WO2024119117A1 - Coiffe en 5' d'arnm modifiée et ses méthodes d'utilisation - Google Patents

Coiffe en 5' d'arnm modifiée et ses méthodes d'utilisation Download PDF

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WO2024119117A1
WO2024119117A1 PCT/US2023/082144 US2023082144W WO2024119117A1 WO 2024119117 A1 WO2024119117 A1 WO 2024119117A1 US 2023082144 W US2023082144 W US 2023082144W WO 2024119117 A1 WO2024119117 A1 WO 2024119117A1
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Prior art keywords
substituted
heterocycloalkyl
cycloalkyl
fluoro
independently
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PCT/US2023/082144
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English (en)
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Umair JAVED
Ekambareswara Rao KANDIMALLA
Mallikarjuna Reddy Putta
Seth Alexander
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Prime Medicine, Inc.
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Publication of WO2024119117A1 publication Critical patent/WO2024119117A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds

Definitions

  • capping of 5’-end of mRNA is an essential structural modification that allows efficient mRNA translation, mitigates immune response, and limits degradation by cellular exonucleases.
  • the 5’-cap of the eukaryotic mRNA has a unique structure that is recognized by polymerases for in-vitro transcription. Distinguishing features of the mRNA 5’-cap help eukaryotic initiation factors (eIFs) and cap binding proteins (CBP) to recruit translation machinery.
  • eIFs eukaryotic initiation factors
  • CBP cap binding proteins
  • the 5’-cap also protects mRNA from degradation by exonucleases stabilizing the mRNA and increasing their half-lives and facilitates nuclear export of mRNA into cytoplasm where translation takes place.
  • the present disclosure provides compounds of Formula (I): or a stereoisomer, tautomer, solvate, or salt thereof, wherein: R 1 is H, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted with 1-3 groups of me
  • R 9 is -H or -CH3; each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ; R 20 is independently selected at each instance from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl; each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil; and B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified , modified , with the proviso that when R 1 is
  • the present disclosure provides a compound selected from: or a stereoisomer, tautomer, solvate, or salt thereof.
  • the present disclosure provides a messenger RNA (mRNA) sequence comprising a 5’-end motif of Formula (III):
  • R 1 is H, C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently at each instance hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified cytosine
  • the present disclosure provides cells comprising a ribonucleic acid (RNA) molecule comprising the mRNA sequence disclosed herein.
  • RNA ribonucleic acid
  • the present disclosure provides cells comprising a polypeptide or protein translated from an RNA molecule comprising the mRNA sequence disclosed herein.
  • the present disclosure provides pharmaceutical compositions comprising an RNA molecule comprising the mRNA sequence disclosed herein.
  • the present disclosure provides methods for synthesizing a ribonucleic acid (RNA) molecule comprising: (a) introducing the mRNA sequence disclosed herein into a mixture comprising an RNA polymerase, and (b) incubating the mixture for a time sufficient to allow for transcription of the RNA molecule.
  • the present disclosure provides methods of gene editing comprising introducing into a cell a ribonucleic acid (RNA) molecule comprising the mRNA sequence disclosed herein, or a pharmaceutical composition disclosed herein, wherein the RNA molecule encodes a therapeutic protein, wherein the RNA molecule is translated in the cell.
  • the present disclosure provides vectors comprising the mRNA sequence disclosed herein.
  • the present disclosure provides ribonucleic acid (RNA) molecules comprising the mRNA sequence disclosed herein, wherein the RNA molecule has a half-life that is at least 1.2 times of that of a corresponding natural RNA molecule in a cellular environment.
  • RNA ribonucleic acid
  • the present disclosure provides methods of producing a protein in a cell, comprising: (a) introducing a modified messenger ribonucleic acid (mRNA) molecule into a cell; and (b) translating the mRNA molecule in the cell to make a protein; wherein the modified mRNA molecule comprises the mRNA sequence disclosed herein, and wherein: (i) the mRNA molecule has a translation efficiency of at least 1.2-fold of that of a corresponding unmodified mRNA molecule, or; (ii) the mRNA molecule persists in the cell at least 20% longer than the corresponding unmodified mRNA molecule, or; (iii) the mRNA molecule produces the protein at least 20% more than the corresponding unmodified mRNA molecule, or; (iv) the mRNA molecule induces an innate immune response at a frequency of at least 20% less than the corresponding unmodified mRNA molecule, or; (v) the mRNA molecule has a
  • the present disclosure provides the mRNA sequence disclosed herein for use as a medicament.
  • the present disclosure provides the vector comprising the mRNA sequence disclosed herein for use as a medicament.
  • the present disclosure provides ribonucleic acid (RNA) molecule comprising the mRNA sequence disclosed herein for use as a medicament.
