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, or modules) “consists essentially of” a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy-terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).
[0022] “Amino” refers to the -NH2 substituent.
[0023] “Aminocarbonyl” refers to the–C(O)NH2 substituent.
[0024] “Carboxyl” refers to the–CO2H substituent.
[0025] “Carbonyl” refers to a–C(O)–,–(CO)– or–C(=O)– group. All notations are used interchangeably within the specification.
[0026] “Cyano” refers to the–CºN substituent.
[0027] “Cyanoalkylene” refers to the -(alkylene)CºN substituent.
[0028] “Acetyl” refers to the–C(O)CH3 substituent.
[0029] “Hydroxy” or“hydroxyl” refers to the -OH substituent.
[0030] “Hydroxyalkylene” refers to the -(alkylene)OH subsituent. [0031] “Oxo” refers to a =O substituent.
[0032] “Thio” or“thiol” refer to a–SH substituent.
[0033] “Alkyl” refers to a saturated, straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, having from one to twelve carbon atoms (C1-C12 alkyl), from one to eight carbon atoms (C1-C8 alkyl) or from one to six carbon atoms (C1-C6 alkyl), and which is attached to the rest of the molecule by a single bond. Exemplary alkyl groups include methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.
[0034] “Lower alkyl” has the same meaning as alkyl defined above but having from one to four carbon atoms (C1-C4 alkyl).
[0035] “Alkenyl” refers to an unsaturated alkyl group having at least one double bond and from two to twelve carbon atoms (C2-C12 alkenyl), from two to eight carbon atoms (C2-C8 alkenyl) or from two to six carbon atoms (C2-C6 alkenyl), and which is attached to the rest of the molecule by a single bond, e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.
[0036] “Alkynyl” refers to an unsaturated alkyl group having at least one triple bond and from two to twelve carbon atoms (C2-C12 alkynyl), from two to ten carbon atoms (C2-C10 alkynyl) from two to eight carbon atoms (C2-C8 alkynyl) or from two to six carbon atoms (C2-C6 alkynyl), and which is attached to the rest of the molecule by a single bond, e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
[0037] “Alkylene” or“alkylene chain” refers to a straight or branched divalent hydrocarbon (alkyl) chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, respectively. Alkylenes can have from one to twelve carbon atoms, e.g., methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule can be through one carbon or any two carbons within the chain.“Optionally substituted alkylene” refers to alkylene or substituted alkylene.
[0038] “Alkenylene” refers to divalent alkene. Examples of alkenylene include without limitation, ethenylene (-CH=CH-) and all stereoisomeric and conformational isomeric forms thereof.“Substituted alkenylene” refers to divalent substituted alkene.“Optionally substituted alkenylene” refers to alkenylene or substituted alkenylene.
[0039] “Alkynylene” refers to divalent alkyne. Examples of alkynylene include without limitation, ethynylene, propynylene.“Substituted alkynylene” refers to divalent substituted alkyne. [0040] “Alkoxy” refers to a radical of the formula -ORa where Ra is an alkyl having the indicated number of carbon atoms as defined above. Examples of alkoxy groups include without limitation–O-methyl (methoxy), -O-ethyl (ethoxy), -O-propyl (propoxy), -O-isopropyl (iso propoxy) and the like.
[0041] “Alkylaminyl” refers to a radical of the formula -NHRa or -NRaRa where each Ra is, independently, an alkyl radical having the indicated number of carbon atoms as defined above.
[0042] “Cycloalkylaminyl” refers to a radical of the formula -NHRa or -NRaRa where Ra is a cycloalkyl radical as defined herein.
[0043] “Alkylcarbonylaminyl” refers to a radical of the formula -NHC(O)Ra or -NRaC(O)Ra, where Ra is an alkyl radical having the indicated number of carbon atoms as defined herein.
[0044] “Cycloalkylcarbonylaminyl” refers to a radical of the formula -NHC(O)Ra, where Ra is a cycloalkyl radical as defined herein.
[0045] “Aryl” refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. Exemplary aryls are hydrocarbon ring system radical comprising hydrogen and 6 to 9 carbon atoms and at least one aromatic ring;
hydrocarbon ring system radical comprising hydrogen and 9 to 12 carbon atoms and at least one aromatic ring; hydrocarbon ring system radical comprising hydrogen and 12 to 15 carbon atoms and at least one aromatic ring; or hydrocarbon ring system radical comprising hydrogen and 15 to 18 carbon atoms and at least one aromatic ring. For purposes of the compounds of the present disclosure, the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.“Optionally substituted aryl” refers to an aryl group or a substituted aryl group.
[0046] “Arylene” denotes divalent aryl, and“substituted arylene” refers to divalent substituted aryl.
[0047] “Aralkyl” or“araalkylene” may be used interchangeably and refer to a radical of the formula -Rb-Rc where Rb is an alkylene chain as defined herein and Rc is one or more aryl radicals as defined herein, for example, benzyl, diphenylmethyl and the like.
[0048] “Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, three to nine carbon atoms, three to eight carbon atoms, three to seven carbon atoms, three to six carbon atoms, three to five carbon atoms, a ring with four carbon atoms, or a ring with three carbon atoms. The cycloalkyl ring may be saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl,
7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like.
[0049] “Cycloalkylalkylene” or“cycloalkylalkyl” may be used interchangeably and refer to a radical of the formula -RbRe where Rb is an alkylene chain as defined herein and Re is a cycloalkyl radical as defined herein. In certain embodiments, Rb is further substituted with a cycloalkyl group, such that the cycloalkylalkylene comprises two cycloalkyl moieties.
Cyclopropylalkylene and cyclobutylalkylene are exemplary cycloalkylalkylene groups, comprising at least one cyclopropyl or at least one cyclobutyl group, respectively.
[0050] “Fused” refers to any ring structure described herein which is fused to an existing ring structure in the compounds or translational enhancers of the present disclosure. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure which becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced with a nitrogen atom.
[0051] “Halo” or“halogen” refers to bromo (bromine), chloro (chlorine), fluoro (fluorine), or iodo (iodine).
[0052] “Haloalkyl” refers to an alkyl radical having the indicated number of carbon atoms, as defined herein, wherein one or more hydrogen atoms of the alkyl group are substituted with a halogen (halo radicals), as defined above. The halogen atoms can be the same or different. Exemplary haloalkyls are trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.
[0053] “Heterocyclyl,”“heterocycle,” or“heterocyclic ring” refers to a stable 3- to 18- membered saturated or unsaturated radical which consists of two to twelve carbon atoms and from one to six heteroatoms, for example, one to five heteroatoms, one to four heteroatoms, one to three heteroatoms, or one to two heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Exemplary heterocycles include without limitation stable 3-15 membered saturated or unsaturated radicals, stable 3-12 membered saturated or unsaturated radicals, stable 3-9 membered saturated or unsaturated radicals, stable 8-membered saturated or unsaturated radicals, stable 7-membered saturated or unsaturated radicals, stable 6-membered saturated or unsaturated radicals, or stable 5-membered saturated or unsaturated radicals. [0054] Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of non-aromatic heterocyclyl radicals include, but are not limited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl,
decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, thietanyl, trithianyl,
tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and
1,1-dioxo-thiomorpholinyl. Heterocyclyls include heteroaryls as defined herein, and examples of aromatic heterocyclyls are listed in the definition of heteroaryls below.
[0055] “Heterocyclylalkyl” or“heterocyclylalkylene” refers to a radical of the formula -RbRf where Rb is an alkylene chain as defined herein and Rf is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to the alkyl radical at the nitrogen atom.
[0056] “Heteroaryl” or“heteroarylene” refers to a 5- to 14-membered ring system radical comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of the compounds or translational enhancers of the present disclosure, the heteroaryl radical may be a stable 5-12 membered ring, a stable 5-10 membered ring, a stable 5-9 membered ring, a stable 5-8 membered ring, a stable 5-7 membered ring, or a stable 6 membered ring that comprises at least 1 heteroatom, at least 2 heteroatoms, at least 3 heteroatoms, at least 4 heteroatoms, at least 5 heteroatoms or at least 6 heteroatoms. Heteroaryls may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, 2 carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized. The heteroatom may be a member of an aromatic or non-aromatic ring, provided at least one ring in the heteroaryl is aromatic. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,
benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl,
benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl).
[0057] “Heteroarylalkyl” or“heteroarylalkylene” refers to a radical of the formula -RbRg where Rb is an alkylene chain as defined above and Rg is a heteroaryl radical as defined above.
[0058] “Thioalkyl” refers to a radical of the formula -SRa where Ra is an alkyl radical as defined above containing one to twelve carbon atoms, at least 1-10 carbon atoms, at least 1-8 carbon atoms, at least 1-6 carbon atoms, or at least 1-4 carbon atoms.
[0059] “Heterocyclylaminyl” refers to a radical of the formula–NHRf where Rf is a heterocyclyl radical as defined above.
[0060] “Thione” refers to a =S group attached to a carbon atom of a saturated or unsaturated (C3-C8)cyclic or a (C1-C8)acyclic moiety.
[0061] “Sulfoxide” refers to a–S(O)- group in which the sulfur atom is covalently attached to two carbon atoms.
[0062] “Sulfone” refers to a–S(O)2- group in which a hexavalent sulfur is attached to each of the two oxygen atoms through double bonds and is further attached to two carbon atoms through single covalent bonds.
[0063] The term“oxime” refers to a–C(Ra)=N-ORa radical where Ra is hydrogen, lower alkyl, an alkylene or arylene group as defined above.
[0064] The compounds or translational enhancers provided in the present disclosure can exist in various isomeric forms, as well as in one or more tautomeric forms, including both single tautomers and mixtures of tautomers. The term“isomer” is intended to encompass all isomeric forms of a compound of the present disclosure, including tautomeric forms of the compound.
[0065] Some compounds or translational enhancers described herein can have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. A compound provided in the present disclosure can be in the form of an optical isomer or a diastereomer. Accordingly, the invention encompasses compounds or translational enhancers provided in the present disclosure and their uses as described herein in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds or translational enhancers provided in the present disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, or via chemical separation of stereoisomers through the employment of optically active resolving agents.
[0066] Unless otherwise indicated,“stereoisomer” means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
[0067] If there is a discrepancy between a depicted structure and a name given to that structure, then the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. In some cases, however, where more than one chiral center exists, the structures and names may be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know if the compounds or translational enhancers are prepared as single enantiomers from the methods used to prepare them.
[0068] In this description, a“pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound of the present disclosure. Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.
[0069] In addition, it should be understood that the individual compounds or translational enhancers, or groups of compounds or translational enhancers, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or translational enhancer or group of compounds or translational enhancers was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure.
[0070] As used herein, the term“derivative” refers to a modification of a compound by chemical or biological means, with or without an enzyme, which modified compound is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. Generally, a“derivative” differs from an“analog” in that a parent compound may be the starting material to generate a“derivative,” whereas the parent compound may not necessarily be used as the starting material to generate an“analog.” A derivative may have different chemical, biological or physical properties from the parent compound, such as being more hydrophilic or having altered reactivity as compared to the parent compound.
Derivatization (i.e., modification) may involve substitution of one or more moieties within the molecule (e.g., a change in functional group). For example, a hydrogen may be substituted with a halogen, such as fluorine or chlorine, or a hydroxyl group (-OH) may be replaced with a carboxylic acid moiety (-COOH). Other exemplary derivatizations include glycosylation, alkylation, acylation, acetylation, ubiqutination, esterification, and amidation.
[0071] The term“derivative” also refers to all solvates, for example, hydrates or adducts (e.g., adducts with alcohols), active metabolites, and salts of a parent compound. The type of salt depends on the nature of the moieties within the compound. For example, acidic groups, such as carboxylic acid groups, can form alkali metal salts or alkaline earth metal salts (e.g., sodium salts, potassium salts, magnesium salts, calcium salts, and also salts with physiologically tolerable quaternary ammonium ions and acid addition salts with ammonia and physiologically tolerable organic amines such as, for example, triethylamine, ethanolamine or tris-(2- hydroxyethyl)amine). Basic groups can form acid addition salts with, for example, inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or with organic carboxylic acids or sulfonic acids such as acetic acid, citric acid, lactic acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, methanesulfonic acid or p-toluenesulfonic acid. Molecules that simultaneously contain a basic group and an acidic group, for example, a carboxyl group in addition to basic nitrogen atoms, can be present as zwitterions. Salts can be obtained by customary methods known to those skilled in the art, for example, by combining a compound with an inorganic or organic acid or base in a solvent or diluent, or from other salts by cation exchange or anion exchange.
[0072] As used herein, the term“eIF4E,” also referred to as“eukaryotic translation initiation factor-4E,” refers to a translation initiation factor that, when part of an eIF4F pre-initiation complex also comprising eIF4A RNA helicase and eIF4G scaffold protein, binds to the 7- methyl-guanosine (m7GpppX) 5’-cap structure on eukaryotic mRNAs and directs ribosomes to the cap structure. The availability of eIF4E as part of the eIF4F complex is a limiting factor in controlling the rate of translation. Interactions of eIF4E and the m7G cap and eIF4G are tightly regulated by key mitogenic signals, such as the PI3K/mTOR and Ras/MAPK signal transduction pathways. There are four different isoforms of eIF4E: isoform 1 is the canonical sequence; isoform 2 contains an alternate in-frame exon in the 3’-coding region compared to isoform 1; isoform 3 uses an alternate 5’-terminal exon, which results in a different 5’-UTR and use of an alternate translation start codon compared to isoform 1; and isoform 4 differs in its 5’-UTR and contains an alternate exon in its 5’-coding region compared to isoform 1. In certain
embodiments, eIF4E refers to eIF4E isoform 1, isoform 2, isoform 3, isoform 4, or any combination thereof. In certain embodiments, eIF4E refers to the canonical eIF4E isoform 1. In particular embodiments, eIF4E refers to human eIF4E.
[0073] As used herein, the terms“attached,”“linked,”“connected,”“bound”,“coupled,” “fused,” or“fusion” are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation and recombinant means.
[0074] As used herein, the term“attached directly” refers to two molecules that are attached or linked or connected or bound or coupled or fused to one another in the absence of an intervening molecule.
[0075] The term“promoter” as used herein refers to a region of dsDNA template that directs and controls the initiation of transcription of a particular DNA sequence (e.g. gene). Promoters are located on the same strand and upstream on the DNA (towards the 5’ region of the sense strand). Promoters are typically immediately adjacent to (or partially overlap with) the DNA sequence to be transcribed. Nucleotide positions in the promoter are designated relative to the transcriptional start site, where transcription of DNA begins (position +1). The initiating oligonucleotide primer is complementary to initiation site of promoter sequence (which, in certain embodiments, is at positions +1 and +2 and, in the case of initiating tetramers, at positions +1, +2 and +3).
[0076] As used herein, the terms“transcription” or“transcription reaction” refers to methods known in the art for enzymatically making RNA that is complementary to DNA template, thereby producing the number of RNA copies of a DNA sequence. The RNA molecule synthesized in transcription reaction called“RNA transcript”,“primary transcript” or “transcript”. Transcription of DNA template may be exponential, nonlinear or linear. A DNA template may be a double stranded linear DNA, a partially double stranded linear DNA, circular double stranded DNA, DNA plasmid, PCR amplicon, a modified nucleic acid template which is compatible with RNA polymerase.
[0077] As used herein, the terms“translation” or“translation reaction” refer to the process or mechanism of synthesizing a protein from a messenger RNA (mRNA). Translation reaction involving the compositions and methods provided herein employs the translational enhancers of the disclosure.
[0078] As used herein, the term“immune response” refers to the action of an immune cell, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement), that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. In certain embodiments, an immune response comprises an antigen-specific T cell response.
[0079] The phrase“inducing an attenuated immune response” refers to causing a decreased stimulation of one or more components of a subject’s innate immune system and/or one or more components of a subject’s adaptive immune system. The assay for detecting cytokine levels (e.g., IL-2, IL-10, IFNg) to determine whether an immune response is induced, enhanced, or attenuated is the multiplex assay described by Dossus et al. (J. Immunol. Methods 350:125, 2009). The assay for detecting T cell proliferation to determine whether an immune response induced or enhanced is the assay described by Liu et al. (Clin. Cancer Res.21:1639, 2015). The assay for determining increased antigen responsiveness is the assay described by Tumeh et al. (Nature 515:568, 2014).
[0080] As used herein,“amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to chemical entities that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical entities that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0081] A“conservative substitution” refers to amino acid substitutions that do not
significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other conservative substitutions groups include: sulfur- containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company. [0082] As used herein, the phrase“cell-free translation extract” refers to an in vitro system used for translating protein from mRNAs. A cell-free translation extract contains all cellular components required for protein expression. Thus, an exogenous mRNA molecule added to the cell-free translation extract is translated to the protein encoded by the mRNA. In certain embodiments, a transcribed mRNA is incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce a desired polypeptide or fragment thereof.
[0083] As used herein,“protein” or“polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers.
[0084] “Nucleic acid molecule” or“polynucleotide” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double stranded. If single stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
[0085] As used herein, the term“agent” refers to any molecule, either naturally occurring or synthetic, e.g., peptide, protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule (e.g., an organic molecule having a molecular weight of less than about 2500 daltons, e.g., less than 2000, less than 1000, or less than 500 daltons), circular peptide, peptidomimetic, antibody, polysaccharide, lipid, fatty acid, inhibitory RNA (e.g., siRNA or shRNA), polynucleotide, oligonucleotide, aptamer, drug compound, or other compound.
[0086] The term“inhibit” or“inhibitor” refers to an alteration, interference, reduction, down regulation, blocking, suppression, abrogation or degradation, directly or indirectly, in the expression, amount or activity of a target gene, target protein, or signaling pathway relative to (1) a control, endogenous or reference target or pathway, or (2) the absence of a target or pathway, wherein the alteration, interference, reduction, down regulation, blocking, suppression, abrogation or degradation is statistically, biologically, or clinically significant. The term “inhibit” or“inhibitor” includes gene“knock out” and gene“knock down” methods, such as by chromosomal editing.
[0087] “Treatment,”“treating” or“ameliorating” refers to medical management of a disease, disorder, or condition of a subject (i.e., patient), which may be therapeutic,
prophylactic/preventative, or a combination treatment thereof. A treatment may improve or decrease the severity at least one symptom of a disease, delay worsening or progression of a disease, or delay or prevent onset of additional associated diseases.“Reducing the risk of developing a disease” refers to preventing or delaying onset of a disease or reoccurrence of one or more symptoms of the disease (e.g., cancer). In certain embodiments, the immune
modulation provided by the translational enhancers of this disclosure aids or augments treatment regimens or aids or augments a host organism’s immune system.
[0088] A“patient” or“subject” includes an animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. The animal can be a mammal, such as a non-primate and a primate (e.g., monkey and human). In some
embodiments, a subject is a human, such as a human infant, child, adolescent or adult.
[0089] Further, a“mammal” includes primates, such as humans, monkeys and apes, and non- primates such as domestic animals, including laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife or the like.
[0090] “Effective amount” or“therapeutically effective amount” refers to that amount of a composition described herein which, when administered to a mammal (e.g., human), is sufficient to aid in treating a disease. The amount of a composition that constitutes a“therapeutically effective amount” will vary depending on the cell preparations, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure. When referring to an individual active ingredient or composition, administered alone, a therapeutically effective dose refers to that ingredient or composition alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients, compositions or both that result in the therapeutic effect, whether
administered serially, concurrently or simultaneously. [0091] The term“therapeutically effective amount”, as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
[0092] As used herein,“hyperproliferative disorder” or“hyperproliferative disease” refers to excessive growth or proliferation as compared to a normal cell or an undiseased cell. Exemplary hyperproliferative disorders include dysplasia, neoplasia, non-contact inhibited or oncogenically transformed cells, tumors, cancers, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis, or the like). In certain embodiments, a cancer being treated by the compositions and methods of this disclosure includes carcinoma (epithelial), sarcoma (connective tissue), lymphoma or leukemia (hematopoietic cells), germ cell tumor (pluripotent cells), blastoma (immature“precursor” cells or embryonic tissue), or any combination thereof. These various forms of hyperproliferative disease are known in the art and have established criteria for diagnosis and classification (e.g., Hanahan and Weinberg, Cell 144:646, 2011; Hanahan and Weinberg Cell 100:57, 2000; Cavallo et al., Canc. Immunol.
Immunother.60:319, 2011; Kyrigideis et al., J. Carcinog.9:3, 2010).
[0093] As used herein, the term“nucleobase” refers to a nitrogen-containing heterocyclic moiety, which is the parts of the nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand in a sequence specific manner. The most common naturally-occurring nucleobases are: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).
[0094] The term“modified nucleobase” refers to a moiety that can replace a nucleobase. The modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner. A modified nucleobase can pair with at least one of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex. The term“modified nucleoside” or“modified nucleotide” refers to a nucleoside or nucleotide that contains a modified nucleobase and/or other chemical modification disclosed herein, such as modified sugar, modified phosphorus atom bridges or modified internucleoside linkage.
[0095] Non-limiting examples of suitable nucleobases include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine optionally having their respective amino groups protected by, e.g., acyl protecting groups, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5- methylcytosine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6- diaminopurine, azacytosine, 2-thiouracil, 2-thiothymine, 2-aminopurine, N9-(2-amino-6- chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8- aza-guanine), N8-(8-aza-7-deazaadenine), pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products). Exemplary modified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol.7, 313.
[0096] As used herein, the term“messenger RNA” (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo. An mRNA has been transcribed from a DNA sequence by an RNA polymerase enzyme, and interacts with a ribosome to synthesize genetic information encoded by DNA. Generally, mRNA are classified into two sub-classes: pre-mRNA and mature mRNA. Precursor mRNA (pre- mRNA) is mRNA that has been transcribed by RNA polymerase but has not undergone any post-transcriptional processing (e.g., 5‘capping, splicing, editing, and polyadenylation). Mature mRNA has been modified via post-transcriptional processing (e.g. , spliced to remove introns and polyadenylated) and is capable of interacting with ribosomes to perform protein synthesis. An mRNA can be isolated from tissues or cells by a variety of methods. For example, a total RNA extraction can be performed on cells or a cell lysate and the resulting extracted total RNA can be purified (e.g. , on a column comprising oligo-dT beads) to obtain extracted mRNA.
[0097] Alternatively, mRNA can be synthesized in a cell-free environment, for example by in vitro transcription (IVT). An“polynucleotide template” as used herein, includes
deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of mRNA. In some embodiments, an IVT template encodes a 5’ untranslated region, contains an open reading frame, and encodes a 3’ untranslated region and a poly A tail. The particular nucleotide sequence composition and length of an IVT template will depend on the mRNA of interest encoded by the template. An“polynucleotide template” as used herein, may also include ribonucleic acid (RNA).
[0098] A“5’ untranslated region (UTR)” refers to a region of an mRNA that is directly upstream (i.e., 5’) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a protein or peptide.
[0099] A“3’ untranslated region (UTR)” refers to a region of an mRNA that is directly downstream (i.e., 3’) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a protein or peptide.
[0100] An“open reading frame” is a continuous stretch of RNA beginning with a start codon (e.g., ATG), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a protein or peptide.
[0101] A“poly-A tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, consecutive adenosine monophosphates. A poly-A tail may contain 10 to 300 adenosine monophosphates. For example, a poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly-A tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo, etc.) the poly-A tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus, and translation.
[0102] The present disclosure provides for polynucleotides comprised of unmodified or modified nucleosides and nucleotides and combinations thereof. As described herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein,“nucleotide” is defined as a nucleoside including a phosphate group. The modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides). The polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. Any combination of base/sugar or linker may be incorporated into the polynucleotides of the disclosure. [0103] As used herein, a“structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural
modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to“AT-5meC-G”. The same polynucleotide may be structurally modified from“ATCG” to“ATCCCG”. Here, the dinucleotide“CC” has been inserted, resulting in a structural modification to the polynucleotide.
[0104] In certain embodiments, polynucleotides of the disclosure may include at least one chemical modification. The polynucleotides described herein can include various substitutions and/or insertions from native or naturally occurring polynucleotides, e.g., in addition to the modification on the 5’ terminal mRNA cap moieties disclosed herein. As used herein, when referring to a polynucleotide, the terms“chemical modification” or, as appropriate,“chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribnucleosides and the internucleoside linkages in one or more of their position, partem, percent or population. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5’-terminal mRNA cap moieties.
[0105] The modifications may be various distinct modifications. In some embodiments, the regions may contain one, two, or more (optionally different) nucleoside or nucleotide
modifications. In some embodiments, a modified polynucleotide introduced to a cell may exhibit reduced degradation in the cell as compared to an unmodified polynucleotide.
[0106] Modifications of the polynucleotides of the disclosure include, but are not limited to those listed in detail below. The polynucleotide may comprise modifications which are naturally occurring, non-naturally occurring or the polynucleotide can comprise both naturally and non- naturally occurring modifications.
[0107] The polynucleotides of the disclosure can include any modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine or purine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). [0108] Non-natural modified nucleotides may be introduced to polynucleotides during synthesis or post-synthesis of the chains to achieve desired functions or properties. The modifications may be on internucleotide lineage, the purine or pyrimidine bases, or sugar. The modification may be introduced at the terminal of a chain or anywhere else in the chain; with chemical synthesis or with a polymerase enzyme. Any of the regions of the polynucleotides may be chemically modified.
[0109] The present disclosure provides for polynucleotides comprised of unmodified or modified nucleosides and nucleotides and combinations thereof. As described herein
“nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein,“nucleotide” is defined as a nucleoside including a phosphate group. The modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides). The polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. Any combination of base/sugar or linker may be incorporated into the polynucleotides of the disclosure.
[0110] Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), including but not limited to chemical modification, that are useful in the compositions and methods of the present disclosure include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6- methyladenosine; 2- methylthio-N6-threonyl carbamoyladenosine; N6- glycinylcarbamoyladenosine; N6- isopentenyladenosine; N6-methyladenosine; N6- threonylcarbamoyladenosine; l, 2’-0- dimethyladenosine; 1 -methyladenosine; 2’-0- methyladenosine; 2’-0-ribosyladenosine
(phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6- hydroxynorvalyl carbamoyladenosine; 2’-0- methyladenosine; 2’-0-ribosyladenosine
(phosphate); Isopentenyladenosine; N6-(cis- hydroxyisopentenyl)adenosine; N6,2’-0- dimethyladenosine; N6,2’-0-dimethyladenosine; N6,N6,2’-0-trimethyladenosine; N6,N6- dimethyladenosine; N6-acetyladenosine; N6- hydroxynorvalylcarbamoyladenosine; N6-methyl- N6-threonylcarbamoyladenosine; 2- methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; Nl-methyl- adenosine; N6, N6 (dimethyl)adenine; N6-cis-hydroxy- isopentenyl-adenosine; a-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2
(methylthio) N6 (isopentenyl)adenine; 2- (alkyl)adenine; 2-(aminoalkyl)adenine; 2- (aminopropyl)adenine; 2-(halo)adenine; 2- (halo)adenine; 2-(propyl)adenine; 2’-Amino-2’- deoxy-ATP; 2’-Azido-2’-deoxy-ATP; 2’-Deoxy- 2’-a-aminoadenosine TP; 2’-Deoxy-2’-a- azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8- (alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8- (thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7- methyladenine; 1- Deazaadenosine TP; 2’Fluoro-N6-Bz-deoxyadenosine TP; 2’-OMe-2- Amino- ATP; 2’0-methyl- N6-Bz-deoxyadenosine TP; 2’-a-Ethynyladenosine TP; 2- aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2’-a-Trifluoromethyladenosine TP; 2- Azidoadenosine TP; 2’-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2’-b- Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2’- Deoxy-2’,2’-difluoroadenosine TP; 2’-Deoxy-2’-a-mercaptoadenosine TP; 2’-Deoxy-2’-a- thiomethoxyadenosine TP; 2’-Deoxy-2’- b-aminoadenosine TP; 2’-Deoxy-2’-b-azidoadenosine TP; 2’-Deoxy-2’-b-bromoadenosine TP; 2’-Deoxy-2’-b-chloroadenosine TP; 2’-Deoxy-2’-b- fiuoroadenosine TP; 2’-Deoxy-2’-b- iodoadenosine TP; 2’-Deoxy-2’-b-mercaptoadenosine TP; 2’- Deoxy-2’-b- thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2- Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3- bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza- 3-iodoadenosine TP; 3-Deazaadenosine TP; 4’-Azidoadenosine TP; 4’-Carbocyclic adenosine TP; 4’-Ethynyladenosine TP; 5’-Homo-adenosine TP; 8-Aza-ATP; 8-bromo- adenosine TP; 8- Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza- 2,6- diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6- diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3- methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4- acetylcytidine; 2’-0-methylcytidine; 2’-0-methylcytidine; 5, 2’-0-dimethylcytidine; 5-formyl-2’- O-methylcytidine; Lysidine; N4, 2’-0-dimethylcytidine; N4-acetyl-2’-0-methylcytidine; N4- methylcytidine; N4,N4-Dimethyl-2 -OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;
Pseudo-iso-cytidine; pyrrolo-cytidine; a-thio-cytidine; 2-(thio)cytosine; 2’-Amino-2’-deoxy- CTP; 2’-Azido-2’-deoxy-CTP; 2’-Deoxy-2’-a-aminocytidine TP; 2’-Deoxy-2’-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3- (methyl)cytidine; 4,2’-0-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5
(propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5- (halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo- cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1 -methyl- 1-deaza-pseudoisocyti dine; 1-methyl-pseudoisocytidine; 2- methoxy- 5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy- 1- methyl- pseudoisocytidine; 4-methoxy-pseudoisocy tidine; 4-thio- 1 -methyl- 1 -deaza- pseudoisocy tidine; 4-thio-l -methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza- zebularine; 5-methyl- zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5 -(2 -Bromo- vinyl)cy tidine TP; 2,2’- anhydro-cytidine TP hydrochloride; 2’Fluor-N4-Bz-cytidine TP;
2’Fluoro-N4-Acetyl-cytidine TP; 2’-0-Methyl-N4-Acetyl-cytidine TP; 2’0-methyl-N4-Bz- cytidine TP; 2’-a-Ethynylcytidine TP; 2’-a-Trifluoromethylcytidine TP; 2’-b-Ethynylcytidine TP; 2’-b-Trifluoromethylcytidine TP; 2’-Deoxy-2’,2’-difluorocytidine TP; 2’-Deoxy-2’-a- mercaptocytidine TP; 2’-Deoxy-2’-a- thiomethoxy cytidine TP; 2’-Deoxy-2’-b-aminocytidine TP; 2’-Deoxy-2’-b-azidocytidine TP; 2’- Deoxy-2’-b-bromocytidine TP; 2’-Deoxy-2’-b- chlorocytidine TP; 2’-Deoxy-2’-b-fluorocytidine TP; 2’-Deoxy-2’-b-iodocytidine TP; 2’-Deoxy- 2’-b-mercaptocytidine TP; 2’-Deoxy-2’-b- thiomethoxy cytidine TP; 2’-0-Methyl-5-(l - propynyl)cytidine TP; 3’-Ethynylcytidine TP; 4’- Azidocytidine TP; 4’-Carbocyclic cytidine TP; 4’-Ethynylcytidine TP; 5-(l -Propynyl)ara- cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5- Aminoallyl-CTP; 5 -Cyanocy tidine TP; 5 - Ethynylara-cy tidine TP; 5 -Ethyny Icy tidine TP; 5’- Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP;
Pseudoisocytidine; 7-methylguanosine; N2,2’-0- dimethylguanosine; N2-methylguanosine; Wyosine; l,2’-0-dimethylguanosine; 1- methylguanosine; 2’-0-methylguanosine; 2’-0- ribosylguanosine (phosphate); 2’-0- methylguanosine; 2’-0-ribosylguanosine (phosphate); 7- aminomethyl-7-deazaguanosine; 7- cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2’-0- trimethylguanosine; N2,N2,7-trimethylguanosine;
N2,N2-dimethylguanosine; N2,7,2’-0- trimethylguanosine; 6-thio-guanosine; 7-deaza- guanosine; 8-oxo-guanosine; Nl -methyl- guanosine; a-thio-guanosine; 2 (propyl)guanine; 2- (alkyl)guanine; 2’-Amino-2’-deoxy-GTP; 2’- Azido-2’-deoxy-GTP; 2’-Deoxy-2’-a- aminoguanosine TP; 2’-Deoxy-2’-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6- (methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8- (thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N- (methyl)guanine; l-methyl-6- thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza- guanosine; 6-thio-7-deaza- guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7- methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1 - Me-GTP; 2’Fluoro-N2- isobut l-guanosine TP; 2’0-methyl-N2-isobutyl-guanosine TP; 2’-a- Ethynylguanosine TP; 2’-a- Trifluoromethylguanosine TP; 2’-b-Ethynylguanosine TP; 2’-b- Trifluoromethylguanosine TP; 2’-Deoxy-2’,2’-difluoroguanosine TP; 2’-Deoxy-2’-a- mercaptoguanosine TP; 2’-Deoxy-2’-a- thiomethoxyguanosine TP; 2’-Deoxy-2’-b- aminoguanosine TP; 2’-Deoxy-2’-b-azidoguanosine TP; 2’-Deoxy-2’-b-bromoguanosine TP; 2’- Deoxy-2’-b-chloroguanosine TP; 2’-Deoxy-2’-b- fluoroguanosine TP; 2’-Deoxy-2’-b- iodoguanosine TP; 2’-Deoxy-2’-b-mercaptoguanosine TP; 2’-Deoxy-2’-b-thiomethoxyguanosine TP; 4’-Azidoguanosine TP; 4’-Carbocyclic guanosine TP; 4’-Ethynylguanosine TP; 5’-Homo- guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1 - methylinosine; Inosine; l ,2’-0-dimethylinosine; 2’-0- methylinosine; 7-methylinosine; 2’-0- methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy - thymidine; 2’-0-methyluridine; 2- thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5- hydroxyuridine; 5-methyluridine; 5- taurinomethyl-2-thiouridine; 5-taurinomethyluridine;
Dihydrouridine; Pseudouridine; (3-(3- amino-3-carboxypropyl)uridine; l -methyl-3-(3-amino-5- carboxypropyl)pseudouridine; 1- methylpseduouridine; 1 -ethyl-pseudouridine; 2’-0- methyluridine; 2’-0-methylpseudouridine; 2’- O-methyluridine; 2-thio-2’-0-methyluridine; 3-(3- amino-3-carboxypropyl)uridine; 3,2’-0- dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4- thiouridine; 5- (carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2’-0- dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl- 2’-0- methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5- carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2’-0-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5- Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2’-0-methyluridine; 5- methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methyluridine,), 5- methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5- methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5- Oxyacetic acid- Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; Nl-methyl-pseudo- uracil; Nl-ethyl-pseudo-uracil; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3- (3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)- 2-thiouridine TP; 5- (iso-Pentenylaminomethyl)-2’-0-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5- propynyl uracil; a-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminocarbonylethylenyl)- 4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)- pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; l -(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil; 1- Methy 1-3 -(3 -amino-3-carboxy propyl) pseudouridine TP; l -Methyl-3-(3-amino-3- carboxypropyl)pseudo-UTP; 1 -Methyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 2
(thio)pseudouracil; 2’ deoxy uridine; 2’ fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2’ methyl, 2’amino, 2’azido, 2’fluro-guanosine; 2’-Amino-2’-deoxy-UTP; 2’-Azido-2’-deoxy- UTP; 2’-Azido- deoxyuridine TP; 2’-0-methylpseudouridine; 2’ deoxy uridine; 2’ fluorouridine; 2’-Deoxy-2’-a- aminouridine TP; 2’-Deoxy-2’-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio )pseudouracil; 4-(thio)uracil; 4- thiouracil; 5 (l ,3-diazole-l -alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2- (thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4
(dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5
(methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5
(propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2- (thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5- (alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5- (cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5- (guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(l,3-diazole-l-alkyl)uracil; 5-(methoxy)uracil; 5- (methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio )uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2- (thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5- (methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2,4(dithio )uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5- aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6- aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; P seudo-UTP-1-2- ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1 -carboxymethyl-pseudouridine; 1 -methyl- 1 - deaza-pseudouridine; 1-propynyl-uridine; 1-taurinomethyl-l -methyl-uridine; l-taurinomethyl- 4- thio-uridine; 1 -taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-l - methyl- 1-deaza-pseudouridine; 2-thio-l -methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio- dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio- pseudouridine; 4-methoxy-pseudouridine; 4-thio-l-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (±)l -(2-Hydroxypropyl)pseudouridine TP; (2R)-l -(2- Hydroxypropyl)pseudouridine TP; (2S)-l -(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2- Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara- uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; l -(2,2,2-Trifluoroethyl)-pseudo-UTP; 1 - (2,2,3, 3,3-Pentafluoropropyl)pseudouridine TP; l-(2,2-Diethoxyethyl)pseudouridine TP; 1- (2,4,6- Trimethylbenzyl)pseudouridine TP; l -(2,4,6-Trimethyl-benzyl)pseudo-UTP; 1 -(2,4,6- Trimethyl-phenyl)pseudo-UTP; l-(2-Amino-2-carboxyethyl)pseudo-UTP; l -(2-Amino- ethyl)pseudo-UTP; l-(2-Hydroxyethyl)pseudouridine TP; l-(2-Methoxyethyl)pseudouridine TP; l-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; l-(3,4-Dimethoxybenzyl)pseudouridine TP; l -(3-Amino-3-carboxypropyl)pseudo-UTP; l -(3-Amino-propyl)pseudo-UTP; l-(3- Cyclopropyl-prop-2-ynyl)pseudouridine TP; l-(4-Amino-4-carboxybutyl)pseudo-UTP; l-(4- Amino-benzyl)pseudo-UTP; l-(4-Amino-butyl)pseudo-UTP; l -(4-Amino-phenyl)pseudo-UTP; l-(4-Azidobenzyl)pseudouridine TP; l-(4-Bromobenzyl)pseudouridine TP; l -(4- Chlorobenzyl)pseudouridine TP; 1 -(4-Fluorobenzyl)pseudouridine TP; l-(4- Iodobenzyl)pseudouridine TP; l -(4-Methanesulfonylbenzyl)pseudouridine TP; l -(4- Methoxybenzyl)pseudouridine TP; l-(4-Methoxy-benzyl)pseudo-UTP; l-(4-Methoxy- phenyl)pseudo-UTP; l -(4-Methylbenzyl)pseudouridine TP; l -(4-Methyl-benzyl)pseudo-UTP; 1- (4-Nitrobenzyl)pseudouridine TP; l -(4-Nitro-benzyl)pseudo-UTP; l (4-Nitro-phenyl)pseudo- UTP; l -(4-Thiomethoxybenzyl)pseudouridine TP; l-(4-Trifluoromethoxybenzyl)pseudouridine TP; l -(4-Trifluoromethylbenzyl)pseudouridine TP; l -(5-Amino-pentyl)pseudo-UTP; l -(6- Amino-hexyl)pseudo-UTP; 1 ,6-Dimethyl-pseudo-UTP; l -[3-(2-{2-[2-(2-Aminoethoxy)- ethoxy]-ethoxy}-ethoxy)-propionyl] pseudouridine TP; l -{3-[2-(2-Aminoethoxy)-ethoxy]- propionyl } pseudouridine TP; 1 -Acetylpseudouridine TP; l-Alkyl-6-(l-propynyl)-pseudo-UTP; l-Alkyl-6-(2-propynyl)-pseudo-UTP; l-Alkyl-6-allyl-pseudo-UTP; l-Alkyl-6-ethynyl-pseudo- UTP; l-Alkyl-6-homoallyl-pseudo-UTP; l-Alkyl-6-vinyl-pseudo-UTP; 1 -Ally lpseudouri dine TP; 1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP; 1- Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1 -Biotinyl-PEG2-pseudouridine TP; 1-Biotiny lpseudouri dine TP; 1-But l-pseudo-UTP; 1-Cyanomethy lpseudouri dine TP; 1- Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP; 1- Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1- Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoally lpseudouri dine TP; 1- Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP; l-Me-2-thio-pseudo-UTP; 1-Me- 4-thio-pseudo-UTP; 1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; l-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl- 6-(4-morpholino)-pseudo-UTP; l-Methyl-6-(4-thiomorpholino)-pseudo-UTP; l-Methyl-6- (substituted phenyl)pseudo-UTP; l-Methyl-6-amino-pseudo-UTP; l-Methyl-6-azido-pseudo- UTP; l-Methyl-6-bromo-pseudo-UTP; l-Methyl-6-butyl-pseudo-UTP; 1 -Methyl-6-chloro- pseudo-UTP; l-Methyl-6-cyano-pseudo-UTP; l-Methyl-6-dimethylamino-pseudo-UTP; 1- Methyl-6-ethoxy-pseudo-UTP; l-Methyl-6-ethylcarboxylate-pseudo-UTP; l-Methyl-6-ethyl- pseudo-UTP; l-Methyl-6-fluoro-pseudo-UTP; l-Methyl-6-formyl-pseudo-UTP; l-Methyl-6- hydroxyamino-pseudo-UTP; l-Methyl-6-hydroxy-pseudo-UTP; l-Methyl-6-iodo-pseudo-UTP; l- Methyl-6-iso-propyl-pseudo-UTP; l-Methyl-6-methoxy-pseudo-UTP; l-Methyl-6- methylamino- pseudo-UTP; 1 -Methyl-6-phenyl-pseudo-UTP; 1 -Methyl-6-propyl-pseudo-UTP; l-Methyl-6- tert-butyl-pseudo-UTP; l-Methyl-6-trifluoromethoxy-pseudo-UTP; l-Methyl-6- trifluoromethyl- pseudo-UTP; l-Mo holinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1- Phenyl-pseudo- UTP; 1-Pivaloy lpseudouri dine TP; 1-Propargy lpseudouri dine TP; 1-Propyl- pseudo-UTP; 1 - propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1- Thiomethoxymethylpseudouridine TP; l-Thiomo holinomethylpseudouridine TP; 1- Trifluoroacety lpseudouri dine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Viny lpseudouri dine TP; 2,2’-anhydro-uridine TP; 2’-bromo-deoxyuridine TP; 2’-F-5-Methyl-2’-deoxy-UTP; 2’-OMe-5- Me-UTP; 2’-OMe-pseudo-UTP; 2’-a-Ethynyluridine TP; 2’-a-Trifluoromethyluridine TP; 2’-b- Ethynyluridine TP; 2’-b-Trifluoromethyluridine TP; 2’-Deoxy-2’,2’-difluorouridine TP; 2’- Deoxy-2’-a-mercaptouridine TP; 2’-Deoxy-2’-a-thiomethoxyuridine TP; 2’-Deoxy-2’-b- aminouridine TP; 2’-Deoxy-2’-b-azidouridine TP; 2’-Deoxy-2’-b-bromouridine TP; 2’-Deoxy- 2’- b-chlorouridine TP; 2’-Deoxy-2’-b-fluorouridine TP; 2’-Deoxy-2’-b-iodouridine TP; 2’- Deoxy-2’- b-mercaptouridine TP; 2’-Deoxy-2’-b-thiomethoxyuridine TP; 2-methoxy-4-thio- uridine; 2- methoxyuridine; 2’-0-Methy 1-5 -(l-propynyl)uri dine TP; 3-Alkyl-pseudo-UTP; 4’- Azidouridine TP; 4’-Carbocyclic uridine TP; 4’-Ethynyluridine TP; 5-(l-Propynyl)ara-uridine TP; 5-(2- Furanyl)uridine TP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5’-Homo- uridine TP; 5- iodo-2’-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl- 6- deuterouridine TP; 5-Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2- Trifluoroethyl)-pseudo-UTP; 6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo- UTP; 6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6- Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6- Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl- pseudo-UTP; 6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6- Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo- UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl- pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo- UTP; 6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine l -(4- methylbenzenesulfonic acid) TP; Pseudouridine 1 -(4-methylbenzoic acid) TP; Pseudouridine TP1- [3 -(2-ethoxy)] propionic acid; Pseudouridine TP l-[3- {2-(2-[2-(2-ethoxy )-ethoxy]-ethoxy )- ethoxy}] propionic acid; Pseudouridine TP l -[3- {2-(2-[2- {2(2-ethoxy )-ethoxy} -ethoxy]- ethoxy )-ethoxy}]propionic acid; Pseudouridine TP l -[3- {2-(2-[2-ethoxy ]-ethoxy)- ethoxy}]propionic acid; Pseudouridine TP l -[3- {2-(2-ethoxy)-ethoxy}] propionic acid;
Pseudouridine TP 1 - methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP- Nl -3-propionic acid; Pseudo-UTP-Nl -4-butanoic acid; Pseudo- UTP-Nl-5-pentanoic acid; Pseudo-UTP-Nl -6-hexanoic acid; Pseudo-UTP-Nl-7-heptanoic acid; Pseudo-UTP-Nl-methyl- p-benzoic acid; Pseudo-UTP-Nl -p-benzoic acid; Wybutosine;
Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4- demethylwyosine; 2,6- (diamino)purine; l -(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl: l ,3-( diaza)-2- ( oxo )-phenthiazin-l- yl;l ,3-(diaza)-2-(oxo)-phenoxazin-l-yl; l ,3,5-(triaza)-2,6-(dioxa)- naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl;2’ methyl, 2’amino, 2’azido, 2’fluro- cytidine;2’ methyl, 2’amino, 2’azido, 2’fluro-adenine;2’methyl, 2’amino, 2’azido, 2’fluro- uridine;2’-amino-2’- deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2’-azido-2’- deoxyribose; 2’fluoro-2’- deoxyribose; 2’-fluoro-modified bases; 2’-0-methyl-ribose; 2-oxo-7- arninopyridopyrirnidin-3-yl; 2- oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3- (methyl)-7- (propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6- (methyl)benzimidazole; 4- (methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5 -nitroindole; 6- (aza)pyrimidine; 6-(azo)thymine; 6- (methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; 7- (aminoalkylhydroxy)-l-(aza)-2-(thio )-3-(aza)-phenthiazin-l-yl; 7- (aminoalkylhydroxy)-l-(aza)- 2- (thio)-3-(aza)-phenoxazin-l -yl; 7-(aminoalkylhydroxy)-l, 3- (diaza)-2-(oxo)-phenoxazin-l -yl; 7-(aminoalkylhydroxy)-l,3-( diaza)-2-( oxo )-phenthiazin-l-yl; 7-(aminoalkylhydroxy)-l,3-( diaza)-2-(oxo)-phenoxazin-l-yl; 7-(aza)indolyl; 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio )- 3- (aza)-phenoxazinl-yl; 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio )-3-(aza)-phenthiazin-l-yl; 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7- (guanidiniumalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(guanidiniumalkyl- hydroxy)- l,3-( diaza)-2-( oxo )-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l,3-(diaza)-2-( oxo )- phenoxazin-l-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7- (aza)indolyl; 7-deaza-inosinyl; 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7- substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho- substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine;
Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2- amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl;
Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06- substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrirnidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl- pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4- triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5’-TP; 2-thio-zebularine; 5-aza-2-thio- zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP;
Formycin A TP; Formycin B TP; Pyrrolosine TP; 2’-OH-ara-adenosine TP; 2’-0H-ara-cytidine TP; 2’-0H-ara-uridine TP; 2’-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(l 9-Amino-pentaoxanonadecyl)adenosine TP. In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
[0111] As used herein, the term“LNA” or“locked nucleic acid” refers to a methylene bridge between the 2Ό and 4’C of the nucleotide monomer and it also refers to a sugar analog, a nucleoside, a nucleotide monomer, or a nucleic acid, each of which contains such bridge.
[0112] “Stable compound” and“stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
[0113] “Isomerism” means compounds or translational enhancers that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed
“diastereoisomers,” and stereoisomers that are non-superimposable mirror images of each other are termed“enantiomers” or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a“racemic mixture.”
[0114] A carbon atom bonded to four nonidentical substituents is termed a“chiral center.” “Chiral isomer” means a compound with at least one chiral center. Compounds or translational enhancers with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit.1966, 5, 385; errata 511; Cahn et al., Angew. Chem.1966, 78, 413; Cahn and Ingold, J. Chem. Soc.1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).
[0115] The term“tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. A tautomer is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.
[0116] It is to be understood that the compounds or translational enhancers of the present disclosure may be depicted as different tautomers. It should also be understood that when compounds or translational enhancers have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present disclosure, and the naming of the compounds or translational enhancers does not exclude any tautomer form. It will be understood that certain tautomers may have a higher level of activity than others.
[0117] The terms“comprising”,“having”,“being of” as in“being of a chemical formula”, “including”, and“containing” are to be construed as open terms (i.e., meaning“including but not limited to”) unless otherwise noted. Additionally whenever“comprising” or another open-ended term is used in various embodiments, it is to be understood that the same embodiment can be more narrowly claimed using the intermediate term“consisting essentially of or the closed term “consisting of.”
[0118] As used herein, the expressions“one or more of A, B, or C,”“one or more A, B, or C,” “one or more of A, B, and C,”“one or more A, B, and C” and the like are used interchangeably and all refer to a selection from the group consisting of A, B, and /or C, i.e., one or more As, one or more Bs, one or more Cs, or any combination thereof.
[0119] Alternative nucleotides can be altered on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases“phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.
[0120] The alternative nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate,
phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged
phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene- phosphonates).
[0121] As used herein, the term“translational enhancer” refers to any compound or biological molecule that increases the rate, or amount, or both of polypeptide or protein production from an mRNA. A translational enhancer of the disclosure provides an induced or boosted polypeptide or protein production in vitro or in the cell from an mRNA comprising the translational enhancer, preferably more efficiently than a natural mRNA or an mRNA not comprising the translational enhancer of the disclosure. A translational enhancer may induce or boost polypeptide or protein production from mRNAs, e.g., by increasing stability/half- life/bioavailability/biodistribution of endogenous and/or exogenous mRNAs, increasing the translation efficiency of endogenous and/or exogenous mRNAs, inducing an attenuated immune response to exogenous mRNAs, and/or enhancing delivery and/or permeability of exogenous mRNA molecules to cells.
[0122] As used herein, the phrases“translational enhancer of the disclosure” and“translational enhancers of the disclosure” are used interchangeably to refer to a compound comprising an eIF4E ligand. In certain aspects, a translational enhancer of the disclosure comprises an eIF4E ligand attached to a dinucleotide. In certain embodiments, the eIF4E ligand is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand is attached to the dinucleotide via a linker. In certain embodiments, the translational enhancers of the disclosure function as a 5’ cap analog or 5’ cap mimetic. In other embodiments, the translational enhancers of the disclosure are a 5’ cap analog or 5’ cap mimetic.
[0123] The terms“eIF4E ligand,”“ligand,” and“compound”are used interchangeably to refer to any compound or biological molecule that binds to the translation initiation factor eIF4E. In certain embodiments, binding of the eIF4E ligand to eIF4E does not affect the interaction of eIF4E with any of the other components of the cellular translational machinery (e.g., other translation initiation factors). In certain embodiments, binding of the eIF4E ligand to eIF4E enhances the interaction of eIF4E with one or more components of the cellular translational machinery.
[0124] As used herein, the term“linker” refers to moieties that connects or attaches the eIF4E ligand and the dinucleotide moiety of a translational enhancer of the disclosure.
[0125] As used herein, the term“5’ cap analog” or“5’ cap mimetic” refers to a compound that mimics the activity of a natural mRNA 5’ cap structure and binds to eIF4E.
[0126] As used herein, the phrases“RNA molecule of the disclosure” and“RNA molecules of the disclosure” are used interchangeably to refer to an RNA molecule (e.g., mRNA molecule) comprising a translational enhancer of the disclosure or a stereoisomer, tautomer, or salt thereof.
[0127] The RNA molecules of the disclosure can be used as therapeutic agents or are therapeutic mRNAs. As used herein, the term“therapeutic mRNA” refers to an mRNA that encodes a therapeutic protein.“Therapeutic protein” refers to a protein that, when administered to a cell or a subject has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. Therapeutic proteins mediate a variety of effects in a host cell or a subject in order to treat a disease or ameliorate the signs and symptoms of a disease. For example, an RNA or a multimeric structure described herein can be
administered to an animal or human subject, wherein the RNA is translated in vivo to produce a therapeutic peptide in the subject in need thereof. Accordingly, provided herein are
compositions, methods, kits, and reagents for treatment or prevention of disease or conditions in humans and other mammals. The active therapeutic agents of the disclosure include RNAs (e.g., mRNAs) disclosed herein, cells containing the RNAs, mRNAs, or polypeptides translated from the mRNAs, polypeptides translated from mRNAs, cells contacted with cells or particles (e.g., nanoparticles or liposomes) containing RNAs, mRNAs, or polypeptides translated therefrom, tissues containing cells or particles (e.g., nanoparticles or liposomes) containing the RNAs described herein and organs containing tissues containing cells or particles (e.g., nanoparticles or liposomes) containing the RNAs described herein.
[0128] As used herein,“treating” or“treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of an active ingredient of the disclosure to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term“treat” can also include treatment of a cell in vitro or an animal model. [0129] An active ingredient of the disclosure, can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes. As used herein,“preventing,”“prevent,” or“protecting against” or“ameliorating,” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.