  • FIG.1A shows an LCMS spectrum of compound PM001_1.
  • FIG.1B shows a proton NMR spectrum of compound PM001_1.
  • FIG.2A shows an LCMS spectrum of compound PM001.
  • FIG.2B shows a proton NMR spectrum of compound PM001.
  • FIG.3A shows an LCMS spectrum of compound PM001_N7_Me.
  • FIG.3B shows a proton NMR spectrum of compound PM001_N7_Me.
  • FIG.4 shows an LCMS spectrum of compound mCAP005.
  • FIG.5A shows an LCMS spectrum of compound PM002_1.
  • FIG.5B shows a proton NMR spectrum of compound PM002_1.
  • FIG.6A shows an LCMS spectrum of compound PM002.
  • FIG.6B shows a proton NMR spectrum of compound PM002.
  • FIG.7A shows an LCMS spectrum of compound PM002_N7_Me.
  • FIG.7B shows a proton NMR spectrum of compound PM002_N7_Me.
  • FIG.8 shows an LCMS spectrum of compound mCAP006.
  • FIG.9 shows an LCMS spectrum of compound mCAP007.
  • FIG.10 shows an LCMS spectrum of compound mCAP008.
  • DETAILED DESCRIPTION OF THE DISCLOSURE [0035]
  • the mRNA capped with a chemically modified 5’-cap analogs provided herein may exhibit or produce enhanced translation efficiency, persistence within a cell, protein production, innate immune response frequency, gene transfer rate, degradation suppression, or any combination thereof.
  • the mRNA capped with a chemically modified 5’-cap analogs provided herein may exhibit or produce reduced immune response compared to an mRNA without a chemically modified 5’-cap analog.
  • references to “some embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a particular feature or characteristic described in connection with the embodiments is included in at least one or more embodiments, but not necessarily all embodiments, of the present disclosure.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value.
  • a “cell” can generally refer to a biological cell.
  • a cell can be the basic structural, functional and/or biological unit of a living organism.
  • a cell can originate from any organism having one or more cells.
  • Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an animal cell, a cell from an invertebrate animal (e.g.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a cell may not originate from a natural organism (e.g., a cell can be synthetically made, sometimes termed an artificial cell).
  • the cell is a human cell.
  • a cell can be of or derived from different tissues, organs, and/or cell types.
  • the cell is a primary cell.
  • the term “primary cell” means a cell isolated from an organism, e.g., a mammal, which is grown in tissue culture (i.e., in vitro) for the first time before subdivision and transfer to a subculture.
  • the cell is a stem cell.
  • mammalian cells, including primary cells and stem cells can be modified through introduction of one or more polynucleotides, polypeptides, and/or prime editing compositions (e.g., through transfection, transduction, electroporation, and the like) and further passaged.
  • Such modified cells include muscle cells (e.g., cardiac muscle cells, smooth muscle cells, hepatocytes), hematopoietic stem cells (HSCs), hematopoietic stem progenitor cells (HSPC)s, fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), muscle cells and precursors of these somatic cell types.
  • the cell is a stem cell.
  • the cell is a progenitor cell.
  • the cell is a pluripotent cell (e.g., a pluripotent stem cell)
  • the cell e.g., a stem cell
  • the cell is an embryonic stem cell, tissue-specific stem cell, mesenchymal stem cell, or an induced pluripotent stem cell.
  • the cell is an induced pluripotent stem cell (iPSC).
  • the cell is an embryonic stem cell (ESC).
  • the cell is a human stem cell.
  • the cell is a human pluripotent stem cell.
  • the cell is a human fibroblast.
  • the cell is an induced human pluripotent stem cell.
  • the cell is a human stem cell. In some embodiments, the cell is a human embryonic stem cell. [0049] In some embodiments, the cell is a CD34 + cell. In some embodiments, the cell is a human CD34 + cell. In some embodiments, the cell is a hematopoietic stem cell (HSC). In some embodiments, the cell is a hematopoietic progenitor cell (HPC). In some embodiments, hematopoietic stem cells and hematopoietic progenitor cells are referred to as hematopoietic stem or progenitor cells (HSPCs). In some embodiments, the cell is a human HSC.
  • the cell is a human HPC. In some embodiments, the cell is a human HSPC. In some embodiments, the cell is a long term (LT)-HSC. In some embodiments, the cell is a short- term (ST)-HSC. In some embodiments, the cell is a myeloid progenitor cell. In some embodiments, the cell is a lymphoid progenitor cell. In some embodiments, the cell is a granulocyte monocyte progenitor cell. In some embodiments, the cell is a megakaryocyte erythroid progenitor cell. In some embodiments, the cell is a multipotent progenitor cell (MPP).