[0130] A“pharmaceutical composition” is a formulation containing the active ingredient of the disclosure in a form suitable for administration to a subject. In some embodiments, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of an active ingredient of the disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In some embodiments, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
[0131] As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, translational enhancers, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0132] “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A“pharmaceutically acceptable excipient” as used in the
specification and claims includes both one and more than one such excipient.
[0133] An“effective amount” of the polynucleotides (e.g., RNA or mRNA) disclosed herein is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the multimeric structures, and other determinants. In general, an effective amount of RNA provides an induced or boosted polypeptide or protein production in the cell, preferably more efficiently than a natural RNA or an RNA not comprising the translational enhancer of the disclosure. Increased polypeptide production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the multimeric structures), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified polynucleotide), or altered polypeptide production in the host cell.
Translational enhancers of the disclosure
[0134] In certain embodiments, the disclosure provides a translational enhancer comprising an eIF4E ligand attached to at least one nucleotide. In certain embodiments, the eIF4E ligand is attached directly to the at least one nucleotide. In other embodiments, the eIF4E ligand is attached to the at least one nucleotide via a linker. In specific embodiments, the disclosure provides a translational enhancer comprising an eIF4E ligand attached to a dinucleotide. In certain aspects, the eIF4E ligand is attached directly to the dinucleotide. In other aspects, the eIF4E ligand is attached to the dinucleotide via a linker.
[0135] In certain aspects, the dinucleotide is selected from the group consisting of an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, and a GG dinucleotide. In certain embodiments, the dinucleotide is an AG dinucleotide. In other embodiments, the dinucleotide is a GG dinucleotide.
[0136] In certain embodiments, one or both of the adenosines in an AA dinucleotide is an N6- methyladenosine (m6A). Thus, in certain aspects, the dinucleotide is an m6A-m6A dinucleotide. In other aspects, the dinucleotide is an A-m6A or an m6A-A dinucleotide. In other embodiments, one or both of the adenosines in an AA dinucleotide is an N6, 2’-O-dimethyladenosine (m6Am). Thus, in certain aspects, the dinucleotide is an m6Am-m6Am dinucleotide. In other aspects, the dinucleotide is an A-m6Am or an m6Am-A dinucleotide. In other instances, the dinucleotide is an m6A-m6Am or an m6Am-m6A dinucleotide.
[0137] In certain embodiments, the adenosine in an AG or a GA dinucleotide is an m6A. In other embodiments, the adenosine in an AG or a GA nucleotide is an m6Am. In certain embodiments, the guanosine in the AG or GA dinucleotides may also be modified to a 2’-O- methylguanosine (Gm).
[0138] Thus, in certain aspects, the dinucleotide is an m6A-G dinucleotide, an m6A-Gm dinucleotide, a G-m6A dinucleotide, a Gm-m6A dinucleotide, an m6Am-G dinucleotide, an m6Am-Gm dinucleotide, a G-m6Am dinucleotide, or a Gm-m6Am dinucleotide. [0139] In other embodiments, the dinucleotide includes, without limitation, an AU dinucleotide, an m6A-U dinucleotide, an m6Am-U dinucleotide, a UA dinucleotide, a U-m6A dinucleotide, a U-m6Am dinucleotide, an AC dinucleotide, an m6A-C dinucleotide, an m6Am-C dinucleotide, a CA dinucleotide, a C-m6A dinucleotide, a C-m6Am dinucleotide, a GU dinucleotide, a Gm-U dinucleotide, a UG dinucleotide, a U-Gm dinucleotide, a GC dinucleotide, a Gm-C dinucleotide, a CG dinucleotide, a C-Gm dinucleotide, a UU dinucleotide, a CC dinucleotide, a CU dinucleotide, or a UC dinucleotide. In certain embodiments, the cytosine or uracil in the above-disclosed dinucleotides may also be modified to a 2’-O-methylcytosine (Cm) or a 2’-O-methyluracil (Um).
[0140] In certain embodiments, the translational enhancer of the disclosure comprises an eIF4E ligand attached directly to the dinucleotide. In other embodiments, the translational enhancer of the disclosure comprises an eIF4E ligand attached to the dinucleotide via a linker. In some embodiments, the linker can be cleavable or non-cleavable. As used herein, the term “linker,” or“linker molecule,” or“linking group” means an organic moiety that connects two parts of a compound (e.g., the eIF4E ligand and the dinucleotide of a translational enhancer of the disclosure). Thus, as used herein, the term“linker” refers to moieties that connect the eIF4E ligand and the dinucleotide moiety of a translational enhancer of the disclosure.
[0141] Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or
unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,
alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,
alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between 1-24 atoms, e.g., 4-24 atoms, 6-18 atoms, 8-18 atoms, or 8- 16 atoms.
[0142] In some embodiments, the linker is a phosphate linker. In certain aspects, the phosphate linker is a monophosphate linker. In other aspects, the phosphate linker is a diphosphate linker. In yet other aspects, the phosphate linker is a triphosphate linker. In other embodiments, the phosphate linker is a tetraphosphate linker. In other aspects, the linker is a phosphonate-diphosphate linker. In yet other aspects, linker is a surrogate linker. In certain aspects, the phosphonate-diphosphate linker is more stable than the monophosphate, diphosphate, triphosphate, and tetraphosphate linkers.
[0143] In the present specification, the structural formula of a translational enhancer of the disclosure represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like, it being understood that not all isomers may have the same level of activity. In addition, in certain cases, a crystal polymorphism may be present for the translational enhancers of the invention. It is noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.
[0144] In some embodiments, the translational enhancer of the disclosure has an improved binding affinity for eIF4E as compared to, e.g., natural mRNA caps (m7GTP). In other embodiments, the translational enhancer of the disclosure has greater than about 1.2 fold, about 1.5 fold, about 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 300 fold, about 400 fold, about 500 fold, or about 1,000 fold higher binding affinity for eIF4E as compared to, e.g., natural mRNA caps (m7GTP). In specific embodiments, the translational enhancer of the disclosure has greater than about 1,000 fold higher binding affinity for eIF4E as compared to, e.g., natural mRNA caps (m7GTP). In certain aspects, the translational enhancer of the disclosure is greater than about 1,000 fold more potent than, e.g., natural mRNA caps (m7GTP) in binding eIF4E. In some aspects the translational enhancer of the disclosure exhibits picomolar cell potency for binding eIF4E. In certain aspects, the translational enhancer of the disclosure has an improved binding affinity for eIF4E as compared to Anti-Reverse Cap Analog (ARCA). In certain embodiments, the translational enhancer of the disclosure has greater than about 1.2 fold, about 1.5 fold, about 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 300 fold, about 400 fold, about 500 fold, or about 1,000 fold higher binding affinity for eIF4E as compared to ARCA. In specific aspects, the translational enhancer of the disclosure has greater than about 1,000 fold higher binding affinity for eIF4E as compared to ARCA. [0145] In certain embodiments, binding of the translational enhancer to eIF4E does not affect the interaction of eIF4E with any of the other components of the cellular translational machinery (e.g., other translation initiation factors). In certain embodiments, binding of the translational enhancer to eIF4E enhances the interaction of eIF4E with one or more components of the cellular translational machinery.
[0146] As used herein, koff is the off-rate, calculated from the dissociation phase, kon is the on- rate, calculated from the association phase; Kd or KD is the binding affinity, which is the ratio of koff/ kon, and the residence time, t, is the inverse of koff.
[0147] In certain embodiments, the translational enhancers of the disclosure with an improved eIF4E binding affinity have a Kd or KD of no more than 10 mM, e.g., using surface plasmon resonance (SPR). For example, Kd of the compound is no more than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.7, 0.5, 0.3, or 0.1 mM. For example, in certain aspects the compound has an eIF4E Kd of no more than 10 mM (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.9, 0.7, 0.5, 0.3, or 0.1 mM) and a t of about 2 seconds or longer (e.g., 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 50 seconds, 75 seconds, 80 seconds, 90 seconds, 100 seconds, or longer). In certain embodiments, the translational enhancers of the disclosure with an improved eIF4E binding affinity have a Kd in the picomolar range.
[0148] In certain embodiments, the translational enhancers of the invention are capable of being co-transcriptionally attached to an RNA molecule. In certain embodiments, the translational enhancers of the invention are capable of being chemically attached to an RNA molecule. In other embodiments, the translational enhancers of the invention are capable of being enzymatically attached to an RNA molecule. In certain embodiments, the translational enhancers of the invention are co-transcriptionally, chemically, or enzymatically attached to the 5’ end of an RNA molecule. In certain aspects, the translational enhancers of the invention act as 5’ RNA cap structures. In certain embodiments, the translational enhancers of the invention are co-transcriptionally, chemically, or enzymatically attached to the 5’ end of RNA molecules to provide 5’ capped RNAs.
[0149] In certain embodiments, the translational enhancers of the disclosure provide capping reagents for in vitro or in vivo transcription of 5’ capped RNA resulting in a Cap 1, Cap 2, Cap 3, or Cap 4 structure. In certain aspects, the translational enhancers of the disclosure provide capping reagents for in vitro transcription of 5’ capped RNA resulting in a Cap 1 structure. Thus, in certain aspects, the translational enhancers of the disclosure provide significant advantages over current methods and compositions involving use of various initiating nucleosides, nucleotides and oligonucleotides or use of polyphosphate dinucleotide derivatives containing Cap 0 structure, such as mCAP and ARCA. In certain embodiments, the translational enhancers of the disclosure are compatible with existing transcription systems and reagents and no additional enzymes or reagents are required for the generation of the Cap 1 RNAs. In other embodiments, the use of the translational enhancers of the disclosure makes several non- enzymatic and enzymatic steps (such as capping and 2’-O-methylation) unnecessary thus reducing complexity of the process and a cost of RNA synthesis.
[0150] In some embodiments, the invention is directed to a translational enhancer having a structure:
Figure imgf000041_0001
,
wherein the eIF4E ligand has a structure according to Formula I:
Figure imgf000041_0002
or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein:
X1 is CR2, -C-L1-Y or N;
X2, X5 and X6 are independently CR2 or N,
wherein X5 and X6 together with 3 or 4 carbon or nitrogen atoms combine to form a 5- or 6-membered cycloalkyl or heterocyclyl,
or when X2 is CR2, R1 and R2 together with the atoms they attached to form a 6- membered aryl or heteroaryl;
X3 is C, or X3 is C or N when X4 is a bond;
X4 is a bond, CR2 or N,
wherein X4 and X5 together with 3 or 4 carbon or nitrogen atoms combine to form a 5- or 6-membered heteroaryl;
Q is H or–L1-Y; L1 is–(CH2)–,–(CH2)2–,–(CH2)3–,–CH((C1-C8)alkyl)(CH2)–,–CH((C1- C8)alkyl)(CH2)2–,–(CH2)2-O–,–CH2CH=CH–,–CH2CºC– or–CH2(cyclopropyl)–;
Y is
Figure imgf000042_0001
Ring B is a six-membered aryl, heteroaryl or heterocyclyl;
R1 is H, OH, halo, CN, (C1-C8)alkyl, (C1-C8)haloalkyl, (C3-C6)cycloalkyl or NR5R5; R2 is independently H, halo, CN, NO, NO2, CºH, (C1-C8)alkyl, (C1-C8)haloalkyl, CH2SR5, OR5, NHR5, NR5R5, [(C1-C8)alkylene]heterocyclyl, [(C1-C8)alkylene]heteroaryl, [(C1-C8)alkylene]NHR5, [(C1-C8)alkylene]NR5R5, [(C1-C8)alkylyne]NR5R5, C(O)R5, C(O)OR5, C(O)NHR5, C(O)NR5R5, SR5, S(O)R5, SO2R5, SO2NHR5, SO2NR5R5, NH(CO)R6, NR5(CO)R6, aryl, heteroaryl, cycloalkyl or heterocyclyl;
R3 is independently OH, halo, CN, NO2, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, CºH, NHR7, NR7R7, CO2H, CO2R7, [(C1-C3)alkylene] (C1-C3)alkoxy, [(C1- C3)alkylene]CO2H, (C3-C5)cycloalkyl, =O. =S, SR7, SO2R7, NH(CO)R7 or NR7(CO)R7;
R4 is H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, SR7 or Z, wherein Z is
Figure imgf000042_0002
Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl;
L2 is -C(R6)(R6)-, -C(R6)(R6)C(R6)(R6)-, -C(R6)=C(R6)-, -N(R5)C(R6)(R6)-, - OC(R6)(R6)-, -C(=O)-, -C(=O)N(R5)C(R6)(R6)- or a bond;
R5 is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C5)cycloalkyl, CO2H, [(C1- C3)alkylene]heteroaryl, [(C1-C3)alkylene]aryl, [(C1-C3)alkylene]CO2H, heterocyclyl, aryl or heteroaryl, or wherein two R5 substituents together with a nitrogen atom form a 4-, 5-, 6- or 7- membered heterocyclyl;
R6 is independently H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, NHR7, NR7R7, CO2H, [(C1-C3)alkylene]CO2H, (C3-C5)cycloalkyl, SR7, NH(CO)R7 or
NR7(CO)R7;
R7 is independently H, (C1-C8)alkyl, (C1-C8)haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
R8 is H, OH, CO2H, CO2R7, CF2C(R6)2OH, C(R6)2OH, C(CF3)2OH, SO2H, SO3H, CF2SO2C(R6)3, CF2SO2N(H)R5, SO2N(H)R5, SO2N(H)C(O)R6, C(O)N(H)SO2R5,
C(O)haloalkyl, C(O)N(H)OR5, C(O)N(R5)OH, C(O)N(H)R5, C(O)NR5C(O)N(R5)2,
P(O)(OR5)OH, P(O)(O)N(H)R5, P(O)(C(R6)3)C(R6)3, B(OH)2, heterocyclyl or heteroaryl;
n is 0, 1, 2 or 3;
p is 0, 1, 2 or 3;
wherein any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH3, SO2CH3, SO2NH2,
SO2NH(C1-C4)alkyl, halogen, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, NH(aryl), C(O)NH2, C(O)NH(alkyl), CH2C(O)NH(alkyl), COOH, COOMe, acetyl, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, thioalkyl, cyanomethylene, alkylaminyl, alkylene-C(O)NH2, alkylene-C(O)-NH(Me), NHC(O)alkyl, CH2-C(O)-(C1- C8)alkyl, C(O)-(C1-C8)alkyl and alkylcarbonylaminyl, or a cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with OH, halogen, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1- C8)alkyl or O(C1-C8)haloalkyl,
wherein when X4 is a bond ring A forms a 5-membered heteroaryl wherein X1, X5 and X6 can in addition to the above defined substituents be NR2, and X1 can in addition be -N-L1-Y; and wherein either Q is–L1-Y, or X1 is -C-L1-Y or -N-L1-Y; and
wherein X is a linker and Y is a dinucleotide.
[0151] In certain embodiments, the present invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure encompassed by the genus of Formula I, or stereoisomers, tautomers or pharmaceutically acceptable salts thereof.
[0152] In one embodiment, the invention is directed to a translational enhancer, wherein the [0153] translational enhancer comprises an eIF4E ligand having a structure according to Formula II:
Figure imgf000044_0001
or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein:
X2 and X5 are independently CR2 or N,
or when X2 is CR2, R1 and R2 together with the atoms they attached to form a 6- membered aryl or heteroaryl;
L1 is–(CH2)–,–(CH2)2–,–(CH2)3–,–CH((C1-C8)alkyl)(CH2)–,–CH((C1- C8)alkyl)(CH2)2–,–(CH2)2-O–,–CH2CH=CH–,–CH2CºC– or–CH2(cyclopropyl)–;
L2 is -C(R6)(R6)-, -C(R6)(R6)C(R6)(R6)-, -C(R6)=C(R6)-, -N(R5)C(R6)(R6)-, - OC(R6)(R6)-, -C(=O)-, -C(=O)N(R5)C(R6)(R6)- or a bond;
Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl;
R1 is H, OH, halo, CN, (C1-C8)alkyl, (C1-C8)haloalkyl, (C3-C6)cycloalkyl or NR5R5; R2 is independently H, halo, CN, NO, NO2, CºH, (C1-C8)alkyl, (C1-C8)haloalkyl, CH2SR5, OR5, NHR5, NR5R5, [(C1-C8)alkylene]heterocyclyl, [(C1-C8)alkylene]heteroaryl, [(C1-C8)alkylene]NHR5, [(C1-C8)alkylene]NR5R5, [(C1-C8)alkylyne]NR5R5, C(O)R5, C(O)OR5, C(O)NHR5, C(O)NR5R5, SR5, S(O)R5, SO2R5, SO2NHR5, SO2NR5R5, NH(CO)R6, NR5(CO)R6, aryl, heteroaryl, cycloalkyl or heterocyclyl;
R3 is independently OH, halo, CN, NO2, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, CºH, NHR7, NR7R7, CO2H, CO2R7, [(C1-C3)alkylene] (C1-C3)alkoxy, [(C1- C3)alkylene]CO2H, (C3-C5)cycloalkyl, =O. =S, SR7, SO2R7, NH(CO)R7 or NR7(CO)R7;
R5 is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C5)cycloalkyl, CO2H, [(C1- C3)alkylene]heteroaryl, [(C1-C3)alkylene]aryl, [(C1-C3)alkylene]CO2H, heterocyclyl, aryl or heteroaryl, or wherein two R5 substituents together with a nitrogen atom form a 4-, 5-, 6-, or 7- membered heterocyclyl;
R6 is independently H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, NHR7, NR7R7, CO2H, [(C1-C3)alkylene]CO2H, (C3-C5)cycloalkyl, SR7, NH(CO)R7 or
NR7(CO)R7;
R7 is independently H, (C1-C8)alkyl, (C1-C8)haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
R8 is H, OH, CO2H, CO2R7, CF2C(R6)2OH, C(R6)2OH, C(CF3)2OH, SO2H, SO3H, CF2SO2C(R6)3, CF2SO2N(H)R5, SO2N(H)R5, SO2N(H)C(O)R6, C(O)N(H)SO2R5,
C(O)haloalkyl, C(O)N(H)OR5, C(O)N(R5)OH, C(O)N(H)R5, C(O)NR5C(O)N(R5)2,
P(O)(OR5)OH, P(O)(O)N(H)R5, P(O)(C(R6)3)C(R6)3, B(OH)2, heterocyclyl or heteroaryl;
m is 0, 1, 2 or 3;
n is 0, 1, 2 or 3;
p is 0, 1, 2 or 3;
wherein any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH3, SO2CH3, SO2NH2,
SO2NH(C1-C4)alkyl, halogen, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, NH(aryl), C(O)NH2, C(O)NH(alkyl), CH2C(O)NH(alkyl), COOH, COOMe, acetyl, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, thioalkyl, cyanomethylene, alkylaminyl, alkylene-C(O)NH2, alkylene-C(O)-NH(Me), NHC(O)alkyl, CH2-C(O)-(C1- C8)alkyl, C(O)-(C1-C8)alkyl and alkylcarbonylaminyl, or a cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with OH, halogen, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1- C8)alkyl or O(C1-C8)haloalkyl.
[0154] In one embodiment, the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula III:
Figure imgf000046_0001
or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein:
L1 is–(CH2)–,–(CH2)2–,–(CH2)3–,–CH((C1-C8)alkyl)(CH2)–,–CH((C1- C8)alkyl)(CH2)2–,–(CH2)2-O–,–CH2CH=CH–,–CH2CºC– or–CH2(cyclopropyl)–;
L2 is -C(R6)(R6)-, -C(R6)(R6)C(R6)(R6)-, -C(R6)=C(R6)-, -N(R5)C(R6)(R6)-,
-OC(R6)(R6)-, -C(=O)-, -C(=O)N(R5)C(R6)(R6)- or a bond;
Ring C is a heteroaryl;
R1 is H, OH, halo, CN, (C1-C8)alkyl, (C1-C8)haloalkyl, (C3-C6)cycloalkyl or NR5R5; R2 is independently H, halo, CN, NO, NO2, CºH, (C1-C8)alkyl, (C1-C8)haloalkyl, CH2SR5, OR5, NHR5, NR5R5, [(C1-C8)alkylene]heterocyclyl, [(C1-C8)alkylene]heteroaryl, [(C1-C8)alkylene]NHR5, [(C1-C8)alkylene]NR5R5, [(C1-C8)alkylyne]NR5R5, C(O)R5, C(O)OR5, C(O)NHR5, C(O)NR5R5, SR5, S(O)R5, SO2R5, SO2NHR5, SO2NR5R5, NH(CO)R6, NR5(CO)R6, aryl, heteroaryl, cycloalkyl or heterocyclyl;
R3 is independently OH, halo, CN, NO2, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, CºH, NHR7, NR7R7, CO2H, CO2R7, [(C1-C3)alkylene] (C1-C3)alkoxy, [(C1- C3)alkylene]CO2H, (C3-C5)cycloalkyl, =O. =S, SR7, SO2R7, NH(CO)R7 or NR7(CO)R7;
R5 is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C5)cycloalkyl or
heterocyclyl;
R6 is independently H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, NHR7, NR7R7, CO2H, [(C1-C3)alkylene]CO2H, (C3-C5)cycloalkyl, SR7, NH(CO)R7 or
NR7(CO)R7;
R7 is independently H, (C1-C8)alkyl, (C1-C8)haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; R8 is H, OH, CO2H, CO2R7, CF2C(R6)2OH, C(R6)2OH, C(CF3)2OH, SO2H, SO3H, CF2SO2C(R6)3, CF2SO2N(H)R5, SO2N(H)R5, SO2N(H)C(O)R6, C(O)N(H)SO2R5,
C(O)haloalkyl, C(O)N(H)OR5, C(O)N(R5)OH, C(O)N(H)R5, C(O)NR5C(O)N(R5)2,
P(O)(OR5)OH, P(O)(O)N(H)R5, P(O)(C(R6)3)C(R6)3, B(OH)2, heterocyclyl or heteroaryl;
R9 is H, (C1-C8)alkyl, (C1-C8)haloalkyl, cycloalkyl or heterocyclyl;
m is 0, 1, or 2;
n is 0, 1, 2 or 3;
p is 0, 1, 2 or 3;
wherein any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH3, SO2CH3, SO2NH2,
SO2NH(C1-C4)alkyl, halogen, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, NH(aryl), C(O)NH2, C(O)NH(alkyl), CH2C(O)NH(alkyl), COOH, COOMe, acetyl, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, thioalkyl, cyanomethylene, alkylaminyl, alkylene-C(O)NH2, alkylene-C(O)-NH(Me), NHC(O)alkyl, CH2-C(O)-(C1- C8)alkyl, C(O)-(C1-C8)alkyl and alkylcarbonylaminyl.
[0155] In another embodiment, the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula IV:
Figure imgf000047_0001
or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein:
X2 and X5 are independently CR2 or N,
or when X2 is CR2, R1 and R2 together with the atoms they attached to form a 6- membered aryl or heteroaryl;
X3 is C, or X3 is C or N when X4 is a bond; X4 is a bond, CR2 or N,
wherein X4 and X5 together with 3 or 4 carbon or nitrogen atoms combine to form a 5- or 6-membered heteroaryl;
L1 is–(CH2)–,–(CH2)2–,–(CH2)3–,–CH((C1-C8)alkyl)(CH2)–,–CH((C1- C8)alkyl)(CH2)2–,–(CH2)2-O–,–CH2CH=CH–,–CH2CºC– or–CH2(cyclopropyl)–;
L2 is -C(R6)(R6)-, -C(R6)(R6)C(R6)(R6)-, -C(R6)=C(R6)-, -N(R5)C(R6)(R6)-,
-OC(R6)(R6)-, -C(=O)-, -C(=O)N(R5)C(R6)(R6)-;
Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl;
R1 is H, OH, halo, CN, (C1-C8)alkyl, (C1-C8)haloalkyl, (C3-C6)cycloalkyl or NR5R5; R2 is independently H, halo, CN, NO, NO2, CºH, (C1-C8)alkyl, (C1-C8)haloalkyl, CH2SR5, OR5, NHR5, NR5R5, [(C1-C8)alkylene]heterocyclyl, [(C1-C8)alkylene]heteroaryl, [(C1-C8)alkylene]NHR5, [(C1-C8)alkylene]NR5R5, [(C1-C8)alkylyne]NR5R5, C(O)R5, C(O)OR5, C(O)NHR5, C(O)NR5R5, SR5, S(O)R5, SO2R5, SO2NHR5, SO2NR5R5, NH(CO)R6, NR5(CO)R6, aryl, heteroaryl, cycloalkyl or heterocyclyl;
R3 is independently OH, halo, CN, NO2, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, CºH, NHR7, NR7R7, CO2H, CO2R7, [(C1-C3)alkylene] (C1-C3)alkoxy, [(C1- C3)alkylene]CO2H, (C3-C5)cycloalkyl, =O. =S, SR7, SO2R7, NH(CO)R7 or NR7(CO)R7;
R5 is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C5)cycloalkyl, CO2H, [(C1- C3)alkylene]heteroaryl, [(C1-C3)alkylene]aryl, [(C1-C3)alkylene]CO2H, heterocyclyl, aryl or heteroaryl,
or wherein two R5 substituents together with a nitrogen atom form a 4-, 5-, 6- or 7- membered heterocyclyl;
R6 is independently H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, NHR7, NR7R7, CO2H, [(C1-C3)alkylene]CO2H, (C3-C5)cycloalkyl, SR7, NH(CO)R7 or
NR7(CO)R7;
R7 is independently H, (C1-C8)alkyl, (C1-C8)haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
R8 is H, OH, CO2H, CO2R7, CF2C(R6)2OH, C(R6)2OH, C(CF3)2OH, SO2H, SO3H, CF2SO2C(R6)3, CF2SO2N(H)R5, SO2N(H)R5, SO2N(H)C(O)R6, C(O)N(H)SO2R5,
C(O)haloalkyl, C(O)N(H)OR5, C(O)N(R5)OH, C(O)N(H)R5, C(O)NR5C(O)N(R5)2,
P(O)(OR5)OH, P(O)(O)N(H)R5, P(O)(C(R6)3)C(R6)3, B(OH)2, heterocyclyl or heteroaryl; n is 0, 1, 2 or 3;
p is 0, 1, 2 or 3;
wherein any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH3, SO2CH3, SO2NH2,
SO2NH(C1-C4)alkyl, halogen, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, NH(aryl), C(O)NH2, C(O)NH(alkyl), CH2C(O)NH(alkyl), COOH, COOMe, acetyl, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, thioalkyl, cyanomethylene, alkylaminyl, alkylene-C(O)NH2, alkylene-C(O)-NH(Me), NHC(O)alkyl, CH2-C(O)-(C1- C8)alkyl, C(O)-(C1-C8)alkyl and alkylcarbonylaminyl, or a cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with OH, halogen, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1- C8)alkyl or O(C1-C8)haloalkyl,
wherein when X4 is a bond, ring A forms a 5-membered heteroaryl wherein X1and X5 can in addition to C be N.
[0156] In another embodiment, the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula V:
Figure imgf000049_0001
or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein:
Q is–L1-Y;
L1 is–(CH2)–,–(CH2)2–,–(CH2)3–,–CH((C1-C8)alkyl)(CH2)–,–CH((C1- C8)alkyl)(CH2)2–,–(CH2)2-O–,–CH2CH=CH–,–CH2CºC– or–CH2(cyclopropyl)–;
Y is
Figure imgf000050_0001
, wherein
Ring B is a six-membered aryl, heteroaryl or heterocyclyl;
R1 is H, OH, halo, CN, (C1-C8)alkyl, (C1-C8)haloalkyl, (C3-C6)cycloalkyl or NR5R5; R2 is independently H, halo, CN, NO, NO2, CºH, (C1-C8)alkyl, (C1-C8)haloalkyl, CH2SR5, OR5, NHR5, NR5R5, [(C1-C8)alkylene]heterocyclyl, [(C1-C8)alkylene]heteroaryl, [(C1-C8)alkylene]NHR5, [(C1-C8)alkylene]NR5R5, [(C1-C8)alkylyne]NR5R5, C(O)R5, C(O)OR5, C(O)NHR5, C(O)NR5R5, SR5, S(O)R5, SO2R5, SO2NHR5, SO2NR5R5, NH(CO)R6, NR5(CO)R6, aryl, heteroaryl, cycloalkyl or heterocyclyl;
R3 is independently OH, halo, CN, NO2, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, CºH, NHR7, NR7R7, CO2H, CO2R7, [(C1-C3)alkylene] (C1-C3)alkoxy, [(C1- C3)alkylene]CO2H, (C3-C5)cycloalkyl, =O. =S, SR7, SO2R7, NH(CO)R7 or NR7(CO)R7;
R4 is H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, SR7 or Z, wherein Z is
Figure imgf000050_0002
Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl;
L2 is -C(R6)(R6)-, -C(R6)(R6)C(R6)(R6)-, -C(R6)=C(R6)-, -N(R5)C(R6)(R6)-, - OC(R6)(R6)-, -C(=O)-, -C(=O)N(R5)C(R6)(R6)- or a bond;
R5 is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C5)cycloalkyl, CO2H, [(C1- C3)alkylene]heteroaryl, [(C1-C3)alkylene]aryl, [(C1-C3)alkylene]CO2H, heterocyclyl, aryl or heteroaryl,
or wherein two R5 substituents together with a nitrogen atom form a 4-, 5-, or 6- membered heterocyclyl; R6 is independently H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, NHR7, NR7R7, CO2H, [(C1-C3)alkylene]CO2H, (C3-C5)cycloalkyl, SR7, NH(CO)R7 or
NR7(CO)R7;
R7 is independently H, (C1-C8)alkyl, (C1-C8)haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
R8 is H, OH, CO2H, CO2R7, CF2C(R6)2OH, C(R6)2OH, C(CF3)2OH, SO2H, SO3H, CF2SO2C(R6)3, CF2SO2N(H)R5, SO2N(H)R5, SO2N(H)C(O)R6, C(O)N(H)SO2R5,
C(O)haloalkyl, C(O)N(H)OR5, C(O)N(R5)OH, C(O)N(H)R5, P(O)(OR5)OH, P(O)(O)N(H)R5, P(O)(C(R6)3)C(R6)3, B(OH)2, heterocyclyl or heteroaryl;
n is 0, 1, 2 or 3;
p is 0, 1, 2 or 3;
q is 0, 1, 2, 3 or 4;
wherein any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH3, SO2CH3, SO2NH2,
SO2NH(C1-C4)alkyl, halogen, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, NH(aryl), C(O)NH2, C(O)NH(alkyl), CH2C(O)NH(alkyl), COOH, COOMe, acetyl, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, thioalkyl, cyanomethylene, alkylaminyl, alkylene-C(O)NH2, alkylene-C(O)-NH(Me), NHC(O)alkyl, CH2-C(O)-(C1- C8)alkyl, C(O)-(C1-C8)alkyl and alkylcarbonylaminyl, or a cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with OH, halogen, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1- C8)alkyl or O(C1-C8)haloalkyl.
[0157] In another embodiment, the invention is directed to a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula VI:
Figure imgf000051_0001
or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein:
Q is–L1-Y; L1 is–(CH2)–,–(CH2)2–,–(CH2)3–,–CH((C1-C8)alkyl)(CH2)–,–CH((C1- C8)alkyl)(CH2)2–,–(CH2)2-O–,–CH2CH=CH–,–CH2CºC– or–CH2(cyclopropyl)–;
Y is
wherein
Figure imgf000052_0001
Ring B is a six-membered aryl, heteroaryl or heterocyclyl;
R1 is H, OH, halo, CN, (C1-C8)alkyl, (C1-C8)haloalkyl, (C3-C6)cycloalkyl or NR5R5; R2 is independently H, halo, CN, NO, NO2, CºH, (C1-C8)alkyl, (C1-C8)haloalkyl, CH2SR5, OR5, NHR5, NR5R5, [(C1-C8)alkylene]heterocyclyl, [(C1-C8)alkylene]heteroaryl, [(C1-C8)alkylene]NHR5, [(C1-C8)alkylene]NR5R5, [(C1-C8)alkylyne]NR5R5, C(O)R5, C(O)OR5, C(O)NHR5, C(O)NR5R5, SR5, S(O)R5, SO2R5, SO2NHR5, SO2NR5R5, NH(CO)R6, NR5(CO)R6, aryl, heteroaryl, cycloalkyl or heterocyclyl;
R3 is independently OH, halo, CN, NO2, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, CºH, NHR7, NR7R7, CO2H, CO2R7, [(C1-C3)alkylene] (C1-C3)alkoxy, [(C1- C3)alkylene]CO2H, (C3-C5)cycloalkyl, =O. =S, SR7, SO2R7, NH(CO)R7 or NR7(CO)R7;
R4 is H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, SR7 or Z, wherein Z is
Figure imgf000052_0002
Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl;
L2 is -C(R6)(R6)-, -C(R6)(R6)C(R6)(R6)-, -C(R6)=C(R6)-, -N(R5)C(R6)(R6)-, - OC(R6)(R6)-, -C(=O)-, -C(=O)N(R5)C(R6)(R6)- or a bond;
R5 is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C5)cycloalkyl, CO2H, [(C1- C3)alkylene]heteroaryl, [(C1-C3)alkylene]aryl, [(C1-C3)alkylene]CO2H, heterocyclyl, aryl or heteroaryl, or wherein two R5 substituents together with a nitrogen atom form a 4-, 5-, or 6- membered heterocyclyl;
R6 is independently H, OH, halo, CN, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-C3)alkoxy, NHR7, NR7R7, CO2H, [(C1-C3)alkylene]CO2H, (C3-C5)cycloalkyl, SR7, NH(CO)R7 or
NR7(CO)R7;
R7 is independently H, (C1-C8)alkyl, (C1-C8)haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
R8 is H, OH, CO2H, CO2R7, CF2C(R6)2OH, C(R6)2OH, C(CF3)2OH, SO2H, SO3H, CF2SO2C(R6)3, CF2SO2N(H)R5, SO2N(H)R5, SO2N(H)C(O)R6, C(O)N(H)SO2R5,
C(O)haloalkyl, C(O)N(H)OR5, C(O)N(R5)OH, C(O)N(H)R5, C(O)NR5C(O)N(R5)2,
P(O)(OR5)OH, P(O)(O)N(H)R5, P(O)(C(R6)3)C(R6)3, B(OH)2, heterocyclyl or heteroaryl;
n is 0, 1, 2 or 3;
p is 0, 1, 2 or 3;
q is 0, 1, 2, 3 or 4;
wherein any alkyl, alkylene, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 groups selected from OH, CN, SH, SCH3, SO2CH3, SO2NH2,
SO2NH(C1-C4)alkyl, halogen, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, NH(aryl), C(O)NH2, C(O)NH(alkyl), CH2C(O)NH(alkyl), COOH, COOMe, acetyl, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, thioalkyl, cyanomethylene, alkylaminyl, alkylene-C(O)NH2, alkylene-C(O)-NH(Me), NHC(O)alkyl, CH2-C(O)-(C1- C8)alkyl, C(O)-(C1-C8)alkyl and alkylcarbonylaminyl, or a cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with OH, halogen, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1- C8)alkyl or O(C1-C8)haloalkyl.
[0158] In one embodiment, X2 of Formulae I, II, and III is N.
[0159] In one embodiment X2 of Formulae I, II, and IV is N.
[0160] In one embodiment X3 of Formulae I and IV is C.
[0161] In one embodiment X4 of Formulae I and IV is CR2 or N.
[0162] In one embodiment X5 of Formulae I and IV is CR2.
[0163] In one embodiment L1 of Formulae I, II, III, IV, V and VI is–(CH2)2-O–,
–CH2CH=CH– or–CH2CºC–. In another embodiment L1 is–(CH2)2-O–.
[0164] In one embodiment L2 of Formulae I, II, III, IV, V and VI is a bond. [0165] In one embodiment Ring B of Formulae I, V and VI is aryl.
[0166] In one embodiment Ring C of Formulae I, II, III, IV, V and VI is heteroaryl.
[0167] In one embodiment Ring C of Formulae I, II, III, IV, V and VI is
Figure imgf000054_0001
.
[0168] In one embodiment Ring C of Formula III is
Figure imgf000054_0002
.
[0169] In one embodiment R1 of Formulae I, II, III, IV, V and VI is H, (C1-C8)alkyl or (C1- C8)haloalkyl.
[0170] In one embodiment R1 of Formula IV is NHR5 or N[(C1-C3)alkyl](R5).
[0171] In one embodiment R2 of Formulae I, II, III, IV, V and VI is halo, CN, (C1-C8)alkyl, (C1-C8)haloalkyl or OR5. In another embodiment R2 is halo, CN or (C1-C8)haloalkyl.
[0172] In one embodiment R3 of Formulae I, II, III, IV, V and VI is halo, CN, (C1-C3)alkyl or (C1-C3)haloalkyl.
[0173] In one embodiment R4 of Formulae I, V and VI is Z, wherein Z is
Figure imgf000054_0003
[0174] In one embodiment R5 of Formulae I, II, III, V and VI is H, (C1-C3)alkyl or (C1- C3)haloalkyl. In another embodiment R5 of Formula IV is aryl.
[0175] In one embodiment R6 of Formulae I, II, III, IV, V and VI is H, OH, halo, CN, (C1- C3)alkyl, (C1-C3)haloalkyl or (C1-C3)alkoxy.
[0176] In one embodiment R7 of Formulae I, II, III, IV, V and VI is H, (C1-C8)alkyl or (C1- C8)haloalkyl. [0177] In one embodiment R8 of Formulae I, II, III, IV, V and VI is CO2H or C(O)N(H)SO2R5.
[0178] In one embodiment R9 of Formula III is (C1-C8)alkyl or (C1-C8)haloalkyl.
[0179] In one embodiment R9 of Formula III is cycloalkyl or heterocyclyl.
[0180] In one embodiment“m” of Formulae I and II = 2 or 3. In another embodiment“n” of Formulae I, II, IV, V and VI = 1 or 2. In yet another embodiment“p” of Formulae I, II, III, IV, V and VI = 0 or 1.
[0181] In one embodiment the optional substituents of alkyl, cycloalkyl, heterocyclyl, heteroaryl or aryl are OH, CN, halogen, (C1-C8)alkyl, O(C1-C8)alkyl, haloalkyl, alkylene- C(O)NH2 or alkylene-C(O)-NH(Me). [0182] In one embodiment the optional substituents of alkyl, cycloalkyl, heterocyclyl, heteroaryl or aryl are cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with OH, halogen, (C1-C8)alkyl, (C1-C8)haloalkyl, O(C1-C8)alkyl or O(C1-C8)haloalkyl.
[0183] In another embodiment, the compounds according to Formulae I, II, III, IV, V and VI are selected from
7-(5-chloro-2-(2-(5-cyano-2-methyl-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(4,4-difluorocyclohexyl)-2-methyl-4-oxopyrido[3,4-d]pyrimidin- 3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-((dimethylamino)methyl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3-2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(2-(5-cyano-2-methyl-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid,
5'-chloro-2'-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)-[1,1'-biphenyl]-3-carboxylic acid,
7-(5-chloro-2-(2-(2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin- 3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(difluoromethoxy)-7-((dimethylamino)methyl)-2-methyl-4- oxoquinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(5-fluoro-2-methylpyridin-3-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid, 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-((4-methylpiperazin-1-yl)methyl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-6-((dimethylamino)methyl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(2-(dimethylamino)ethyl)-2-methyl-4-oxoquinazolin-3(4H)- yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-6-(2-(dimethylamino)ethyl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)pyrido[3,2- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid, 3-(2-(4-chloro-2-(thieno[3,2-b]pyridin-7-yl)phenoxy)ethyl)-2-methyl-6-(4-methylpiperazin-1- yl)-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5-carbonitrile,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxopyrido[3,4-d]pyrimidin- 3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(1-methylcyclopropyl)-4-oxopyrido[3,4-d]pyrimidin- 3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 3-(2-(4-chloro-2-(5-methylthieno[3,2-b]pyridin-7-yl)phenoxy)ethyl)-2-methyl-6-(4- methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5-carbonitrile, 7-(5-chloro-2-(2-(5-cyano-2-methyl-4-oxo-6-(1-(trifluoromethyl)cyclopropyl)pyrido[3,4- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid 7-(5-chloro-2-(3-(5-cyano-2-methyl-4-oxo-6-(4-(2,2,2-trifluoroethyl)piperazin-1-yl)pyrido[3,4- d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(4,4-difluorocyclohex-1-en-1-yl)-2-methyl-4-oxopyrido[3,4- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(3-(2,2-difluoroethoxy)azetidin-1-yl)-2-methyl-4-oxopyrido[3,4- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(2-(5-cyano-6-ethyl-2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)- yl)ethoxy)phenyl)-5-ethylthieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-ethylthieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-2-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((trans-4-(3,3-difluoroazetidin-1-yl)cyclohexyl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-2-methyl-6-(methyl(1-(2,2,3,3-tetrafluoropropyl)piperidin-4- yl)amino)-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine- 3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluoroethyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluoro-3-hydroxy-3-methylbutyl)piperidin-4- yl)(methyl)amino)-2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1- yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-((1r,3r)-3-(difluoromethoxy)cyclobutyl)piperidin-4- yl)(methyl)amino)-2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1- yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-(2-(2,2-difluoroethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-(2-(2,2-difluoropropyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-6-(2-(dimethylamino)ethyl)-2-methyl-4-oxoquinazolin-3(4H)- yl)ethoxy)phenyl)-5-methyl-N-(methylsulfonyl)thieno[3,2-b]pyridine-3-carboxamide,
7-(5-chloro-2-(2-(5-cyano-6-((1-(2,2-difluoropropyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(3-(5-cyano-2-methyl-6-(methyl(1-(2,2,2-trifluoroethyl)piperidin-4-yl)amino)-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluoropropyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2-fluoro-2-methylpropyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluoropropyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)-N-(pyridin-4- ylsulfonyl)thieno[3,2-b]pyridine-3-carboxamide,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluoropropyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)-N-(pyridin-3- ylsulfonyl)thieno[3,2-b]pyridine-3-carboxamide,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxopyrido[3,4-d]pyrimidin- 3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid
7-(5-chloro-2-(2-(5-cyano-2,8-dimethyl-4-oxo-6-(2-(trifluoromethyl)phenyl)pyrido[3,4- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(3-hydroxypyrrolidin-1-yl)-2,8-dimethyl-4-oxopyrido[3,4- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid
7-(5-Chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-7-(methylsulfonyl)-4- oxoquinazolin-3(4H)-yl)ethoxy)phenyl)-2,5-dimethylthieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-N-(methylsulfonyl)thieno[3,2-b]pyridine- 3-carboxamide,
7-(5-chloro-2-(2-(5-cyano-6-((1s,3s)-3-methoxycyclobutyl)-2-methyl-4-oxopyrido[3,4- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(2-(5-cyano-2-methyl-4-oxo-6-(2,2,2-trifluoroethoxy)-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 5'-Chloro-2'-(3-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)prop-1-yn-1-yl)-[1,1'-biphenyl]-3-carboxylic acid, 7-(5-chloro-2-(2-(2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)pyrido[3,2- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)-N-(methylsulfonyl)thieno[3,2-b]pyridine-3-carboxamide, 7-(5-chloro-2-(2-(5-cyano-2-methyl-4-oxo-7-(trifluoromethyl)-6-(4-(3,3,3- trifluoropropyl)piperazin-1-yl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(methyl(1-(2,2,2-trifluoroethyl)piperidin-4-yl)amino)-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)ethoxy)phenyl)-N-(methylsulfonyl)thieno[3,2-b]pyridine- 3-carboxamide,
7-(5-chloro-2-(2-(5-cyano-6-(6-cyclopropyl-2,6-diazaspiro[3.3]heptan-2-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-Chloro-2-(2-(5-cyano-6-(4-cyclopropylpiperazin-1-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(3-(5-cyano-2-methyl-4-oxo-6-(4-(3,3,3-trifluoropropyl)piperazin-1-yl)pyrido[3,4- d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-2-methyl-4-oxo-6-(4-(2-(trifluoromethoxy)ethyl)piperazin-1- yl)pyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-(4-cyclopropylpiperazin-1-yl)-2-methyl-4-oxopyrido[3,4- d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-(4-(3,3-difluorocyclobutyl)piperazin-1-yl)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-6-(4-(2,3-difluoro-2-methylpropyl)piperazin-1-yl)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(3-(5-cyano-6-((1-(3,3-difluorocyclobutyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluorocyclobutyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-(4-((1-fluorocyclopropyl)methyl)piperazin-1-yl)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-6-((1-(2,2-difluorobutyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(3-(5-cyano-6-((1-((1-fluorocyclopropyl)methyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-2-methyl-6-(methyl(1-(2-(trifluoromethoxy)ethyl)piperidin-4- yl)amino)-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine- 3-carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-6-((1-cyclopropylpiperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(3-(difluoromethoxy)cyclobutyl)piperidin-4-yl)(methyl)amino)- 2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-2-methyl-6-(methyl(1-((1s,3s)-3- (trifluoromethoxy)cyclobutyl)piperidin-4-yl)amino)-4-oxopyrido[3,4-d]pyrimidin-3(4H)- yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-((1-fluorocyclobutyl)methyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-2-methyl-6-(methyl(1-((1R,2R)-2- (trifluoromethyl)cyclopropyl)piperidin-4-yl)amino)-4-oxopyrido[3,4-d]pyrimidin-3(4H)- yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-6-(ethyl(1-(2,2,2-trifluoroethyl)piperidin-4-yl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-6-((1-(2,2-difluoropropyl)piperidin-4-yl)(ethyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-(6-cyclopropyl-2,6-diazaspiro[3.3]heptan-2-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(4-cyclopropylpiperazin-1-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-N-(methylsulfonyl)thieno[3,2-b]pyridine- 3-carboxamide,
7-(5-Chloro-2-(3-(5-cyano-6-(4,4-difluoro-[1,4'-bipiperidin]-1'-yl)-2-methyl-4-oxopyrido[3,4- d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(3-(5-cyano-6-((1-((1r,3r)-3-fluorocyclobutyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-6-((1-(3-(difluoromethyl)oxetan-3-yl)piperidin-4-yl)(methyl)amino)- 2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluoropropyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)-N-(oxetan-3-ylsulfonyl)thieno[3,2- b]pyridine-3-carboxamide,
7-(5-chloro-2-(3-(5-cyano-6-((1-(3,3-difluorocyclobutyl)piperidin-4-yl)(ethyl)amino)-2-methyl- 4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-((3-fluorooxetan-3-yl)methyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-6-(ethyl(1-(oxetan-3-yl)piperidin-4-yl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-(4-(3-(difluoromethoxy)azetidin-1-yl)piperidin-1-yl)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(3,3-difluorocyclobutyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)-N- (methylsulfonyl)thieno[3,2-b]pyridine-3-carboxamide,
7-(5-Chloro-2-(3-(5-cyano-6-((1-((3-(difluoromethoxy)cyclobutyl)methyl)piperidin-4- yl)(methyl)amino)-2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1- yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-6-((1-(3,3-difluorobutyl)piperidin-4-yl)(methyl)amino)-2-methyl-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-6-(ethyl(1-(2,2,3,3-tetrafluoropropyl)piperidin-4-yl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylic acid, 7-(5-Chloro-2-(3-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(3-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)prop-1-yn-1-yl)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(4-(2,2-difluoroethyl)piperazin-1-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic 7-(5-chloro-2-(2-(5-cyano-6-((1-(2-cyclopropyl-2,2-difluoroethyl)piperidin-4- yl)(methyl)amino)-2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2- b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(3-(5-cyano-2-methyl-6-(methyl(1-(3-methyloxetan-3-yl)piperidin-4-yl)amino)-4- oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
Methyl 7-(5-chloro-2-(3-(5-cyano-2-methyl-4-oxo-6-(4-(2-(trifluoromethoxy)ethyl)piperazin-1- yl)pyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3-carboxylate, Methyl 7-(5-chloro-2-(3-(5-cyano-6-((1-(2,2-difluoroethyl)piperidin-4-yl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine-3- carboxylate,
Methyl 7-(5-chloro-2-(3-(5-cyano-2-methyl-6-(methyl(1-(2,2,2-trifluoroethyl)piperidin-4- yl)amino)-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)thieno[3,2-b]pyridine- 3-carboxylate,
Methyl 7-(5-chloro-2-(2-(5-cyano-6-(4-cyclopropylpiperazin-1-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylate, 7-(5-chloro-2-(2-(5-cyano-2,8-dimethyl-4-oxo-6-(4-(2,2,2-trifluoroethyl)piperazin-1- yl)pyrido[3,4-d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid 7-(5-chloro-2-(2-(5-cyano-6-(4-(2-fluoroethyl)piperazin-1-yl)-2-methyl-4-oxopyrido[3,4- d]pyrimidin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(4-(2,2-difluoroethyl)piperazin-1-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(4-(2-fluoroethyl)piperazin-1-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid, 7-(5-Chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-ethylthieno[3,2-b]pyridine-3-carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-ethyl-2-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-6-(4-(2-methoxyethyl)piperazin-1-yl)-2-methyl-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-(fluoromethyl)-2-methylthieno[3,2- b]pyridine-3-carboxylic acid,
7-(5-Chloro-2-(2-(5-cyano-2-methyl-6-(4-(oxetan-3-yl)piperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridine-3-carboxylic acid, 7-(5-Chloro-2-(2-(5-cyano-2-methyl-6-(4-(oxetan-3-yl)piperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-methylthieno[3,2-b]pyridine-3- carboxylic acid,
7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)-5-(methoxymethyl)-2-methylthieno[3,2- b]pyridine-3-carboxylic acid, and
7-(5-chloro-2-(3-(5-cyano-6-((trans-4-(3,3-difluoroazetidin-1-yl)cyclohexyl)(methyl)amino)-2- methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)prop-1-yn-1-yl)phenyl)-N- (methylsulfonyl)thieno[3,2-b]pyridine-3-carboxamide, or
any combination of two to four of the compounds.
[0184] In some embodiments, the present disclosure provides a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula I, II, III, IV, V or VI. In other embodiments, the present disclosure provides a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand of Formula I, II, III, IV, V or VI attached to a dinucleotide. In certain embodiments, the eIF4E ligand of Formula I, II, III, IV, V or VI is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand of Formula I, II, III, IV, V or VI is attached to the dinucleotide via a linker.
[0185] In specific embodiments, the translational enhancer of the disclosure comprises an eIFE ligand, wherein the eIF4E ligand has a structure according to Formula I. In other embodiments, the translational enhancer of the disclosure comprises an eIF4E ligand attached to a dinucleotide, wherein the eIF4E ligand has a structure according to Formula I. In certain aspects, the eIF4E ligand of Formula I is attached directly to the dinucleotide. In other aspects, the eIF4E ligand of Formula I is attached to the dinucleotide via a linker.
[0186] In some embodiments, the translational enhancer has a structure:
Figure imgf000064_0001
,
wherein the eIF4E ligand has a structure according to Formula I, II, III, IV, V, or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide.
[0187] In certain embodiments, the translational enhancer has a structure:
Figure imgf000064_0002
[0188] In other embodiments, the translational enhancer has a structure:
Figure imgf000065_0001
[0189] In yet other embodiments, the translational enhancer has a structure:
Figure imgf000065_0002
RNA molecules
[0190] In certain embodiments, the translational enhancers of the disclosure are used to couple to RNA sequences of interest. In certain aspects, the translational enhancers of the disclosure are coupled to the 5’ end of RNA sequences of interest. In certain embodiments, coupling of the translational enhancers of the disclosure to the 5’ end of RNA sequences of interest generates 5’ capped RNA molecules. Thus, in certain aspects, the translational enhancer functions as a 5’ cap structure. In other aspects, the translational enhancer functions as a 5’ cap analog or 5’ cap mimetic. In certain aspects, the translational enhancer is a 5’ cap analog or 5’ cap mimetic. In certain instances, the 5’ capped RNA molecules are Cap 0, Cap 1, Cap 2, Cap 3, or Cap 4 RNA molecules. In specific instances, the 5’ capped RNA molecules are Cap 1 RNA molecules.
[0191] Thus, in certain embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer of the disclosure. In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand. In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer consists of or consists essentially of an eIF4E ligand. In certain embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to at least one nucleotide. In certain aspects, the eIF4E ligand is attached directly to the at least one nucleotide. In other aspects, the eIF4E ligand is attached to the at least one nucleotide via a linker. In other embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to a dinucleotide. In certain embodiments, the eIF4E ligand is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand is attached to the dinucleotide via a linker. In specific embodiments, a translational enhancer of the disclosure is attached to the 5’ end of an RNA molecule. In certain aspects, the translational enhancer is attached directly to the 5’ end of the RNA molecule. In other aspects, the translational enhancer is attached to the 5’ end of theRNA molecule via the at least one nucleotide.
[0192] In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand of Formula I, II, III, IV, V or VI. In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer consists of or consists essentially of an eIF4E ligand of Formula I, II, III, IV, V or VI. In other embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand of Formula I, II, III, IV, V or VI attached to a dinucleotide. In certain embodiments, the eIF4E ligand is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand is attached to the dinucleotide via a linker. In specific embodiments, the translational enhancer is attached to the 5’ end of an RNA molecule. In specific aspects, the translational enhancer is attached to the 5’ end of an RNA molecule via the Y moiety.
[0193] In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer comprising an eIF4E ligand, wherein the eIF4E ligand has a structure according to Formula I. In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer consisting of or consisting essentially of an eIF4E ligand, wherein the eIF4E ligand has a structure according to Formula I. In other embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, comprising an eIF4E ligand attached to a dinucleotide, wherein the eIF4E ligand has a structure according to Formula I. In certain embodiments, the eIF4E ligand is attached directly to the dinucleotide. In other embodiments, the eIF4E ligand is attached to the dinucleotide via a linker. In specific embodiments, the translational enhancer is attached to the 5’ end of an RNA molecule. In specific aspects, the translational enhancer is attached to the 5’ end of an RNA molecule via the Y moiety.
[0194] In certain embodiments, coupling of the RNA molecules of the disclosure to the translational enhancer does not affect the interaction of the translational enhancer with any of the other components of the cellular translational machinery (e.g., eIF4E). In other embodiments, coupling of the RNA molecules of the disclosure to the translational enhancer enhances the interaction of the translational enhancer with one or more components of the cellular
translational machinery (e.g., eIF4E).
[0195] In certain aspects, the dinucleotide is selected from the group consisting of an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, and a GG dinucleotide. In certain embodiments, the dinucleotide is an AG dinucleotide. In other embodiments, the dinucleotide is a GG dinucleotide. In certain embodiments, one or both of the adenosines in an AA
dinucleotide is an N6-methyladenosine (m6A). Thus, in certain aspects, the dinucleotide is an m6A-m6A dinucleotide. In other aspects, the dinucleotide is an A-m6A or an m6A-A
dinucleotide. In other embodiments, one or both of the adenosines in an AA dinucleotide is an N6, 2’-O-dimethyladenosine (m6Am). Thus, in certain aspects, the dinucleotide is an m6Am- m6Am dinucleotide. In other aspects, the dinucleotide is an A-m6Am or an m6Am-A
dinucleotide.
[0196] In certain embodiments, the adenosine in an AG or a GA dinucleotide is an m6A. In other embodiments, the adenosine in an AG or a GA nucleotide is an m6Am. In certain embodiments, the guanosine in the AG or GA dinucleotides may also be modified to a 2’-O- methylguanosine (Gm).
[0197] Thus, in certain aspects, the dinucleotide is an m6A-G dinucleotide, an m6A-Gm dinucleotide, a G-m6A dinucleotide, a Gm-m6A dinucleotide, an m6Am-G dinucleotide, an m6Am-Gm dinucleotide, a G-m6Am dinucleotide, or a Gm-m6Am dinucleotide.
[0198] In other embodiments, the dinucleotide includes, without limitation, an AU
dinucleotide, an m6A-U dinucleotide, an m6Am-U dinucleotide, a UA dinucleotide, a U-m6A dinucleotide, a U-m6Am dinucleotide, an AC dinucleotide, an m6A-C dinucleotide, an m6Am-C dinucleotide, a CA dinucleotide, a C-m6A dinucleotide, a C-m6Am dinucleotide, a GU dinucleotide, a Gm-U dinucleotide, a UG dinucleotide, a U-Gm dinucleotide, a GC dinucleotide, a Gm-C dinucleotide, a CG dinucleotide, a C-Gm dinucleotide, a UU dinucleotide, a CC dinucleotide, a CU dinucleotide, or a UC dinucleotide. In certain embodiments, the cytosine or uracil in the above-disclosed dinucleotides may also be modified to a 2’-O-methylcytosine (Cm) or a 2’-O-methyluracil (Um).
[0199] In some embodiments, the linker is a phosphate linker. In certain aspects, the phosphate linker is a monophosphate linker. In other aspects, the phosphate linker is a diphosphate linker. In yet other aspects, the phosphate linker is a triphosphate linker. In other embodiments, the phosphate linker is a tetraphosphate linker. In other aspects, the linker is a phosphonate-diphosphate linker. In yet other aspects, linker is a surrogate linker. In certain aspects, the phosphonate-diphosphate linker is more stable than the monophosphate,
diphosphate, triphosphate, and tetraphosphate linkers.
[0200] In other embodiments, the linkers of the invention typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as a, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,
alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl,
alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl,
alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,
alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl,
alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between 1-24 atoms, e.g., 4-24 atoms, 6-18 atoms, 8-18 atoms, or 8-16 atoms.
[0201] In specific embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand having a structure according to Formula I. [0202] In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer having a structure:
Figure imgf000069_0001
,
wherein the eIF4E ligand has a structure according to Formula I, II, II, IV, V or VI, X is a linker; Y is a dinucleotide, and wherein the translational enhancer is attached to the 5’ end of the RNA molecule. In certain embodiments, the dinucleotide is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide. In certain aspects, the translational enhancer is attached to the 5’ end of the RNA molecule via the Y moiety.
[0203] In certain embodiments, the translational enhancers of the invention are co- transcriptionally attached to the 5’ end of an RNA molecule. Thus, in certain embodiments is provided a method of making an RNA molecule comprising a translational enhancer of the disclosure or a stereoisomer, tautomer, or salt thereof (“the RNA molecule of the disclosure”), comprising reacting a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, and a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into one or more RNA molecules, whereby at least one RNA molecule incorporates at its 5’ end the translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof to generate an RNA molecule of the invention. In certain cases, the method further comprises purifying the RNA molecule of the invention from unreacted components of the reaction. In other cases, the RNA molecules of the invention, thus produced, are used in downstream reactions without further purification.
[0204] In other embodiments are provided a method of making a capped RNA molecule comprising: (a) reacting a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase and under conditions conducive to transcription by the RNA polymerase to generate at least one RNA molecule from the polynucleotide template; and (b) co-transcriptionally coupling to a 5’ end of the at least one RNA molecule a translational enhancer of the disclosure.
[0205] In further embodiments are provided a method of making a capped RNA molecule comprising: (a) reacting a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase and under conditions conducive to transcription by the RNA polymerase to generate at least one RNA molecule from the polynucleotide template; and (b) co-transcriptionally coupling to a 5’ end of the at least one RNA molecule a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof , according to the structure:
Figure imgf000070_0001
,
wherein the eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide, thereby generating a capped RNA molecule.
[0206] In certain embodiments, Y is an AG dinucleotide. In other embodiments, Y is a GG dinucleotide. In certain cases, the linker is a phosphate linker. In other aspects, the linker is a phosphonate-diphosphate linker. In certain aspects, the RNA molecule is a single-stranded RNA molecule. In other aspects, the single-stranded RNA molecule is an mRNA.
[0207] In certain aspects, the polynucleotide template is a complementary DNA (cDNA). In certain aspects, the polynucleotide template comprises any nucleotide base at its 5’ terminal. In other aspects, the polynucleotide template comprises at least one guanine nucleobase at its 5’ terminal. In some cases, the polynucleotide template comprises two guanine nucleobases at its 5’ terminal. In other cases, the polynucleotide template comprises three guanine nucleobases at its 5’ terminal. In certain embodiments, the presence of one or more guanine nucleobases results in efficient transcription of the polynucleotide template by the RNA polymerase to generate the RNA molecule.
[0208] In certain embodiments, the RNA polymerase includes, without limitation, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, eukaryotic RNA polymerase II, a thermostable RNA polymerase, and an RNA-dependent RNA polymerase. In certain aspects, the RNA polymerase is T7 RNA polymerase.
[0209] In other embodiments, the translational enhancers of the invention are chemically attached to the 5’ end of an RNA molecule. Thus, in certain cases is provided a method of making an RNA of the invention comprising chemically synthesizing at least one RNA molecule having attached at its 5’ end a translational enhancer of the disclosure. In other embodiments are provided a method of making an RNA molecule of the invention comprising: (a) providing the RNA molecule; and (b) chemically coupling to a 5’ end of the RNA molecule a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, thereby producing a 5’ capped RNA molecule. In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand. In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer consists of or consists essentially of an eIF4E ligand. In certain embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer, wherein the translational enhancer comprises an eIF4E ligand attached to at least one nucleotide. In certain cases, the method further comprises purifying the RNA molecule of the invention from unreacted components of the reaction. In other cases, the RNA molecules of the invention, thus produced, are used in downstream reactions without further purification. In certain embodiments, the RNAs used in the chemical reaction are double stranded RNAs. In other embodiments, the RNAs used in the chemical reaction are single stranded RNAs including, without limitation, mRNAs. In other
embodiments, the RNAs used in the chemical reaction are non-mammalian RNAs, including, without limitation, viral RNAs. In certain embodiments, the RNAs used in the chemical reaction include, without limitation, short interfering RNAs (siRNAs), inhibitory RNAs (RNAi), micro RNAs (miRNAs), short hairpin-loop RNAs (shRNAs), small nuclear RNAs (snRNAs), single guide RNAs (sgRNAs), or a derivative thereof. In specific embodiments, the RNAs used in the chemical reaction are mRNAs.
[0210] In yet other embodiments, the translational enhancers of the invention are
enzymatically attached to the 5’ end of an RNA molecule. Thus, in certain cases is provided a method of making an RNA of the invention comprising reacting a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, and a RNA molecule in the presence one or more capping enzymes, whereby at least one RNA molecule incorporates at its 5’ end the translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof to generate an RNA molecule of the invention. In certain cases, the method further comprises purifying the RNA molecule of the invention from unreacted components of the reaction. In other cases, the RNA molecules of the invention, thus produced, are used in downstream reactions without further purification. In certain embodiments, the RNAs used in the enzymatic reaction are double stranded RNAs. In other embodiments, the RNAs used in the enzymatic reaction are single stranded RNAs including, without limitation, mRNAs. In other embodiments, the RNAs used in the enzymatic reaction are non-mammalian RNAs, including, without limitation, viral RNAs. In certain embodiments, the RNAs used in the enzymatic reaction include, without limitation, short interfering RNAs (siRNAs), inhibitory RNAs (RNAi), micro RNAs (miRNAs), short hairpin-loop RNAs (shRNAs), small nuclear RNAs (snRNAs), or a derivative thereof. In specific embodiments, the RNAs used in the enzymatic reaction are mRNAs. [0211] In certain embodiments, the capping efficiency of the method is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 990%, or 100%.
[0212] In certain embodiments, the RNA molecule of the disclosure has enhanced resistance to degradation by one or more cellular exonucleases. In certain embodiments, the one or more cellular exonucleases are 5’-3’ exonucleases. In certain embodiments, the RNA molecule of the disclosure has enhanced resistance to degradation by one or more cellular exonucleases compared to an RNA molecule not comprising the translational enhancer. In certain aspects, the one or more cellular exonucleases are 5’-3’exonucleases. Thus, in certain aspects, the RNA molecule of the disclosure has enhanced resistance to degradation by one or more cellular 5’- 3’exonucleases compared to an RNA molecule not comprising the translational enhancer.
[0213] In certain embodiments, the RNA molecule of the disclosure has an increased half-life in a cellular environment compared to that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain aspects, the RNA molecule of the disclosure has a half-life in a cellular environment that is at least 1.2 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In other embodiments, the half-life of the RNA molecule of the invention in a cellular environment is at least 1.5, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising the translational enhancer of the disclosure. In specific embodiments, the RNA molecule of the disclosure has half-life in a cellular environment that is at least 1.2 times of that of an RNA molecule not comprising the translational enhancer.
[0214] In other embodiments, when administered to a subject, the RNA molecule of the disclosure has an increased half-life compared to that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain embodiments, when administered to a subject, the RNA molecule of the disclosure has a half- life in a cellular environment that is at least 1.2 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In other embodiments, when administered to a subject, the half-life of the RNA molecule of the invention is at least 1.5, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times of that of a corresponding natural RNA molecule or an RNA molecule not comprising the translational enhancer of the disclosure. In specific embodiments, when administered to a subject, the RNA molecule of the disclosure has half-life that is at least 1.2 times of that of an RNA molecule not comprising the translational enhancer.
[0215] In certain aspects, the RNA molecule of the disclosure has an enhanced biodsitribution when administered to a subject compared to the biodistribution of a natural RNA molecule or an RNA molecule not comprising the translational enhancer of the disclosure.
[0216] In all organisms, RNA molecules can be decapped in a process known as RNA decapping. The process of mRNA decapping consists of hydrolysis of the 5’ cap structure on the RNA by enzymes dcp1 and dcp2, exposing a 5’ monophosphate. In eukaryotes, this 5’ monophosphate is a substrate for the 5’ exonulease Xrn1 and the mRNA is quickly destroyed. In certain embodiments, the RNA molecule of the disclosure is not recognized by one or more de-capping enzymes. In other embodiments, the RNA molecule of the disclosure is recognized by one or more de-capping enzymes to a lesser extent compared to an RNA molecule not comprising a translational enhancer of the disclosure. In some embodiments, the RNA molecule of the disclosure is resistant to activity of one or more de-capping enzymes. In other embodiments, the RNA molecule of the disclosure has enhanced resistance to activity of one or more de-capping enzymes compared to an RNA molecule not comprising a translational enhancer of the disclosure. In certain aspects, the one or more de-capping enzymes include, but are not limited to, dcp1 and dcp2. In certain embodiments, the RNA molecule of the disclosure is resistant to activity of one or more de-capping enzymes selected from the group consisting of dcp1 and dcp2.
[0217] In some embodiments, the present disclosure provides an RNA molecule comprising a translational enhancer having an improved eIF4E binding affinity, enhanced resistance to degradation, or both, as compared to, e.g., natural mRNA molecules and RNA molecules not comprising a translational enhancer of the disclosure.
[0218] In some embodiments, the RNA molecules of the disclosure have improved binding affinity for eIF4E compared to an RNA molecule not comprising a translational enhancer of the disclosure. In other embodiments, RNA molecules of the disclosure have greater than about 1.2 fold, about 1.5 fold, about 2 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 300 fold, about 400 fold, about 500 fold, or about 1,000 fold higher binding affinity for eIF4E compared to an RNA molecule not comprising a translational enhancer of the disclosure. In specific embodiments, the RNA molecules of the disclosure have greater than about 1,000 fold higher binding affinity for eIF4E as compared to an RNA molecule not comprising a translational enhancer of the disclosure. In certain aspects, the RNA molecules of the disclosure are greater than about 1,000 fold more potent in binding eIF4E than an RNA molecule not comprising a translational enhancer of the disclosure.
[0219] In certain embodiments, binding of the RNA molecules of the disclosure to eIF4E does not affect the interaction of eIF4E with any of the other components of the cellular translational machinery (e.g., other translation initiation factors). In certain embodiments, binding of the RNA molecules of the disclosure to eIF4E enhances the interaction of eIF4E with one or more components of the cellular translational machinery.
[0220] In certain embodiments, the RNA molecule of the disclosure is a double stranded RNA. In other embodiments, the RNA molecule of the disclosure is a single stranded RNA. In other embodiments, the RNA molecules of the disclosure include, without limitation, short interfering RNAs (siRNAs), inhibitory RNAs (RNAi), micro RNAs (miRNAs), short hairpin- loop RNAs (shRNAs), small nuclear RNAs (snRNAs), single guide RNAs (sgRNAs), or a derivative thereof.
[0221] In specific embodiments, the RNA molecule of the disclosure is a single-stranded RNA molecule. In specific aspects, the single-stranded RNA molecule is a messenger RNA (mRNA) molecule. Thus, in specific embodiments, the RNA molecule of the disclosure is an mRNA molecule.
[0222] In certain embodiments, the mRNA molecule of the disclosure comprises (a) a first region of linked nucleosides encoding a polypeptide of interest; (b) a first terminal region located 5’ relative to said first region comprising a 5’ untranslated region (UTR); (c) a second terminal region located 3’ relative to said first region; and (d) a poly (A) tail region.
[0223] In other embodiments, the RNA molecule of the disclosure comprises a 5’ UTR element, an open reading frame, a 3’ UTR element, a poly-A tail and/or a polyadenylation signal. In certain embodiments, the open reading frame is codon optimized for expression in a non-mammalian subject. In other embodiments, the open reading frame is codon optimized for expression in a mammalian subject. In certain aspects, the open reading frame is codon optimized for expression in a human subject.
[0224] In yet other embodiments, the RNA molecule of the disclosure comprises a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5’-terminus of the first region (e.g., a 5’-UTR), a second flanking region located at the 3’-terminus of the first region (e.g., a 3’-UTR), at least one 5’-cap region, and a 3’- stabilizing region. In some embodiments, an RNA molecule of the disclosure further comprises a poly-A region or a Kozak sequence (e.g., in the 5’-UTR). In some cases, an RNA molecule of the disclosure may contain one or more intronic nucleotide sequences capable of being excised from the RNA molecule. In some embodiments, an RNA molecule of the disclosure (e.g., an mRNA) comprises a 5’ cap structure, a chain terminating nucleotide, a stem loop, a poly-A sequence, and/or a polyadenylation signal.
[0225] In some embodiments, any one of the regions of the RNA molecule of the disclosure comprises at least one alternative nucleoside. For example, the 3’-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2’-0-methyl nucleoside and/or the coding region, 5’-UTR, 3’-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5-methoxyuridine), a 1 -substituted
pseudouridine (e.g., 1-methyl-pseudouridine), and/or a 5-substituted cytidine (e.g., 5-methyl- cytidine).
[0226] In some embodiments, the RNA molecule of the disclosure includes from about 200 to about 3,000 nucleotides (e.g., from about 200 to about 500, from about 200 to about 1,000, from about 200 to about 1,500, from about 200 to about 3,000, from about 500 to about 1,000, from about 500 to about 1,500, from about 500 to about 2,000, from about 500 to about 3,000, from about 1,000 to about 1,500, from about 1,000 to about 2,000, from about 1,000 to about 3,000, from about 1,500 to about 3,000, or from about 2,000 to about 3,000 nucleotides).
[0227] In certain embodiments, the mRNA molecule of the disclosure is transcribed from a polynucleotide template having a sequence complementary to the RNA molecule. In certain embodiments, the polynucleotide template is cloned into a vector downstream of a viable promoter. In certain aspects, the polynucleotide template is a complementary DNA (cDNA). In certain aspects, the polynucleotide template comprises any nucleotide base at its 5’ terminal. In other aspects, the polynucleotide template comprises at least one guanine nucleobase at its 5’ terminal. In some cases, the polynucleotide template comprises two guanine nucleobases at its 5’ terminal. In other cases, the polynucleotide template comprises three guanine nucleobases at its 5’ terminal. In certain embodiments, the presence of one or more guanine nucleobases results in efficient transcription of the polynucleotide template by the RNA polymerase to generate the RNA molecule.
[0228] In other embodiments, the polynucleotide template is genomic DNA. In yet other embodiments, the polynucleotide template is an RNA. In certain embodiments, the
polynucleotide template is a complementary DNA (cDNA). In certain aspects, the promoter is recognized by an RNA polymerase promoter. In certain aspects, the RNA polymerase binds to the promoter and transcribes the polynucleotide template in the presence of a translational enhancer of the disclosure to generate an RNA molecule of the disclosure. [0229] In certain instances, the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, eukaryotic RNA polymerase II, an RNA polymerase from other sources including a thermostable RNA polymerase, and an RNA-dependent RNA polymerase. In some instances, the RNA polymerase is T7 RNA polymerase.
[0230] In certain aspects, translation of an RNA molecule of the disclosure can be in vivo, ex vivo, in culture, or in vitro. In some embodiments, are provided methods of inducing translation of an RNA molecule of the disclosure to produce a polypeptide in a cell-free extract. In other embodiments, are provided methods of inducing translation of an RNA molecule of the disclosure to produce a polypeptide in a cell population.
[0231] In some embodiments, the RNA molecule of the disclosure is an in vitro transcribed RNA molecule (IVT RNA). In certain embodiments, the RNA molecule of the disclosure is an mRNA molecule. In other embodiments, an RNA molecule is recombinantly expressed in cells, purified, and co-transcriptionally, chemically, or enzymatically coupled to a translational enhancer of the disclosure to generate an RNA molecule of the disclosure. In certain aspects, the cell is a bacterial cell. In other aspects, the cell is an eukaryotic cell including, but limited to, a yeast cell, a non-mammalian cell, a mammalian cell, a human cell, a cancer cell, an
immortalized cell line, or an insect cell. In other embodiments, an RNA molecule is purified from natural sources and chemically or enzymatically coupled to a translational enhancer of the disclosure to generate an RNA molecule of the disclosure. In yet other embodiments, an RNA molecule is chemically synthesized and chemically or enzymatically coupled to a translational enhancer of the disclosure to generate an RNA molecule of the disclosure.
[0232] In certain aspects, IVT mRNA molecules disclosed herein may function as mRNA but are distinguished from natural mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics. For example, in some cases, the IVT mRNA may be structurally modified or chemically modified.
[0233] In certain aspects, the length of the RNA molecule of the disclosure (e.g., IVT mRNA) encoding a polypeptide of interest is generally greater than about 30 nucleotides (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). [0234] In some embodiments, the RNA molecule of the disclosure comprises from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, or from 2,000 to 100,000 nucleotides).
[0235] In some embodiments, when introduced to a cell, the RNA molecules of the disclosure exhibit reduced degradation as compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure. In other embodiments, when administered to a subject, the RNA molecules of the disclosure exhibit reduced degradation as compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure.
[0236] In certain embodiments, the RNA molecules of the disclosure do not induce an innate immune response when administered to a subject. In other embodiments, the RNA molecules of the disclosure do not substantially induce an innate immune response when administered to a subject. In certain aspects, when administered to a subject, the immune response induced by the RNA molecules of the disclosure is attenuated compared to the immune response induced by a natural RNA or an RNA molecule not comprising the translational enhancer of the disclosure. In certain aspects, the immune response is an innate immune response.
[0237] In certain embodiments, the RNA molecules of the disclosure do not induce an immune response of a cell into which the RNA molecule is introduced. In other embodiments, the RNA molecules of the disclosure do not substantially induce an immune response of a cell into which the RNA molecule is introduced. In some embodiments induce an attenuated immune response of a cell into which the RNA molecule is introduced compared to the immune response induced by a natural RNA or an RNA molecule not comprising the translational enhancer of the disclosure. In certain aspects, the immune response is an innate immune response. [0238] Features of an induced innate immune response include 1) increased expression of pro- inflammatory cytokines, 2) activation of innate immune sensors (e.g., intracellular PRRs such as RIG-I, MDA5, etc.) termination or reduction in protein translation.
[0239] Thus, in certain embodiments, the RNA molecules of the disclosure do not activate one or more innate immune sensors when administered to a subject. In other embodiments, the RNA molecule of the disclosure activates one or more innate immune sensors to a lesser extent when administered to a subject compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure. In other embodiments, the RNA molecule of the disclosure is not recognized by one or more innate immune sensors when administered to a subject. In yet other embodiments, the RNA molecule of the disclosure is recognized to a lesser extent by one or more innate immune sensors when administered to a subject compared to a natural RNA or an RNA molecule not comprising a translational enhancer of the disclosure.
[0240] In specific embodiments, the RNA molecule of the disclosure does not activate one or more innate immune sensors when administered to a subject. In certain aspects, the one or more innate immune sensors include, without limitation, RIG-I, MDA5, IFIT-1, IFIT-5, protein kinase R (PKR), Toll-like receptor 3 (TLR 3), TLR 7, or TLR 8. In specific aspects, the one or more innate immune sensors comprise RIG-I.
[0241] In specific embodiments, the RNA molecule of the disclosure does not activate one or more innate immune sensors when administered to a subject, wherein the one or more innate immune sensors are selected from the group consisting of RIG-I, MDA5, IFIT-1, protein kinase R (PKR), Toll-like receptor 3 (TLR 3), TLR 7, and TLR 8.
[0242] In certain embodiments, the mRNA molecules of the disclosure encode a protein.
[0243] In certain embodiments, the RNA molecule of the disclosure exhibits increased cell permeability compared to an RNA molecule not comprising the translational enhancer. In some embodiments, the RNA molecule of the disclosure is translated with greater efficiency compared to an RNA molecule not comprising the translational enhancer. In other embodiments, the RNA molecule of the disclosure is translated with less efficiency compared to an RNA molecule not comprising the translational enhancer. In some embodiments, the RNA molecule of the disclosure is translated with greater efficiency in vitro compared to an RNA molecule not comprising the translational enhancer. In other embodiments, the RNA molecule of the disclosure is translated with greater efficiency in vivo or ex vivo, compared to an RNA molecule not comprising the translational enhancer of the disclosure. [0244] In certain embodiments, the RNA molecules of the disclosure are translated with greater efficiency in vitro in a cell-free translation extract compared to an RNA molecule not comprising a translational enhancer of the disclosure. In other embodiments, the RNA molecules of the disclosure are translated with greater efficiency in a cellular environment compared to an RNA molecule not comprising a translational enhancer of the disclosure. In yet other embodiments, the RNA molecules of the disclosure are translated with greater efficiency when administered to a subject compared to an RNA molecule not comprising a translational enhancer of the disclosure. In certain aspects, the RNA molecules of the disclosure are preferentially translated in vitro in a cell-free translation extract over an RNA molecule not comprising a translational enhancer of the disclosure. In other aspects, the RNA molecules of the disclosure are preferentially translated in a cellular environment over an RNA molecule not comprising a translational enhancer of the disclosure. In certain instances, the RNA molecules of the disclosure are more stable in a cell-free extract compared to an RNA molecule not comprising a translational enhancer of the disclosure. In other instances, the RNA molecules of the disclosure are more stable in a cellular environment compared to an RNA molecule not comprising a translational enhancer of the disclosure.
[0245] In certain embodiments, the RNA molecules of the disclosure are translated to produce a protein. In certain aspects, the protein is a functional protein. In certain instances, the protein is a therapeutic protein. In certain embodiments, the mRNA molecules of the disclosure encode a therapeutic protein. As used herein,“therapeutic protein” refers to a protein that, when administered to a cell or a subject has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, the protein is an enzyme. In other cases, the protein is a structural protein. In other embodiments, the protein is a regulatory protein.
[0246] In certain embodiments, the shortest length of an RNA molecule of the invention is generally the length of the RNA sequence that is sufficient to encode for a dipeptide. In other embodiments, the length of the RNA molecule of the invention is sufficient to encode for a tripeptide. In some embodiments, the length of the RNA molecule of the invention is sufficient to encode for a tetrapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a pentapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a hexapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a heptapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for an octapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a nonapeptide. In other embodiments, the length of the polynucleotide sequence is sufficient to encode for a decapeptide. Examples of dipeptides that the alternative polynucleotide sequences can encode for include, but are not limited to, carnosine and anserine.
[0247] In certain embodiments, the cell in which the RNA molecules of the disclosure are translated is a bacterial cell. In other aspects, the cell is an eukaryotic cell including, but limited to, a yeast cell, a non-mammalian cell, a mammalian cell, a human cell, a cancer cell, an immortalized cell line, or an insect cell.
[0248] In some cases, the RNA molecules of the disclosure are greater than about 30 nucleotides in length. In other cases, the RNA molecule of the disclosure is greater than about 35, greater than about 50 nucleotides, greater than about 100 nucleotides, greater than about 200 nucleotides, greater than about 300 nucleotides, greater than about 400 nucleotides, greater than about 500 nucleotides, greater than about 600 nucleotides, greater than about 700 nucleotides, greater than about 800 nucleotides, greater than about 900 nucleotides, greater than about 1,000 nucleotides, greater than about 2,000 nucleotides, greater than about 3,000 nucleotides, greater than about 4,000 nucleotides, or greater than about 5,000 nucleotides in length.
[0249] In certain embodiments, a polynucleotide is at least about 30 nucleotides in length. In other embodiments, the length is at least about 35 nucleotides. In other embodiments, the length is at least about 40 nucleotides. In other cases, the length is at least about 45 nucleotides. In certain aspects, the length is at least about 50 nucleotides. In other embodiments, the length is at least about 55 nucleotides. In other aspects the length is at least 60 nucleotides. In other embodiments, the length is at least about 80 nucleotides. In other embodiments, the length is at least about 90 nucleotides. In other embodiments, the length is at least about 100 nucleotides. In other embodiments, the length is at least about 120 nucleotides. In other embodiments, the length is at least about 140 nucleotides. In other embodiments, the length is at least about 160 nucleotides. In other embodiments, the length is at least about 180 nucleotides. In other embodiments, the length is at least about 200 nucleotides. In other embodiments, the length is at least about 250 nucleotides. In other embodiments, the length is at least about 300 nucleotides. In other embodiments, the length is at least about 350 nucleotides. In other embodiments, the length is at least 400 nucleotides. In other embodiments, the length is at least about 450 nucleotides. In other embodiments, the length is at least about 500 nucleotides. In other embodiments, the length is at least about 600 nucleotides. In other embodiments, the length is at least about 700 nucleotides. In other embodiments, the length is at least about 800 nucleotides. In other embodiments, the length is at least about 900 nucleotides. In other embodiments, the length is at least about 1000 nucleotides. In other embodiments, the length is at least about 1100 nucleotides. In other embodiments, the length is at least about 1200 nucleotides. In other embodiments, the length is at least about 1300 nucleotides. In other embodiments, the length is at least about 1400 nucleotides. In other embodiments, the length is at least about 1500 nucleotides. In other embodiments, the length is at least about 1600 nucleotides. In other embodiments, the length is at least about 1800 nucleotides. In other embodiments, the length is at least about 2000 nucleotides. In other embodiments, the length is at least about 2500 nucleotides. In other embodiments, the length is at least about 3000 nucleotides. In other embodiments, the length is at least about 4000 nucleotides. In other embodiments, the length is at least about 5000 nucleotides.
[0250] In certain embodiments, the RNA molecules of the disclosure comprise one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In some embodiments, all or substantially of the nucleotides comprising (a) the 5’-UTR, (b) the open reading frame (ORF), (c) the 3’-UTR, (d) the poly A tail, and any combination of (a, b, c or d above) comprise naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U
(uridine), or T (thymidine).
[0251] In some embodiments, the RNA molecule of the disclosure, except for the 5’ end cap thereof, is an unmodified RNA molecule which has the same sequence and structure as that of a natural RNA molecule. In other embodiments, the RNA molecule of the disclosure, in addition to the modifications on the 5’ end cap disclosed herein, may include at least one chemical modification as described herein.
[0252] In certain embodiments, the RNA molecules of the disclosure comprises one or more modifications. In certain aspects, the one or more modifications comprise one or more phosphorothioate backbone linkages. In other aspects, the one or more modifications comprise one or more modified nucleobases.
[0253] In certain embodiments, the RNA molecules of the disclosure comprise one or more alternative components (e.g., in a 3’-stabilizing region), which impart useful properties including increased stability and/or the lack of a substantial induction of the innate immune response of a cell into which the RNA molecule is introduced. In some cases, a modified (e.g., altered or alternative) RNA molecule of the disclosure exhibits reduced degradation in a cell into which the RNA molecule is introduced, relative to a corresponding natural RNA molecule or an unaltered RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain cases, the RNA molecules of the disclosure enhance the efficiency of protein production, intracellular retention of the RNA molecules, and/or viability of contacted cells, as well as possess reduced immunogenicity.
[0254] In certain cases, the RNA molecules of the disclosure may be naturally or non-naturally occurring. In some cases, the RNA molecules of the disclosure include one or more modified (e.g., altered or alternative) nucleobases, nucleosides, nucleotides, or combinations thereof. In certain aspects, the RNA molecules of the disclosure include any suitable modification or alteration, such as to the nucleobase, the sugar, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). In certain
embodiments, alterations (e.g., one or more alterations) are present in each of the nucleobase, the sugar, and the internucleoside linkage of the RNA molecules of the disclosure. In certain embodiments, alterations according to the present disclosure may be alterations of RNAs, e.g., the substitution of the 2’-OH of the ribofuranosyl ring to 2’-H.
[0255] In other cases, the RNA molecules of the disclosure are not uniformly altered along the entire length of the molecule. For example, in some aspects, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly altered in an RNA molecule of the disclosure, or in a given predetermined sequence region thereof. In some instances, all nucleotides X in an RNA molecule of the disclosure (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
[0256] In certain embodiments, different sugar alterations and/or internucleoside linkages (e.g., backbone structures) exist at various positions in the RNA molecules of the disclosure. One of ordinary skill in the art will appreciate that the nucleotide analogs or other alteration(s) may be located at any position(s) of a RNA molecule such that the function of the RNA molecule is not substantially decreased. In certain cases, an alteration may also be a 5’- or 3’- terminal alteration. In some embodiments, the RNA molecules of the disclosure include an alteration at the 3’-terminus. In certain aspects, the RNA molecules of the disclosure contain from about 1 % to about 100% alternative nucleotides (either in relation to overall RNA content, or in relation to one or more types of RNA, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1 % to 20%, from 1 % to 25%, from 1 % to 50%, from 1% to 60%, from 1% to 70%, from 1 % to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of A, G, U, or C.
[0257] In some embodiments, the RNA molecules of the disclosure contain at a minimum one and at maximum 100% alternative nucleotides, or any intervening percentage, such as at least about 5% altemative nucleotides, at least about 10% alternative nucleotides, at least about 25% alternative nucleotides, at least about 50% altemative nucleotides, at least about 80% altemative nucleotides, or at least about 90% altemative nucleotides. For example, in certain aspects, the RNA molecules of the disclosure contain an alternative pyrimidine such as an alternative uracil or cytosine. In some embodiments, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 80%, at least about 90% or about 100% of the uracil in the polynucleotide is replaced with an alternative uracil (e.g., a 5-substituted uracil). In certain cases, the alternative uracil is replaced by a nucleobase having a single unique structure, or is replaced by a plurality of nucleobases having different structures (e.g., 2, 3, 4 or more unique structures).
[0258] In some embodiments, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 80%, at least about 90% or about 100% of the cytosine in the RNA molecules of the disclosure is replaced with an alternative cytosine (e.g., a 5-substituted cytosine). In certain embodiments, the alternative cytosine is replaced by a nucleobase having a single unique structure, or is replaced by a plurality of nucleobases having different structures (e.g., 2, 3, 4 or more unique structures).
[0259] In certain embodiments, the RNA molecules of the disclosure comprise one or more alternative nucleosides and nucleotides. In certain instances, the alternative nucleosides and nucleotides include an alternative nucleobase. A nucleobase of a nucleic acid is an organic base such as a purine or pyrimidine or a derivative thereof. A nucleobase may be a canonical base (e.g., adenine, guanine, uracil, thymine, and cytosine). In certain cases, these nucleobases are altered or wholly replaced to provide RNA molecules having enhanced properties, e.g., increased stability such as resistance to nucleases. Non-canonical or modified bases may include, for example, one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and/or thio substitutions; one or more fused or open rings; oxidation; and/or reduction. [0260] In certain embodiments, the RNA molecules of the disclosure comprise one or more alternative nucleotide base pairings. In some case, alternative nucleotide base pairing encompasses not only the standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and/or alternative nucleotides including non-standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non- standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.
[0261] In other cases, alternative nucleotides are altered on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and“phosphodiester” are used interchangeably. In certain aspects, backbone phosphate groups are altered by replacing one or more of the oxygen atoms with a different substituent.
[0262] In certain embodiments, the alternative nucleotides include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. In certain aspects, the phosphate linker is altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene- phosphonates).
[0263] In certain instances, the alternative nucleosides and nucleotides include the
replacement of one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl, and/or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., the alpha (a), beta (b) or gamma (g) position) can be replaced with a sulfur (thio) and a methoxy.
[0264] In certain embodiments, the replacement of one or more of the oxygen atoms at the phosphate moiety (e.g., a thio phosphate) is provided to confer stability (such as against exonucleases and endonucleases) to RNA molecules of the disclosure through the unnatural phosphorothioate backbone linkages. In certain embodiments, the phosphorothioate RNA molecules have increased nuclease resistance and subsequently a longer half-life in a cellular environment. [0265] Other inter nucleoside linkages known in the art that may be employed according to the present disclosure, including inter nucleoside linkages which do not contain a phosphorous atom, are also contemplated.
[0266] In other embodiments, the RNA molecules of the disclosure contain an internal ribosome entry site (IRES). An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. In certain aspects, an RNA molecule of the disclosure containing more than one functional ribosome binding site encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multicistronic mRNA). Thus, in certain aspects, when RNA molecules of the disclosure are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from
picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot- and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
[0267] In certain cases, the RNA molecules of the invention comprise a stem loop structure such as, but not limited to, a histone stem loop. The stem loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length. In some embodiments, the histone stem loop is located 3’-relative to the coding region (e.g., at the 3’-terminus of the coding region). In some cases, the stem loop may be located at the 3’-UTR of an RNA molecule of the disclosure. In other cases, the stem loop may be located at the 5’-UTR of an RNA molecule of the disclosure. In some cases, an RNA molecule of the disclosure includes more than one stem loop (e.g., two stem loops). Examples of such stem loop sequences are well known in the art.
[0268] In certain embodiments, the RNA molecules of the invention comprise a poly-A tail. In some embodiments, the poly-A region is generally at least about 30 nucleotides in length. In other embodiments, the poly-A region is at least about 35 nucleotides in length. In other embodiments, the length is at least about 40 nucleotides. In other embodiments, the length is at least about 45 nucleotides. In other embodiments, the length is at least about 55 nucleotides. In other embodiments, the length is at least about 60 nucleotides. In other embodiments, the length is at least about 70 nucleotides. In other embodiments, the length is at least about 80
nucleotides. In other embodiments, the length is at least about 90 nucleotides. In other embodiments the length is at least about 100 nucleotides. In other embodiments, the length is at least about 120 nucleotides. In other embodiments, the length is at least about 140 nucleotides. In other embodiments, the length is at least about 160 nucleotides. In other embodiments, the length is at least about 180 nucleotides. In other embodiments, the length is at least about 200 nucleotides. In other embodiments, the length is at least about 250 nucleotides. In other embodiments, the length is at least about 300 nucleotides. In other embodiments, the length is at least 350 nucleotides. In other embodiments, the length is at least about 400 nucleotides. In other embodiments, the length is at least about 450 nucleotides. In other embodiments, the length is at least about 500 nucleotides. In other embodiments, the length is at least about 600 nucleotides. In other embodiments, the length is at least about 700 nucleotides. In other embodiments, the length is at least about 800 nucleotides. In other embodiments, the length is at least about 900 nucleotides. In other embodiments, the length is at least about 1000 nucleotides. In other embodiments, the length is at least about 1100 nucleotides. In other embodiments, the length is at least about 1200 nucleotides. In other embodiments, the length is at least about 1300 nucleotides. In other embodiments, the length is at least about 1400 nucleotides. In other embodiments, the length is at least about 1500 nucleotides. In other embodiments, the length is at least about 1600 nucleotides. In other embodiments, the length is at least about 1700 nucleotides. In other embodiments, the length is at least about 1800 nucleotides. In other embodiments, the length is at least about 1900 nucleotides. In other embodiments, the length is at least about 2000 nucleotides. In other embodiments, the length is at least about 2500 nucleotides. In other embodiments, the length is at least about 3000 nucleotides.
[0269] In certain cases, engineered binding sites and/or the conjugation of RNA molecules of the disclosure for poly-A binding protein may be used to enhance expression. In some embodiments, the engineered binding sites are sensor sequences which operate as binding sites for ligands of the local microenvironment of the RNA molecules. In certain cases, the RNA molecules of the disclosure include at least one engineered binding site to alter the binding affinity of poly-A binding protein (PABP) and analogs thereof. In some cases, the incorporation of at least one engineered binding site increases the binding affinity of the PABP and analogs thereof.
[0270] In certain embodiments, a poly-A- region may be used to modulate translation initiation. While not wishing to be bound by theory, the poly-A region recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis.
[0271] In other cases, a poly-A region may also be used to protect the RNA molecules of the disclosure against 3’-5’-exonuclease digestion, thereby further increasing the stability of the RNA molecule. [0272] Thus, in certain embodiments are provided a method of improving the stability of an RNA molecule comprising: (a) providing the RNA molecule; and (b) coupling to a 5’ end of the RNA molecule a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, according to the structure:
Figure imgf000087_0001
,
wherein the eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide, thereby generating at least one capped RNA molecule that exhibits improved stability compared to an RNA molecule not comprising a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof.
Therapeutic Uses
[0273] In certain embodiments, the present invention also contemplates the production of RNA molecules of the disclosure for use as therapeutic agents in a pharmaceutical composition, or the introduction of the RNA molecules of the disclosure into cells that utilize those RNAs to produce proteins that may have a therapeutic effect on the host cells, or the introduction of the RNA molecules of the disclosure into cells of a subject to treat a medical condition.
[0274] In certain aspects, a cell population is contacted with an effective amount of a composition containing an RNA molecule of the disclosure, and a translatable region encoding a polypeptide. In some case the cell population is contacted under conditions such that the polynucleotide is localized into one or more cells of the cell population and the polypeptide is translated in the cell from the RNA molecule.
[0275] In certain embodiments, an effective amount of the composition of an RNA of the disclosure is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA molecule (e.g., size, and extent of modified nucleosides), and other determinants. In general, an effective amount of the composition provides efficient protein production in the cell, preferably more efficient than a composition containing a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain embodiments, increased efficiency may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the polynucleotide), increased protein translation from the RNA molecule, decreased degradation of the RNA molecule (as demonstrated, e.g., by increased duration of protein translation from the RNA molecule), reduced innate immune response of the host cell to the RNA molecule, or improve therapeutic utility of the RNA molecule.
[0276] In certain aspects, the present disclosure is directed to methods of inducing in vivo translation of an RNA of the disclosure in a subject in need thereof. In certain instances, an effective amount of a composition containing the RNA molecule of the disclosure and a translatable region encoding a polypeptide of interest is administered to the subject using the delivery methods described herein. In certain cases, the RNA molecule of the disclosure also comprises at least one modified nucleoside. In certain embodiments, the RNA molecule of the disclosure is provided in an amount and under other conditions such that the RNA molecule is localized into a cell or cells of the subject and the polypeptide of interest is translated in the cell from the RNA molecule. In some embodiments, the cell in which the RNA molecule is localized, or the tissue in which the cell is present, is targeted with one or more than one rounds of administration of the RNA molecule of the disclosure. In certain aspects, the subject is a mammal. In other aspects, the subject is a human.
[0277] Other aspects of the present disclosure relate to transplantation of cells comprising the RNA molecules of the disclosure to a subject. Administration of cells to subjects is known to those of ordinary skill in the art, such as local implantation (e.g., topical or subcutaneous administration), organ delivery or systemic injection (e.g., intravenous injection or inhalation), as is the formulation of cells in pharmaceutically acceptable carrier. In certain embodiments, compositions comprising the RNA molecules of the disclosure are formulated for administration intramuscularly, transarterially, intraperitoneally, intravenously, intranasally, subcutaneously, endoscopically, transdermally, or intrathecally. In some embodiments, the compositions are formulated for extended release.
[0278] In certain aspects, the subject to whom the RNA molecule of the disclosure is administered suffers from or is at risk of developing a disease, disorder, or deleterious condition. In some embodiments, methods of identifying, diagnosing, and classifying subjects on these bases include clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art.
[0279] In certain embodiments, the administered RNA molecule of the disclosure directs production of one or more polypeptides that provide a functional activity which is substantially absent in the cell in which the polypeptide is translated. For example, the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
[0280] Thus, in certain embodiments, the RNA molecules of the disclosure encode a protein. In certain embodiments, the protein is a therapeutic protein. In other embodiments, the protein is an enzyme. In other embodiments, the protein is a structural protein. In yet other embodiments, the protein is a regulatory protein. In yet other embodiments, the protein acts as a vaccine when expressed in a subject.
[0281] In other embodiments, the administered RNA molecule of the disclosure directs production of one or more polypeptides that replace a polypeptide (or multiple polypeptides) that is substantially absent in the cell in which the one or more polypeptides are translated. In certain cases, such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof. In other embodiments, the administered RNA molecule of the disclosure directs production of one or more polypeptides to supplement the amount of polypeptide (or multiple polypeptides) that is present in the cell in which the one or more polypeptides are translated. In yet other embodiments, the administered RNA molecule of the disclosure directs production of one or more polypeptides, which function to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. Examples of antagonized biological moieties include, without limitations, proteins (e.g., cancer), lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a polynucleotide, a carbohydrate, or a small molecule toxin.
[0282] In certain embodiments, the translated proteins are engineered for localization within the cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
[0283] In certain aspects, a useful feature of the RNA molecules of the disclosure of the disclosure is the capacity to reduce, evade, avoid or eliminate the innate immune response of a cell to an exogenous RNA. In some embodiments, the RNA molecules of the disclosure of the disclosure induces an attenuated immune response when administered to a subject compared to an RNA molecule not comprising a translational enhancer of the disclosure. In some
embodiments, the RNA molecules of the disclosure of the disclosure induces an attenuated immune response in a cell or a population of cells compared to an RNA molecule not comprising a translational enhancer of the disclosure. Thus, in certain embodiments are provided methods for performing the titration, reduction or elimination of the immune response in a cell or a population of cells. Such methods are well known in the art.
[0284] Also provided herein are methods for treating or preventing a symptom of diseases characterized by missing or aberrant protein activity, by replacing the missing protein activity or overcoming the aberrant protein activity. In certain embodiments, because of the rapid initiation of protein production following introduction of the RNA molecules of the disclosure into cells, as compared to viral DNA vectors, the RNA molecules of the disclosure are particularly advantageous in treating acute diseases such as sepsis, stroke, and myocardial infarction. In other embodiments, the lack of transcriptional regulation of the mRNA molecules of the disclosure is advantageous in that accurate titration of protein production is achievable. Multiple diseases are characterized by missing (or substantially diminished such that proper protein function does not occur) protein activity. Such proteins may not be present, are present in very low quantities or are essentially non-functional. In certain aspects, the present disclosure provides a method for treating such conditions or diseases in a subject by introducing the RNA molecules of the disclosure or cell-based therapeutics containing the RNA molecules of the disclosure, wherein the RNA molecules encode for a protein that replaces the protein activity missing from the target cells of the subject.
[0285] In certain aspects, diseases characterized by dysfunctional or aberrant protein activity include, but not limited to, hyperproliferative diseases (e.g., cancer), genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, inflammatory diseases, viral infections, cardiovascular diseases, and metabolic diseases.
[0286] In certain embodiments, the present disclosure provides a method for treating such conditions or diseases in a subject by introducing the RNA molecules of the disclosure or cell- based therapeutics containing the RNA molecules of the disclosure, wherein the RNA molecules encode for a protein that antagonizes or otherwise overcomes the aberrant protein activity present in the cell of a subject.
[0287] In certain embodiments, the RNA molecule of the disclosure is capable of treating, preventing, or ameliorating a disease when administered to a subject in need thereof. In some embodiments, the disease includes, but is not limited to, hyperproliferative disease, genetic disease, autoimmune disease, diabetes, neurodegenerative disease, inflammatory disease, viral infection, cardiovascular disease, and metabolic disease.
[0288] In other embodiments, the disease is selected from the group consisting of
hyperproliferative disease, inflammatory disease, viral infection, cardiovascular disease, genetic disease, autoimmune disease. In certain aspects, the genetic disease is cystic fibrosis.
[0289] In other aspects, the hyperproliferative disease is cancer. In certain cases, the cancer includes, without limitation, solid tumor, melanoma, non-small cell lung cancer, renal cell carcinoma, renal cancer, a hematological cancer, prostate cancer, castration-resistant prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck cancer, thyroid cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, lung cancer, urothelial cancer, pancreatic cancer, glioblastoma, hepatocellular cancer, myeloma, multiple myeloma, leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, myelodysplastic syndrome, brain cancer, CNS cancer, malignant glioma, or any combination thereof.
[0290] Specific examples of a dysfunctional protein are the missense or nonsense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional or nonfunctional, respectively, protein variant of CFTR protein, which causes cystic fibrosis.
[0291] Thus, in certain aspects are provided methods of treating cystic fibrosis in a subject by contacting a cell of the subject with an RNA molecule of the disclosure having a translatable region that encodes a functional CFTR polypeptide, under conditions such that an effective amount of the CFTR polypeptide is produced in the cell.
[0292] In some aspects are provided a method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject a therapeutically effective amount of an mRNA molecule of the disclosure. In certain aspects, the disease includes, but is not limited to, hyperproliferative disease, inflammatory disease, viral infection, cardiovascular disease, genetic disease, and autoimmune disease.
[0293] In other aspects are provided a method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject an mRNA molecule comprising a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof:
Figure imgf000091_0001
, wherein the eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide, wherein the translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof is attached to the 5’ end of the mRNA molecule. In certain aspects, the mRNA molecule expresses a therapeutic protein when administered to the subject. In other aspects, expression of the therapeutic protein results in treating, preventing, or ameliorating the disease.
[0294] In certain embodiments, the subject is a non-mammalian subject. In other
embodiments, the subject is a mammalian subject. In certain aspects, the subject is a human. [0295] In certain embodiments, the target cells are epithelial cells, such as the lung, and methods of administration are determined in view of the target tissue; i.e., for lung delivery, the RNA molecules are formulated for administration by inhalation.
[0296] In certain instances, methods of the present disclosure enhance polynucleotide delivery into a cell population, in vivo, ex vivo, or in culture. For example, in some cases, a cell culture containing a plurality of host cells (e.g., eukaryotic cells such as yeast or mammalian cells) is contacted with a composition that contains an RNA molecule of the disclosure. The
composition also generally contains a transfection reagent or other compound that increases the efficiency of RNA uptake into the host cells. In certain aspects, the RNA molecules of the disclosure exhibit enhanced retention in the cell population, relative to a corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In certain instances, the retention of the RNA of the disclosure is greater than the retention of the corresponding natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In some embodiments, the retention of the RNA of the disclosure is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greater than the retention of the natural RNA molecule or an RNA molecule not comprising a translational enhancer of the disclosure. In some cases, such retention advantage may be achieved by one round of transfection with the RNA of the disclosure, or may be obtained following repeated rounds of transfection.
[0297] In some embodiments, the RNA of the disclosure is delivered to a target cell population with one or more additional polynucleotides. In certain cases, such delivery may be at the same time, or the RNA of the disclosure is delivered prior to delivery of the one or more additional polynucleotides. In certain embodiments, the additional one or more polynucleotides may be RNA molecules of the disclosure or natural polynucleotides. It is understood that the initial presence of the RNA of the disclosure does not substantially induce an innate immune response of the cell population and, moreover, that the innate immune response will not be activated by the later presence of the natural polynucleotides. In this regard, in certain aspects, the RNA of the disclosure may not itself contain a translatable region, if the protein desired to be present in the target cell population is translated from the natural polynucleotides.
Pharmaceutical Compositions
[0298] In certain embodiments are provided pharmaceutical compositions comprising the RNA molecules of the disclosure, and, optionally, one or more pharmaceutically acceptable carriers, diluents, or excipients. In other embodiments are provided pharmaceutical compositions comprising the RNA molecules of the disclosure and one or more
pharmaceutically acceptable carriers, diluents, or excipients.
[0299] In some embodiments, pharmaceutical compositions optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances. In certain cases, pharmaceutical compositions of the present disclosure are sterile and/or pyrogen- free. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
[0300] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.
[0301] In specific embodiments, an RNA molecule of the disclosure comprising a translational enhancer of the disclosure is formulated as a pharmaceutical composition in an amount effective to treat a particular disease or condition of interest (e.g., cancer, cardiovascular disease, or autoimmune disease) upon administration of the pharmaceutical composition to a subject. In certain aspects, the subject is a mammal. In certain aspects, the mammal is a human.
[0302] Further, a“mammal” includes primates, such as humans, monkeys and apes, and non- primates such as domestic animals, including laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife or the like.
[0303] In particular embodiments, a pharmaceutical composition comprises an RNA molecule of the disclosure and a pharmaceutically acceptable carrier, diluent or excipient. In this regard, a “pharmaceutically acceptable carrier, diluent or excipient” includes any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
[0304] In certain cases, a pharmaceutical composition of the disclosure is prepared by combining or formulating an RNA molecule of the disclosure with an appropriate
pharmaceutically acceptable carrier, diluent or excipient. In certain aspects, a pharmaceutical composition of the disclosure is formulated into preparations in solid, semi-solid, liquid or gaseous forms, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
[0305] Exemplary routes of administering such pharmaceutical compositions include oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. Thus, in certain embodiments, a pharmaceutical composition of the disclosure is formulated to be administered by routes selected from the group consisting of oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In certain aspects, pharmaceutical compositions of the disclosure are formulated to allow the active ingredients contained therein to be bioavailable upon administration to a subject or patient.
[0306] In certain aspects, pharmaceutical compositions that will be administered to a subject or patient take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container of an RNA molecule of the disclosure in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). A composition to be administered will, in any event, contain a therapeutically effective amount of an RNA molecule of the disclosure, or a pharmaceutically acceptable salt thereof, to aid in treatment of a disease or condition of interest in accordance with the teachings herein.
[0307] In certain instances, a pharmaceutical composition of an RNA molecule of the disclosure may be in the form of a solid or liquid. In some aspects, the carrier(s) are particulate so that the compositions are, for example, in tablet or powder form. In other aspects, the carrier(s) are liquid, with a composition being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, a pharmaceutical composition of an RNA molecule of the disclosure is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
[0308] In certain aspects, as a solid composition for oral administration, a pharmaceutical composition of an RNA molecule of the disclosure may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. In some instances, such a solid composition will typically contain one or more inert diluents or edible carriers. In certain embodiments, one or more of the following may be additionally present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
[0309] In some aspects, when the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
[0310] In other aspects, the pharmaceutical composition is in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. In certain embodiments, the liquid may be for oral administration or for delivery by injection. In certain embodiments, when intended for oral administration, preferred compositions contain, in addition to an RNA molecule of the disclosure, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In certain aspects, in a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
[0311] In certain cases, the liquid pharmaceutical compositions of RNA molecules of the disclosure, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. In some cases, the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some aspects, physiological saline is a preferred adjuvant. In some embodiments, an injectable pharmaceutical composition is preferably sterile.
[0312] A liquid pharmaceutical composition of the disclosure intended for either parenteral or oral administration should contain an amount of an RNA molecule of the disclosure such that a suitable dosage will be obtained.
[0313] In other embodiments, a pharmaceutical composition of an RNA molecule of the disclosure may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. In certain aspects, the base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. In other aspects, thickening agents may be present in a pharmaceutical composition for topical administration. In certain embodiments, if intended for transdermal administration, a composition of an RNA molecule of the disclosure may be included with a transdermal patch or iontophoresis device.
[0314] In yet other embodiments, the pharmaceutical composition of an RNA molecule of the disclosure is intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the RNA molecule. In certain instances, a composition for rectal administration contains an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter or polyethylene glycol.
[0315] In other aspects, the pharmaceutical composition of an RNA molecule of the disclosure includes various materials that modify the physical form of a solid or liquid dosage unit. For example, in certain aspects, the composition includes materials that form a coating shell around the active ingredients. In some instances, the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. In other instances, the active ingredients are encased in a gelatin capsule.
[0316] In yet other aspects, the pharmaceutical composition of this disclosure in solid or liquid form include an agent that binds to an RNA molecule of the disclosure and thereby assist in the delivery of the RNA molecule. In certain cases, suitable agents that act in this capacity include a protein or a liposome.
[0317] In other aspects, a pharmaceutical composition of an RNA molecule of the disclosure consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. In certain embodiments, delivery is accomplished by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. In some embodiments, aerosols of the RNA molecules of the disclosure may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). In other
embodiments, delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation, may determine preferred aerosol formulations and delivery modes.
[0318] A pharmaceutical composition of this disclosure may be prepared by methodology well-known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining an RNA molecule of the disclosure with a sterile solvent so as to form a solution. In certain embodiments, a surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are molecules that non-covalently interact with a compound of this disclosure so as to facilitate dissolution or homogeneous suspension of the compound in an aqueous delivery system.
[0319] In certain embodiments, the pharmaceutical compositions of the disclosure comprise one or more additional therapeutically active substances. In other embodiments, a
therapeutically effective dose of the pharmaceutical compositions of the disclosure is administered to a subject in need thereof in combination with one or more additional therapeutically active substances. As used herein, a“combination” refers to a combination comprising an RNA molecule of the disclosure and one or more additional therapeutically active substances, each of which may be administered serially (sequentially), concurrently or simultaneously.
[0320] For the purposes of the present disclosure, the phrase“active ingredient” refers to an RNA molecule of the disclosure, and salts thereof. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formulation or concurrently in separate formulations). The most effective doses may generally be determined using experimental models and/or clinical trials. Design and execution of pre- clinical and clinical studies for a therapeutic agent (including when administered for
prophylactic benefit) described herein are well within the skill of a person skilled in the relevant art.
[0321] In certain aspects, relative amounts of the active ingredient (i.e., an RNA molecule of the disclosure), the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the pharmaceutical composition may comprise between 0.1 % and 100% (w/w), e.g., between 0.1% and 99%, between 0.5 and 50%, between 1-30%, between 5-80%, or at least about 80% (w/w) of the active ingredient.
[0322] In some embodiments, the RNA molecules of the disclosure are formulated using one or more excipients, for example, to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo. In certain aspects, in addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with multimeric structures, hyaluronidase, nanoparticle mimics and
combinations thereof.
[0323] In certain embodiments, the RNA molecules of the disclosure are formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, pharmaceutical compositions of the nucleic acids or multimeric nucleic acid molecules include lipid
nanoparticles (LNPs). In some embodiments, lipid nanoparticles are MC3-based lipid nanoparticles.
[0324] In some aspects, the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle ranges from about 1 RNA molecule to about 100 RNA molecules. In other embodiments, the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle ranges from about 50 to about 500 RNA molecules. In other aspects, the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle ranges from about 250 to about 1000 RNA molecules. In yet other embodiments, the number of RNA molecules of the disclosure encapsulated by a lipid nanoparticle is greater than 1000 RNA molecules.
[0325] In certain embodiments, the RNA molecules of the disclosure are formulated in a lipid- poly cation complex. The formation of the lipid-poly cation complex may be accomplished by methods known in the art. As a non-limiting example, the poly cation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or
polyarginine. In other embodiments, the RNA molecules of the disclosure are formulated in a lipid-poly cation complex which further includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).
[0326] In certain embodiments, the RNA molecules of the disclosure are formulated in a nanoparticle. In some embodiments, the nanoparticle comprises at least one lipid. In some cases, the lipid is selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC 3 -DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In other aspects, the lipid is a cationic lipid such as, but not limited to, DLin- DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. In some embodiments, nanoparticle compositions also includes one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components known in the art. In other embodiments, nanoparticle compositions includes any substance useful in pharmaceutical compositions. In other aspects, nanoparticle compositions includes a lipid component and one or more additional components, such as an additional therapeutic agent.
[0327] In certain aspects, the amount of a therapeutic agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic agent. For example, the amount of an RNA molecule of the disclosure useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA molecule. In certain cases, the relative amounts of a therapeutic agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic agent in a nanoparticle composition may be from about 5 : 1 to about 60: 1 , such as 5 : 1, 6: 1, 7: 1 , 8: 1, 9: 1 , 10: 1, 11 : 1, 12: 1, 13: 1 , 14: 1 , 15: 1 , 16: 1 , 17: 1, 18: 1, 19: 1, 20: 1, 25 : 1, 30: 1, 35 : 1 , 40: 1 , 45: 1 , 50: 1 , and 60: 1. In certain aspects, the wt/wt ratio of the lipid component to a therapeutic agent may be from about 10: 1 to about 40:1. In specific aspects, the wt/wt ratio is about 20: 1.
Polypeptides
[0328] In certain embodiments, the RNA molecules of the disclosure are designed to encode polypeptides of interest selected from any of several target categories including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.
[0329] In certain embodiments, the RNA molecules of the disclosure encode a protein. In certain embodiments, the protein is a therapeutic protein. In other embodiments, the protein is an enzyme. In other embodiments, the protein is a structural protein. In yet other embodiments, the protein is a regulatory protein. As used herein,“therapeutic protein” refers to a protein that, when administered to a cell or a subject has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
[0330] The term“therapeutically effective amount”, as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. In certain aspects, the precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. In certain cases, therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In some aspects, the disease or condition to be treated is cancer. In another aspect, the disease or condition to be treated is a cell proliferative disorder.
Doses
[0331] Generally, a therapeutic agent is administered at a therapeutically effective amount or dose. A therapeutically effective amount or dose will vary according to several factors, including the chosen route of administration, formulation of the composition, patient response, severity of the condition, the subject’s weight, and the judgment of the prescribing physician. The dosage can be increased or decreased over time, as required by an individual subject. In certain instances, a patient initially is given a low dose, which is then increased to an efficacious dosage tolerable to the subject. In addition, a patient may be given a plurality of doses over a determined period of time and in particular time increments (such as daily, weekly, biweekly, monthly, quarterly, biannually, annually or the like). Determination of an effective amount or dosing regimen is well within the capability of those skilled in the art.
[0332] The route of administration of a composition in accordance with the disclosure can be oral, intraperitoneal, transdermal, subcutaneous, by intravenous or intramuscular injection, by inhalation, topical, intralesional, infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art.
[0333] In certain embodiments, compositions in accordance with the disclosure is
administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging. In certain embodiments, the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the desired dosage is delivered using multiple
administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
Assays [0334] In certain embodiments, the translational enhancers and RNA molecules of the disclosure, once produced, can be characterized using a variety of assays known to those skilled in the art to determine whether they have biological activity. For example, the RNA molecules of the disclosure can be characterized by conventional assays, including but not limited to protein production assays (e.g., cell-free translation assays or cell based expression assays), degradation assays, cell culture assays (e.g., of neoplastic cells), animal models (e.g., rats, mice, rabbits, dogs, or pigs) and the like, to determine whether they have a predicted activity, e.g., binding activity and/or binding specificity, and stability.
[0335] In some embodiments, high-throughput screening can be used to speed up analysis using such assays. As a result, it can be possible to rapidly screen the molecules described herein for activity, using high-throughput screening. General methodologies for performing high- throughput screening are well known in the art.
Kits
[0336] In certain aspects, kits including, a translational enhancer of the disclosure for performing transcription are also contemplated. In certain instances, kits comprise all transcription reagents for synthesis of common RNAs (e.g., FLuc mRNA). More specifically, in certain cases, a kit contains: a polynucleotide template having a sequence complementary to the RNA molecule, a translational enhancer of the disclosure, a container marked for transcription, instructions for performing RNA synthesis, and one or more reagents selected from the group consisting of one or more modified or unmodified initiating capped oligonucleotide primers, one or more unmodified NTPs, one or more modified NTPs (e.g., pseudouridine 5’-triphosphate), an RNA polymerase, other enzymes, a reaction buffer, and magnesium.
[0337] In certain embodiments, is provided a kit for capping an RNA molecule comprising: (a) a polynucleotide template having a sequence complementary to the RNA molecule; (b) an RNA polymerase; and (c) a translational enhancer of the disclosure.
[0338] In certain embodiments, is provided a kit for capping an RNA molecule comprising: (a) a polynucleotide template having a sequence complementary to the RNA molecule; (b) an RNA polymerase; and (c) a translational enhancer of the disclosure, or a stereoisomer, tautomer, or salt thereof, according to the structure:
Figure imgf000101_0001
, wherein the eIF4E ligand has a structure according to Formula I, II, III, IV, V or VI, X is a linker; and Y is an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, or a GG dinucleotide.
[0339] In certain embodiments, Y is an AG dinucleotide. In other embodiments, Y is a GG dinucleotide. In certain cases, the linker is a phosphate linker. In other aspects, the linker is a phosphonate-diphosphate linker. In certain aspects, the RNA molecule is a single-stranded RNA molecule. In other aspects, the single-stranded RNA molecule is an mRNA.
[0340] In certain aspects, the polynucleotide template is a complementary DNA (cDNA). In certain aspects, the polynucleotide template comprises any nucleotide base at its 5’ terminal. In other aspects, the polynucleotide template comprises at least one guanine nucleobase at its 5’ terminal. In some cases, the polynucleotide template comprises two guanine nucleobases at its 5’ terminal. In other cases, the polynucleotide template comprises three guanine nucleobases at its 5’ terminal. In certain embodiments, the presence of one or more guanine nucleobases results in efficient transcription of the polynucleotide template by the RNA polymerase to generate the RNA molecule.
[0341] In certain embodiments, the kit comprises nucleotides. In other embodiments, the kit further comprises a ribonuclease inhibitor. In other embodiments, the kit further comprises a buffer.
[0342] In certain embodiments, a number of other RNA polymerases known in the art for use in transcription reactions may be utilized with the compositions and methods of the present invention. In certain aspects, other enzymes, including natural or mutated variants that may be utilized include, for example, SP6 and T3 RNA polymerases, eukaryotic RNA polymerase II, RNA polymerases from other sources including thermostable RNA polymerases, and RNA- dependent RNA polymerases.
[0343] Thus, in certain embodiments, the RNA polymerase includes, without limitation, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, eukaryotic RNA polymerase II, a thermostable RNA polymerase, and an RNA-dependent RNA polymerase. In certain aspects, the RNA polymerase is T7 RNA polymerase.
[0344] Various aspects of the present invention are illustrated by the following non-limiting examples. The examples are for illustrative purposes and are not a limitation on any practice of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the invention. One of ordinary skill in the art readily knows how to synthesize or commercially obtain the reagents and components described herein.
Examples
[0345] In the synthetic schemes described below, unless otherwise indicated all temperatures are set forth in degrees Celsius and all parts and percentages are by weight. Reagents were purchased from commercial suppliers and were used without further purification unless otherwise indicated. All solvents were purchased from commercial suppliers and were used as received.
[0346] The reactions set forth below were done generally under a positive pressure of nitrogen or argon at an ambient temperature (unless otherwise stated) in anhydrous solvents, and the reaction vessels were fitted with rubber septa (for flasks) or caps (for vials) for the introduction of substrates and reagents via syringe. Glassware was oven dried and/or heat dried. The reactions were assayed by TLC and/or analyzed by LC-MS and terminated as judged by the consumption of starting material. Occasionally, reactions were terminated early as desired products start to decompose.
[0347] Analytical thin layer chromatography (TLC) may be performed on glass-plates precoated with silica gel 60 F254 0.25 mm plates (EMD Chemicals), and visualized with UV light (254 nm) and/or iodine on silica gel and/or heating with TLC stains such as ethanolic phosphomolybdic acid, ninhydrin solution, potassium permanganate solution or ceric sulfate solution.
[0348] 1H-NMR spectra was recorded on a Varian spectrometer operating at 400 MHz.
NMR spectra were obtained as CDCl3 solutions (reported in ppm), using chloroform as the reference standard (7.27 ppm for the proton and 77.00 ppm for carbon), CD3OD solutions using 3.4 ppm and 4.8 ppm as reference standards for the protons and 49.3 ppm as a reference standard for carbon, DMSO-d6 (2.49 ppm for proton), or internally tetramethylsilane (0.00 ppm) when appropriate. Other NMR solvents were used as needed. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broadened), bs (broad singlet), dd (doublet of doublets), dt (doublet of triplets). Coupling constants, when given, are reported in Hertz (Hz).
[0349] In the below methods it is understood that if protecting groups are used during the synthesis of intermediates, or if Formulae I, II, III, IV, V or VI compounds contain one or more protecting groups, then such protecting groups are removed by methods known in the chemical art. Other functional group transformations, such as the conversion of atom X from S to S(O)2, from C=O to C=CR6R7, from NH to N(C1-C8)alkyl, and the conversion of an intermediate or compound of Formulae I, II, III, IV and V to a pharmaceutically acceptable salt are carried out using conventional methods known in the chemical art.
[0350] Examples of eIF4E ligands that are Formulae I, II, III, IV, V or VI compounds are disclosed in U.S. Provisional Application No.62/869,662, wherein the compounds and synthetic methods disclosed therein are incorporated herein by reference in their entirety.
Example 1 (Cpd. No.185F) (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-((((((((7-(5-chloro-2-(2-(5- cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3- yl)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)- 4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.185F)
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Synthesis of 1,2-dibromo-5-fluoro-3-(trifluoromethyl)benzene (2) [0351] A suspension of copper (I) bromide (89.8 g, 620.1 mmol) and tert-butyl nitrite (63.8 mL, 620.1 mmol) in acetonitrile (2500 mL) was heated at 65 °C for 15 min. A solution of 2- bromo-4-fluoro-6-(trifluoromethyl)aniline (1, 100 g, 387.6 momol) in acetonitrile was added and heated the reaction mixture at 65°C for 1 h. After completion, the reaction mass was quenched with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get the crude product. The crude compound was purified by column chromatography using 0-5% ethyl acetate in hexanes over silica gel (100-200 mesh) to afford 1,2-dibromo-5-fluoro-3-(trifluoromethyl)benzene (2) as an off-white solid. Yield: 70.0 g, 55%; no ionization in LCMS; 1H NMR (400 MHz, CDCl3) d 7.60 (dd, J= 7.32, 2.84, 1H), 7.43 (dd, J= 8.24, 2.80, 1H).
Synthesis of 2,3-dibromo-6-fluoro-4-(trifluoromethyl)benzoic acid (3)
[0352] To a solution of 2,2,6,6-tetramethylpiperidine (7.09 mL, 43.61 mmol) in dry tetrahydrofuran (100 mL) was added n-butyllithium (1.2 M, 27.25 mL, 32.71 mmol) dropwise at -78°C under argon atmosphere. The reaction mixture was warmed to 0°C and stirred for 30 min It was again cooled to -78°C and a solution of 1,2-dibromo-5-fluoro-3-(trifluoromethyl)benzene (2 , 10.0 g, 31.15 mol) in dry tetrahydrofuran (70 mL) was added at -100°C and the reaction mixture was stirred for 45 min at -100°C. Carbon dioxide gas was bubbled through the reaction mass at this temperature for 15 min and it was gradually warmed to room temperature in 2 h. After completion, the reaction mixture was quenched with water and washed with diethyl ether. The aqueous layer was acidified to pH ~3-2 with 6N aqueous hydrochloric acid, extracted with ethyl acetate, washed the organic layer, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford 2,3-dibromo-6-fluoro-4-(trifluoromethyl)benzoic acid (3) as a brown solid. Yield: 10.0 g, crude, 87%; MS (ESI) m/z 362.9[M-1]-.
Synthesis of 2,3-dibromo-6-fluoro-N-(1-iminoethyl)-4-(trifluoromethyl)benzamide (4)
[0353] To a solution of 2,3-dibromo-6-fluoro-4-(trifluoromethyl)benzoic acid (3, 27.0 g, 75.8 mmol) and acetamidine hydrochloride (4, 9.31 g, 98.5 mmol) in N,N-dimethylformamide (180 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (36.5 g, 98.5 mmol) and N,N-diisopropylethyl amine (32.25 mL, 221.9 mmol) were added at-10°C and stirred for 3 hours at 0°C. After completion, the reaction mass was quenched with ice-water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The residue was washed with pentanes to afford 2,3-dibromo-6-fluoro-N-(1-iminoethyl)-4-(trifluoromethyl)benzamide (4) as a brown gum. Yield: 30 g (crude); MS (ESI) ) m/z 404.8 [M+1] +.
Synthesis of 5,6-dibromo-2-methyl-7-(trifluoromethyl)quinazolin-4(3H)-one (5) [0354] To a solution of 2,3-dibromo-6-fluoro-N-(1-iminoethyl)-4-(trifluoromethyl)benzamide (4, 30.0 g, 74.4 mmol) in tetrahydrofuran (250 mL), sodium hydride (60%) (5.9 g, 148.8 mmol) was added at 0 oC, warmed to room temperature and continued to stir at room temperature for 16 h. After completion, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The residue was washed with diethyl ether to afford 5,6-dibromo-2- methyl-7-(trifluoromethyl)quinazolin-4(3H)-one (5) as a white solid. Yield: 9.95 g, 35%; MS (ESI) m/z 383.01 [M-1]-.
Synthesis of 6-bromo-2-methyl-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5-carbonitrile (6)
[0355] To a stirred solution of 5,6-dibromo-2-methyl-7-(trifluoromethyl)quinazolin-4(3H)-one (5, 6.0 g, 15.62 mmol) in dimethylformamide (90 mL), copper (I) cyanide (1.53 g, 17.18 mmol) was added at room temperature and the reaction mixture was heated and stirred at 90 °C for 2 h. After completion, the reaction mass was cooled to room temperature, diluted with ethyl acetate and washed with water and 1N aqueous hydrochloric acid. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The crude compound was purified by CombiFlash (40g, Redi Sep column) using 70% ethyl acetate in hexanes as eluent. The desired fractions were concentrated under reduced pressure to afford 6-bromo-2-methyl-4- oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5-carbonitrile (6) as a yellow solid. Yield: 3.2 g, 62.7%; MS (ESI) m/z 330.06 [M-1]-.
Synthesis of 2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (7)
[0356] To solution of 6-bromo-2-methyl-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5- carbonitrile (6, 500 mg, 1.51 mmol) in N-methyl-2-pyrrolidone (6 mL) were added 1- methylpiperazine (0.50 mL, 0.45 g, 4.5 mmol), 1,10-phenanthroline (108 mg, 0.599 mmol), and copper iodide (57 mg, 0.30 mmol) at room temperature under argon. The mixture was stirred at 145 oC for 24h. Then, the mixture was diluted with ethyl acetate and filtered with celite. The filtrate was concentrated in vacuo, and the residue (N-methyl-2-pyrrolidone solution) was loaded onto a 5g Phenomenex strata ion exchange column. The cartridge was washed with water, acetonitrile, and MeOH (some product found in the combined filtrate/rinsing solution). Then, product was eluted with 5% NH4OH in MeOH, and concentrate in vacuo to give 498 mg of a brown oil.466 mg of this material were repurified by HPLC (C18, acetonitrile/water + 0.1% trifluoroacetic acid) to give 172 mg (0.370 mmol, 24.5%; TFA salt) of the TFA salt of 7 as a slightly brownish solid; LCMS: MS (ESI) m/z 352.3 [M+1]+; 1H-NMR (400 MHz, d6-dimethyl sulfoxide) d / ppm = 12.94-12.81 (b, 1H), 9.80-9.66 (b, 1H), 8.16 (s, 1H), 3.90-3.81 (m, 2H), 3.57-3.49 (m, 2H), 3.23-3.15 (m, 2H), 3.12-2.99 (m, 2H), 2.89 (bs, 3H), 2.40 (s, 3H). The filtrate/rinsing solution of the first Phenomenex strata ion exchange extraction was subjected to a second Phenomenex strata ion exchange extraction (same procedure as above) to give another 188 mg (0.535 mmol, 35.5% yield, free base) of product 2-methyl-6-(4-methylpiperazin-1-yl)-4- oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5-carbonitrile (7) as a brown, glassy solid; MS (ESI) m/z 352.0 [M+1]+; 1H-NMR (400 MHz, d6-dimethyl sulfoxide) d / ppm = 12.84-12.47 (b, 1H), 8.09 (s, 1H), 3.80-3.40 (b, 2H), 3.10-2.50 (b, 4H), 2.37 (s, 3H), 2.30-2.10 (b, 2H), 2.23 (s, 3H). Combined yield of TFA salt and free base: 60%.
Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2- isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl (4- chlorophenyl) hydrogen phosphate (9)
[0357] A mixture of N-(9-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-6-oxo- 6,9-dihydro-1H-purin-2-yl)isobutyramide (8, 1.00 g, 1.49 mmol) and 4-chlorophenyl
phosphorodichloridate (8a, 1.83 g, 7.47 mmol) in dry pyridine (10 mL) was stirred at room temperature for 0.5 h. After completion, 1M buffer solution of triethylammonium hydrogen carbonate (25 mL) was added and reaction mixture was cooled to 0 °C for 10 minutes. After 10 minutes, chloroform was added and the mixture was washed with 0.1 M solution of
triethylammonium hydrogen carbonate solution. The organic layer was evaporated under reduced pressure, the residue obtained was co-evaporated with toluene two to three times and dried in vacuum to afford (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5- (2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl (4- chlorophenyl) hydrogen phosphate (9) as a brown solid. Yield: 1.5 g (crude), MS (ESI) m/z 858.15 [M-1]-. The crude obtained was used as such for next reaction.
Synthesis of (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)- 4-methoxytetrahydrofuran-3-yl)oxy)(4-chlorophenoxy)phosphoryl)oxy)methyl)-5-(2- isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diyl bis(2- methylpropanoate) (11)
[0358] The mixture of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5- (2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl (4- chlorophenyl) hydrogen phosphate (9, 1.0 g, 1.16 mmol) and (2R,3R,4R,5R)-2- (hydroxymethyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (10, 0.72 g, 1.16 mmol) were dried by co-evaporation with anhydrous acetonitrile. Reaction mixture was cooled to 0 °C and a solution of 2,4,6- triisopropylbenzenesulfonyl chloride (10a) and N-methyl imidazole in dry acetonitrile (10 mL) was added to reaction mixture. The reaction mixture was allowed to react for 16 h. After completion, reaction mixture was concentrated in vacuum and crude obtained was purified by flash column chromatography using silica gel (230-400 mesh) and 70-100% ethyl acetate in hexanes as eluents to afford (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)-4-methoxytetrahydrofuran-3-yl)oxy)(4-chlorophenoxy)phosphoryl)oxy)methyl)-5-(2- isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diyl bis(2- methylpropanoate) (11) as a brown solid. Yield: 0.29 g, 19 %; MS (ESI) m/z 1333.32 [M-1]-. Synthesis of (2R,3R,4R,5R)-2-((((4-chlorophenoxy)(((2R,3R,4R,5R)-2-(hydroxymethyl)-5-(2- isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-methoxytetrahydrofuran-3- yl)oxy)phosphoryl)oxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (12)
[0359] To a solution of (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)-4-methoxytetrahydrofuran-3-yl)oxy)(4-chlorophenoxy)phosphoryl)oxy)methyl)-5-(2- isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diyl bis(2- methylpropanoate) (11, 0.38 g, 0.284 mmol) in dichloromethane (3.8 mL) was added 2% trifluoro acetic acid in dichloromethane (3.8 mL). Reaction mixture was then stirred at room temperature for 6 h. After completion, reaction mixture was cooled down and quenched by addition of saturated solution sodium bicarbonate and extracted with dichloromethane. Organic layer obtained was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude compound. Crude compound obtained was purified by flash column chromatography using silica gel (230-400 mesh) and 6 to 7 % methanol in dichloromethane as eluents. The desired fractions were concentrated under reduced pressure to afford
(2R,3R,4R,5R)-2-((((4-chlorophenoxy)(((2R,3R,4R,5R)-2-(hydroxymethyl)-5-(2- isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-methoxytetrahydrofuran-3- yl)oxy)phosphoryl)oxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (12) as a white solid. Yield: 0.22 g, 75 %, MS (ESI) m/z 1033.15 [M+1]+. Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4- methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (13)
[0360] A solution of (2R,3R,4R,5R)-2-((((4-chlorophenoxy)(((2R,3R,4R,5R)-2- (hydroxymethyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4- methoxytetrahydrofuran-3-yl)oxy)phosphoryl)oxy)methyl)-5-(2-isobutyramido-6-oxo-1,6- dihydro-9H-purin-9-yl)tetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (12, 0.10 g, 0.096 mmol) in 25 % aqueous ammonia (3.0 mL) is stirred at room temperature for 3 days. After completion, the reaction mixture is cooled down and extracted with dichloromethane to remove impurities. The aqueous layer is concentrated and purified by Preparative HPLC to afford (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4- methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (13).
Synthesis of ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)- 5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)oxidophosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl phosphate (14)
[0361] To a stirred solution of phosphorus oxychloride (0.043 mL, 0.46 mmol) and trimethyl phosphite (1.0 mL) at 0 °C under argon atmosphere, (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6- dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (13, 0.10 g, 0.15 mmol) is added and the reaction mixture is stirred for 4 h at 0 °C. After 4 h, water is added to the reaction mixture. The resulting reaction mixture is washed with ethyl acetate (2 X 5 mL) to remove phosphorylating agent. The collected aqueous solution is adjusted to pH 1.5 and allowed to stir at 4 °C for 15 h. After 15 h, the aqueous solution is adjusted to pH 5.5 and purified by preparative HPLC to afford ((2R,3R,4R,5R)-5-(2-amino-6- oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)oxidophosphoryl)oxy)-4- methoxytetrahydrofuran-2-yl)methyl phosphate (14).
Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(((hydroxy(1H- imidazol-1-yl)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (15)
[0362] A stirred solution of ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)oxidophosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl phosphate (14, 0.10 g, 0.13 mmol), imidazole (0.044 g, 0.65 mmol), 2,2’-dithiodipyridine (0.057 g,0.26 mmol) and triethylamine (0.018 mL, 0.13 mmol) are mixed in anhydrous N,N-dimethylformamide (1.0 mL). After 20 min, triphenyl phosphine (0.068 g, 0.26 mmol) is added and the mixture is stirred at room temperature for 8 h. The product is precipitated from the reaction mixture with a solution of anhydrous sodium perchlorate in dry acetone. After cooling at 4°C, the precipitate is filtered, washed repeatedly with cold, dry acetone and dried to afford (2R,3R,4R,5R)-5-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(((hydroxy(1H-imidazol-1- yl)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo- 1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (15).
Synthesis of triethylammonium salt of ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin- 9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)oxidophosphoryl)oxy)-4-methoxytetrahydrofuran-2- yl)methyl diphosphate (16)
[0363] To a stirred solution of (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 2-(((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methyl) phosphate (15, 0.10 g, 0.13 mmol) and zinc chloride (0.035 g, 0.26 mmol) in dry N,N-dimethylformamide (1.0 mL), 3.0 mL of 1M tris(tributylammonium) phosphate in N,N- dimethylformamide is added under argon atmosphere. The reaction mixture is stirred at room temperature for 5 h. After 5 h, the reaction mixture is diluted with water. The resulting reaction mixture is washed with ethyl acetate to remove phosphorylating agent. The collected aqueous solution is adjusted to pH 5.5 and purified by Preparative HPLC to afford triethylammonium salt of ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)oxidophosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl diphosphate (16). Synthesis of diethyl (7-chlorothieno[3,2-b]pyridin-3-yl)phosphonate (18)
[0364] To a solution of 3-bromo-7-chlorothieno[3,2-b]pyridine (17, 11.0 g, 44.354 mmol) in acetonitrile (110 mL), diethyl phosphate (17a, 6.1 mL, 44.354 mmol) and triethylamine (9 mL, 66.532 mmol) were added and reaction mixture was degassed using argon gas for 20 minutes. Tetrakis(triphenylphosphine)palladium(0) (5.10 g, 4.43 mmol) was added and reaction mixture was stirred at 90 °C for 16 h. After completion, the reaction mixture cooled down and filtered through celite bed and washed with ethyl acetate (2×100 ml). Filtrate obtained was concentrated under reduced pressure to get crude compound. Crude compound obtained was purified by column chromatography using silica gel (100-200 mesh) and 10-40% ethyl acetate in hexanes as eluents. The desired fractions were concentrated under reduced pressure to afford diethyl (7- chlorothieno[3,2-b]pyridin-3-yl)phosphonate (18) as a yellow liquid. Yield: 9.00 g, 66%; MS (ESI) m/z 305.75[M+1]+.
Synthesis of diethyl (7-(5-chloro-2-hydroxyphenyl)thieno[3,2-b]pyridin-3-yl)phosphonate (19)
[0365] To a solution of diethyl (7-chlorothieno[3,2-b]pyridin-3-yl)phosphonate (18, 9.00 g, 29.508 mmol) and (5-chloro-2-hydroxyphenyl)boronic acid (18a, 10.1 g, 59.016 mmol) in 1,4- dioxane (144.0 mL), 2M aqueous solution of potassium carbonate (10.1 g, 73.770 mmol) dissolved in water (36.0 mL) was added and reaction mixture was degassed using argon gas for 10 min.1,1'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (2.40 g, 2.950 mmol) was added to the reaction mixture at room temperature and heated to 100 o C for 16 h. After completion, reaction mixture was cooled to room temperature, diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The crude residue obtained was purified by column chromatography using silica gel (100-200 mesh) and 0-60% ethyl acetate in hexanes to afford diethyl (7-(5-chloro-2-hydroxyphenyl)thieno[3,2-b]pyridin-3-yl)phosphonate (19) as a brownish sticky gum. Yield: 6.00 g, 51%; MS (ESI) m/z 398.10 [M+1]+.
Synthesis of diethyl (7-(2-(2-bromoethoxy)-5-chlorophenyl)thieno[3,2-b]pyridin-3- yl)phosphonate (20)
[0366] To a solution of diethyl (7-(5-chloro-2-hydroxyphenyl)thieno[3,2-b]pyridin-3- yl)phosphonate (19, 4.00 g, 10.07 mmol) in acetone (40.0 mL), potassium carbonate (4.17 g, 30.226 mmol) and 1,2-dibromoethane (19a, 8.64 mL, 100.07 mmol) were added and reaction mixture was heated at 50 °C for 16 h. After completion, reaction mixture was poured onto ice cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to get crude product, which was purified by flash column chromatography using silica gel (230-400 mesh) and 0-50% ethyl acetate in dichloromethane as eluents. The desired fractions were concentrated under reduced pressure to afford diethyl (7-(2- (2-bromoethoxy)-5-chlorophenyl)thieno[3,2-b]pyridin-3-yl)phosphonate (20) as a brown solid. Yield: 3.50 g, 69 %; MS (ESI) m/z 503.90 [M+1]+.
Synthesis of diethyl (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)phosphonate (21) [0367] To a solution of 2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (7, 0.104 g, 0297 mmol) in N, N dimethylformamide (3.0 mL), potassium carbonate (0.12 g, 0.892 mmol) and diethyl (7-(2-(2-bromoethoxy)-5- chlorophenyl)thieno[3,2-b]pyridin-3-yl)phosphonate (20, 0.15 g, 0.297 mmol) were added and reaction mixture was heated at 60 °C for 16 h. After completion, reaction mixture was cooled down, poured onto ice cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to get crude product, which was purified by preparative HPLC to afford diethyl (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4- methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2- b]pyridin-3-yl)phosphonate (21) as a pale yellow solid. Yield: 0.024 g, 10 %; MS (ESI) m/z 775.07 [M+1]+.
Synthesis of (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)phosphonic acid (22)
[0368] To a solution of diethyl (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1- yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3- yl)phosphonate (21, 0.50 g, 0.64 mmol) in dichloromethane (5.0 mL) at 0 °C,
bromotrimethylsilane (0.85 mL, 6.45 mmol) is added and reaction mixture is stirred at room temperature for 16 h. After completion, the reaction mixture is concentrated to get crude product which is purified by preparative HPLC to afford (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4- methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2- b]pyridin-3-yl)phosphonic acid (22).
Synthesis of (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)(1H-imidazol-1- yl)phosphinic acid (23)
[0369] To a stirred solution of (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1- yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3- yl)phosphonic acid (22, 0.50 g, 0.69 mmol) in dry N,N dimethylformamide (5 mL), imidazole (0.23 g, 3.48 mmol), triphenyl phosphine (0.36 g, 1.38 mmol), aldrithiol (0.304 g, 1.38 mmol) and triethylamine (0.1 mL, 0.69 mmol) are added. The reaction mixture is stirred under argon atmosphere at room temperature for 15 h. The reaction mixture is cooled to 0 °C and sodium perchlorate (0.40 g) in 20 mL acetone is added and resulting solid is filtered and dried to afford (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)(1H-imidazol-1- yl)phosphinic acid (23). Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-((((((((7-(5-chloro- 2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.185F)
[0370] To a stirred solution of (7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1- yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)(1H- imidazol-1-yl)phosphinic acid (23, 0.10 g, 0.13 mmol) and triethyl ammonium salt of
((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)oxidophosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl diphosphate (16, 0.104 g, 0.104 mmol) in dry N,N dimethylformamide (10.0 mL), zinc chloride (0.070 g, 0.52 mmol) is added under argon atmosphere and the reaction mixture is stirred at room temperature for 60 h. After 60 h, the reaction mixture is added to a solution of EDTA disodium in water at 0° C. The resulting aqueous solution is adjusted to pH 5.5 and purified by preparative HPLC to afford (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-((((((((7-(5-chloro-2-(2- (5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3- yl)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4- methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.185F) as a white solid. Yield: 0.015 g, 7%; MS (ESI) m/z 1502.2 [M+1]+.
Example 2 (Cpd. No.186F)
(2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(((((((((7-(5-chloro-2-(2-(5- cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy) phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.186F)
Figure imgf000117_0001
Figure imgf000118_0001
Synthesis of 7-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thieno[3,2-b]pyridine (2) [0371] To a solution of the 3-bromo-7-chlorothieno[3,2-b]pyridine (1, 10.0 g, 40.65 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (1a, 12.39 g, 48.78 mmol) and triphenylphosphine (0.63g, 2.43 mmol) in toluene (200 mL), potassium acetate (5.98 g, 60.97 mmol) is added at room temperature under argon atmosphere and reaction mixture is purged with argon gas for 10 minutes. Then, bis(triphenylphosphine)palladium(II) dichloride (0.856 g, 1.21 mmol) is added and the reaction mixture is heated at 100 °C for 18 h. Reaction mixture is cooled down and quenched with addition of water and extracted with ethyl acetate. Ethyl acetate layer obtained is dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude compound. Crude compound obtained is column purified by flash column chromatography using silica gel (230- 400 mesh) and 0 to 40 % ethyl acetate in hexanes as eluents to afford 7-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thieno[3,2-b]pyridine (2).
Synthesis of 7-chlorothieno[3,2-b]pyridin-3-ol (3)
[0372] To a solution of 7-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thieno[3,2- b]pyridine (2, 5 g, 16.94 mmol) in dry tetrahydrofuran (50 mL), aqueous sodium hydroxide solution (2.5 M, 13.5 mL, 33.88 mmol) and hydrogen peroxide 30 percent solution in water (3.45 mL, 33.88 mmol) are added at 0 °C. The reaction mixture is stirred at room temperature for 45 min. Then, the pH of the reaction mixture is adjusted to 2 by the addition of aqueous 2 N hydrochloric acid. The mixture is extracted with dichloromethane. The organic layer is dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel (230-400 mesh) and using 30 to 70% ethyl acetate in hexanes as eluent. The desired fractions are combined and evaporated under reduced pressure to afford 7-chlorothieno[3,2-b]pyridin-3-ol (3).
Synthesis of 7-chloro-3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridine (4)
[0373] To a solution of 7-chlorothieno[3,2-b]pyridin-3-ol (3, 2.0 g, 10.86 mmol) in N,N- dimethylformamide (20 mL), imidazole (2.21 g, 32.60 mmol) and triisopropylsilyl chloride (3.43 mL, 16.29 mmol) are added at 0 °C. The reaction mixture is stirred at room temperature for 18 h. The reaction mixture is cooled down and quenched by addition of water and extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure. The residue obtained is purified by flash chromatography on silica gel (230-400 mesh) and using 30 to 50% ethyl acetate in hexanes as eluents. The desired fractions are combined and evaporated under reduced pressure to afford 7-chloro-3- ((triisopropylsilyl)oxy)thieno[3,2-b]pyridine (4).
Synthesis of 4-chloro-2-(3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridin-7-yl)phenol (5) [0374] To a solution of 7-chloro-3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridine (4, 2.00 g, 5.86 mmol) and (5-chloro-2-hydroxyphenyl)boronic acid (4a, 10.1 g, 11.72 mmol) in 1,4- dioxane (30.0 mL), 2M aqueous solution of potassium carbonate (2.02 g, 14.65 mmol) dissolved in water (10 mL) is added and the reaction mixture is degassed using argon gas for 10 min. Then, 1,1'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (0.47 g, 0.58 mmol) is added to the reaction mixture at room temperature. The reaction mixture is heated to 100 oC for 16 h. After completion, the reaction mixture is cooled to room
temperature, diluted with water and extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The crude residue obtained is purified by column chromatography using silica gel (100-200 mesh) and 30-60% ethyl acetate in hexanes to afford 4-chloro-2-(3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridin-7- yl)phenol (5) as a brownish sticky gum.
Synthesis of 7-(2-(2-bromoethoxy)-5-chlorophenyl)-3-((triisopropylsilyl)oxy)thieno[3,2- b]pyridine (6)
[0375] To a solution of 4-chloro-2-(3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridin-7-yl)phenol (5, 1.30 g, 3.00 mmol) in acetone (15.0 mL), potassium carbonate (1.24 g, 9.00 mmol) and 1,2- dibromoethane (5a, 2.58 mL, 30.0 mmol) are added and the reaction mixture is heated at 50 °C for 16 h. After completion, the reaction mixture is poured onto ice cold water and extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulphate, filtered and
concentrated to get crude product, which is purified by flash column chromatography using silica gel (230-400 mesh) and 0-50% ethyl acetate in hexane as eluents. The desired fractions are combined and concentrated under reduced pressure to afford 7-(2-(2-bromoethoxy)-5- chlorophenyl)-3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridine (6).
Synthesis of 3-(2-(4-chloro-2-(3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridin-7- yl)phenoxy)ethyl)-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (7)
[0376] To a solution of 2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (6a, 1.00 g, 2.84 mmol) in N,N-dimethylformamide (10.0 mL), potassium carbonate (0.78 g, 5.68 mmol) and 7-(2-(2-bromoethoxy)-5-chlorophenyl)-3- ((triisopropylsilyl)oxy)thieno[3,2-b]pyridine (6, 1.53 g, 2.84 mmol) are added and the reaction mixture is heated at 60 °C for 16 h. After completion, reaction mixture is cooled down, poured onto ice cold water and extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulphate, filtered and concentrated to get crude product, which is purified by flash column chromatography using silica gel (230-400 mesh) and 60-90% ethyl acetate in hexanes as eluents to afford 3-(2-(4-chloro-2-(3-((triisopropylsilyl)oxy)thieno[3,2-b]pyridin-7- yl)phenoxy)ethyl)-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (7).
Synthesis of 3-(2-(4-chloro-2-(3-hydroxythieno[3,2-b]pyridin-7-yl)phenoxy)ethyl)-2-methyl-6- (4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5-carbonitrile (8)
[0377] To a stirred solution of 3-(2-(4-chloro-2-(3-((triisopropylsilyl)oxy)thieno[3,2- b]pyridin-7-yl)phenoxy)ethyl)-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)- 3,4-dihydroquinazoline-5-carbonitrile (7, 1.00 g, 1.23 mmol) in dry tetrahydrofuran (20.0 mL) at 0 °C, 1 M solution of tetra-n-butylammonium fluoride in tetrahydrofuran (1.85 mL, 1.85 mmol) is added and the reaction mixture is stirred at room temperature for 5 h. After completion, the reaction mixture is diluted with water and extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulphate, filtered and concentrated to get crude product, which is purified by flash column chromatography using silica gel (230-400 mesh) and 2-5 % methanol in dichloromethane as eluents to afford 3-(2-(4-chloro-2-(3-hydroxythieno[3,2-b]pyridin-7- yl)phenoxy)ethyl)-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4- dihydroquinazoline-5-carbonitrile (8).
Synthesis of 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl diethyl phosphate (9)
[0378] To a solution of 3-(2-(4-chloro-2-(3-hydroxythieno[3,2-b]pyridin-7-yl)phenoxy)ethyl)- 2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)-3,4-dihydroquinazoline-5- carbonitrile (8, 0.30 g, 0.45 mmol) ) and diethyl phosphorochloridate (8a, 0.092 g, 0.54 mmol) in anhydrous dichloromethane (15 mL), triethylamine (0.13 mL, 0.90 mmol) is added and the reaction mixture is stirred at room temperature for 5 h. After completion, the reaction mixture is concentrated under reduced pressure and purified by flash chromatography using silica gel (230- 400 mesh) and 40-70% ethyl acetate in hexanes as eluents to afford 7-(5-chloro-2-(2-(5-cyano-2- methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl diethyl phosphate (9).
Synthesis of 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl dihydrogen phosphate (10)
[0379] To a solution 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl diethyl phosphate (9, 0.20 g, 0.25 mmol) in dichloromethane (3.0 mL), bromotrimethylsilane (0.33 mL, 2.50 mmol) is added at 0 °C. The reaction mixture is stirred at room temperature for 16 h. After completion, the reaction mixture is concentrated to get crude product which is purified by preparative HPLC to afford 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4- oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl dihydrogen phosphate (10).
Synthesis of 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl hydrogen (1H- imidazol-1-yl)phosphonate (11)
[0380] To a stirred solution of 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1- yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl dihydrogen phosphate (10, 0.050 g, 0.069 mmol) in dry N,N-dimethylformamide (1 mL), imidazole (0.023 g, 0.35 mmol), triphenyl phosphine (0.036 g, 0.138 mmol), aldrithiol (0.0304 g, 0.138 mmol) and triethylamine (0.01 mL, 0.069 mmol) are added. The reaction mixture is stirred under argon atmosphere at room temperature for 15 h. After this time, the reaction mixture is cooled to 0 °C. Then, sodium perchlorate in acetone is added and the resulting solid is filtered and dried to afford 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4- oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl hydrogen (1H-imidazol-1-yl)phosphonate (11).
Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(((((((((7-(5-chloro- 2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)
phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl
(((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methyl) phosphate (Cpd. No.186F)
[0381] To a stirred solution of 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1- yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl hydrogen (1H-imidazol-1-yl)phosphonate (11, 0.10 g, 0.127 mmol) and triethyl ammonium salt of ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)oxidophosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl diphosphate (11a, 0.102 g, 0.102 mmol) in dry N,N-dimethylformamide (10.0 mL), zinc chloride (0.069 g, 0.51 mmol) is added under argon atmosphere and the reaction mixture is stirred at room temperature for 60 h. After 60 h, the reaction mixture is added to a solution of ethylenediaminetetraacetic acid disodium salt in water at 0 °C. The resulting aqueous solution is adjusted to pH 5.5 and purified by preparative HPLC to afford (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H- purin-9-yl)-2-(((((((((7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7- (trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3- yl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)- 4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.186F).
Example 3 (Cpd. No.187F)
(2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(((((((7-(5-chloro-2-(2-(5- cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)oxy)(hydroxy)phosphoryl)oxy)
(hydroxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2- amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.187F)
Figure imgf000123_0001
Figure imgf000124_0001
Synthesis of (2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(((((((7-(5-chloro- 2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)- yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)
phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6- dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.187F)
[0382] To a solution of 7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4-methylpiperazin-1-yl)-4-oxo- 7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2-b]pyridin-3-yl hydrogen (1H- imidazol-1-yl)phosphonate (1, 0.10 g, 0.127 mmol) and triethyl ammonium salt of
((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino- 6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)oxidophosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl hydrogen phosphate (1a, 0.084 g, 0.102 mmol) in dry N,N-dimethylformamide (5.0 mL), zinc chloride (0.069 g, 0.51 mmol) is added under argon atmosphere and the reaction mixture is stirred at room temperature for 60 h. After 60 h, the reaction mixture is added to a solution of ethylenediaminetetraacetic acid disodium salt (0.36 g, 0.970 mmol) in water at 0 °C. The resulting aqueous solution is adjusted to pH 5.5 and purified by preparative HPLC to afford (2R,3R,4R,5R)-5-(2-amino-6- oxo-1,6-dihydro-9H-purin-9-yl)-2-(((((((7-(5-chloro-2-(2-(5-cyano-2-methyl-6-(4- methylpiperazin-1-yl)-4-oxo-7-(trifluoromethyl)quinazolin-3(4H)-yl)ethoxy)phenyl)thieno[3,2- b]pyridin-3-yl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4- methoxytetrahydrofuran-3-yl (((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)- 3,4-dihydroxytetrahydrofuran-2-yl)methyl) phosphate (Cpd. No.187F).
Example 188
Co-Transcriptional Synthesis of 5’ Capped Firefly Luciferase (Fluc) mRNA by In Vitro
Transcription
[0383] Mono- and bicistronic reporter mRNAs containing a 5’ CAP1 mimetic and/or a cap- independent translation element were generated using translational enhancer Compound 185F. A DNA template consisting of a T7 RNA polymerase reporter upstream of the Renilla or Firefly luciferase (Fluc) open reading frame (ORF) was generated with and without a downstream, cap- independent cistron composed of the opposite luciferase ORF. The following components were added in the order specified:
Figure imgf000125_0001
[0384] The reaction was incubated at 37oC for 2 hours to generate a 5’ CAP1 Fluc mRNA (FIG.1). As shown in FIG.1, T7 RNA polymerase is recruited to the template DNA via the specific promoter sequence. Once bound, the polymerase will incorporate nucleotides that base pair with the template DNA. The sequence near the transcription initiation site can influence the efficiency of RNA production with a GG or AG dinucleotide yielding the best results.
[0385] The transcription reaction was carried out to completion. The transcription reaction containing the 5’ CAP1 Fluc mRNA was directly added to a cell-free translation extract, incubated at 37oC for an additional 30 minutes, leading to the synthesis of the Firefly luciferase protein from the mRNA. Luciferase protein expression was directly assayed by the measuring the light signal in the reaction mixture. [0386] Additionally, purified mRNAs containing either a 5’ ARCA or“compound 185F” cap and a cap-independent control reporter were delivered into HEK-293t cells by lipid mediated RNA transfection. Cells were cultured for 4-8 hours under normal conditions. Subsequently, cells were washed and immediatedly lysed in luciferase assay buffer. Following a 15 minute incubation at room temperature, the Firefly luciferase (Fluc) signal was read on a luminometer. Then, the FLuc signal was quenched prior to reading the Renilla luciferase signal. The ratio of the Renilla:Firefly luciferase signal will be calculated for each reporter (ARCA and compound 185F derived, and compared to each other to determine the fold increase in translation of the compound 185F derived reporter over the ARCA reporter. Alternatively, 5’ARCA and compound 185F capped reporter signal is read as above and normalized to the amount of reporter RNA present in the cells as measured by reverse-transcription followed by quantitative- PCR.
[0387] Although the above example demonstrates the co-transcriptional synthesis of Fluc mRNA using the novel translational enhancer, compound 185F, the method disclosed herein is applicable to any translational enhancer of the invention and any RNA molecule, and can therefore be used to generate any RNA molecule of the invention.
[0388] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further
embodiments.
[0389] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS What is claimed is: 1. A translational enhancer comprising an eukaryotic initiation factor 4E (eIF4E) ligand attached to at least one nucleotide.
2. The translational enhancer of claim 1, wherein the eIF4E ligand is attached to the at least one nucleotide via a linker.
3. The translational enhancer of claim 2, wherein the translational enhancer has a structure:
Figure imgf000127_0001
,
wherein X is a linker and Y is a dinucleotide, wherein Y is selected from the group consisting of an AA dinucleotide, an AG dinucleotide, a GA dinucleotide, and a GG dinucleotide.
4. The translational enhancer of claim 3, wherein Y is a GG dinucleotide.
5. The translational enhancer of claim 3, wherein X is a phosphate linker.
6. The translational enhancer of claim 3, wherein X is a phosphonate-diphosphate linker.
7. The translational enhancer of claim 3, wherein the eIF4E ligand has a structure according to Formula I, II, II, IV, V or VI.
8. The translational enhancer of claim 7, wherein the eIF4E ligand has a structure according to Formula I.
9. The translational enhancer of claim 8, wherein the translational enhancer has a structure:
Figure imgf000128_0001
.
10. An RNA molecule comprising a translational enhancer, wherein the translational
enhancer comprises an eIF4E ligand attached to at least one nucleotide.
11. The RNA molecule of claim 10, wherein the eIF4E ligand is attached to the at least one nucleotide via a linker.
12. The RNA molecule of claim 10, wherein the translational enhancer is attached to the 5’ end of the RNA molecule.
13. The RNA molecule of claim 12, wherein the translational enhancer functions as a 5’ cap structure.
14. The RNA molecule of claim 10, wherein the RNA molecule is a single-stranded RNA molecule.
15. The RNA molecule of claim 14, wherein the single-stranded RNA molecule is a
messenger RNA (mRNA) molecule.
16. The RNA molecule of claim 15, wherein the RNA molecule encodes a therapeutic protein.
17. The RNA molecule of claim 10, wherein RNA molecule exhibits increased cell
permeability compared to an RNA molecule not comprising the translational enhancer.
18. The RNA molecule of claim 10, wherein the RNA molecule is resistant to activity of one or more de-capping enzymes selected from the group consisting of dcp1 and dcp2.
19. The RNA molecule of claim 10, wherein the RNA has enhanced resistance to degradation by one or more cellular 5’-3’exonucleases compared to an RNA molecule not comprising the translational enhancer.
20. The RNA molecule of claim 10, wherein the RNA molecule has a half-life in a cellular environment that is at least 1.2 times of that of an RNA molecule not comprising the translational enhancer.
21. The RNA molecule of claim 10, wherein, when administered to a subject, the RNA
molecule has a half-life that is at least 1.2 times of that of an RNA molecule not comprising the translational enhancer.
22. The RNA molecule of claim 10, wherein the RNA molecule does not activate one or more innate immune sensors when administered to a subject, wherein the one or more innate immune sensors are selected from the group consisting of RIG-I, MDA5, IFIT-1, protein kinase R (PKR), Toll-like receptor 3 (TLR 3), TLR 7, and TLR 8.
23. The RNA molecule of claim 10, wherein the RNA molecule is translated with greater efficiency compared to an RNA molecule not comprising the translational enhancer.
24. The RNA molecule of claim 10, wherein the RNA molecule induces an attenuated
immune response when administered to a subject compared to an RNA molecule not comprising the translational enhancer.
25. A method of making a capped RNA molecule comprising:
a. reacting a polynucleotide template having a sequence complementary to the RNA molecule in the presence of an RNA polymerase and under conditions conducive to transcription by the RNA polymerase to generate at least one RNA molecule from the polynucleotide template; and
b. co-transcriptionally coupling to a 5’ end of the at least one RNA molecule the translational enhancer of claim 1.
26. A method of treating, preventing, or ameliorating a disease in a subject comprising administering to the subject a therapeutically effective amount of an RNA molecule of claim 10.
27. The method of claim 26, wherein the disease is selected from a group consisting of hyperproliferative disease, inflammatory disease, viral infection, cardiovascular disease, genetic disease, and autoimmune disease.
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