  • MPP multipotent progenitor cell
  • the cell is a hematopoietic stem cell (HSC) or a hematopoietic stem and progenitor cell.
  • HSC hematopoietic stem cell
  • the HSC is from bone marrow or mobilized peripheral blood.
  • the cell is a hematopoietic progenitor cell, multipotent progenitor cell, lymphoid progenitor cell, a myeloid progenitor cell, a megakaryocyte-erythroid progenitor cell, a granulocyte-megakaryocyte progenitor cell, a granulocyte, a promyelocyte, a neutrophil, an eosinophil, a basophil, an erythrocyte, a reticulocyte, a thrombocyte, a megakaryoblast, a platelet-producing megakaryocyte, a monocyte, a macrophage, a dendritic cell, a microglia, an osteoclast,
  • the cell edited by prime editing can be differentiated into, or give rise to recovery of a population of cells, e.g., common lymphoid progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, granulocyte-megakaryocyte progenitor cells, granulocytes, promyelocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, thrombocytes, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, lymphocytes, such as NK cells, B-cells or T-cells.
  • a population of cells e.g., common lymphoid progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, granulocyte-megakaryocyte progen
  • the cell edited by prime editing can be differentiated into or give rise to recovery of a population of cells, e.g., neutrophils, platelets, red blood cells, monocytes, macrophages, antigen-presenting cells, microglia, osteoclasts, dendritic cells, inner ear cell, inner ear support cell, cochlear cell and/or lymphocytes.
  • the cell is in a subject, e.g., a human subject.
  • a cell is not isolated from an organism but forms part of a tissue or organ of an organism, e.g., a mammal.
  • mammalian cells include formed elements of the blood (e.g., lymphocytes, bone marrow cells), precursors of any of these somatic cell types, and stem cells.
  • the cell is a sensory ciliated cell, e.g., a human sensory ciliated cell.
  • the cell is a retinal cell, e.g., a human retinal cell.
  • the cell is a rod cell, e.g., a human rod cell.
  • the cell is a cone cell, e.g., a human cone cell.
  • the cell is a hair cell, e.g., a human hair cell.
  • a cell is isolated from an organism. In some embodiments, a cell is derived from an organism. In some embodiments, a cell is a differentiated cell. In some embodiments, the cell is a fibroblast. In some embodiments, the cell is differentiated from an induced pluripotent stem cell. In some embodiments, the cell is differentiated from an HSC or an HPSC. In some embodiments, the cell is differentiated from an induced pluripotent stem cell (iPSC). In some embodiments, the cell is differentiated from an embryonic stem cell (ESC). [0052] In some embodiments, the cell is a differentiated human cell. In some embodiments, cell is a human fibroblast.
  • the cell is differentiated from an induced human pluripotent stem cell. In some embodiments, the cell is differentiated from a human iPSC or a human ESC. [0053] In some embodiments, the cell comprises a prime editor, a prime editing guide RNA (PEgRNA), or a prime editing composition disclosed herein. In some embodiments, the cell further comprises a nicking single guide RNA (ngRNA). In some embodiments, the cell is from a human subject. In some embodiments, the human subject has a disease or condition, or is at a risk of developing a disease or a condition associated with a mutation to be corrected by prime editing, for example, non-syndromic retinitis pigmentosa (nsRP) or Usher syndrome.
  • prime editing for example, non-syndromic retinitis pigmentosa (nsRP) or Usher syndrome.
  • the cell is from a human subject, and comprises a prime editor or a prime editing composition for correction of the mutation. In some embodiments, the cell is from the human subject and the mutation has been edited or corrected by prime editing. In some embodiments, the cell is in a human subject, and comprises a prime editor, a PEgRNA, or a prime editing composition for correction of the mutation. In some embodiments, the cell is from the human subject and the mutation has been edited or corrected by prime editing. In some embodiments, the cell is in a subject, e.g., a human subject. In some embodiments, the cell is obtained from a subject prior to editing. [0054] The term “substantially” as used herein can refer to a value approaching 100% of a given value.
  • the term can refer to an amount that can be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term can refer to an amount that can be about 100% of a total amount.
  • protein and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three-dimensional conformation.
  • a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined by covalent bonds (e.g., amide bonds). In some embodiments, a protein comprises at least two amide bonds. In some embodiments, a protein comprises multiple amide bonds. In some embodiments, a protein comprises an enzyme, enzyme precursor proteins, regulatory protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody. In some embodiments, a protein can be a full-length protein (e.g., a fully processed protein having certain biological function).
  • a protein can be a variant or a fragment of a full-length protein.
  • a Cas9 protein domain comprises an H840A amino acid substitution compared to a naturally occurring S. pyogenes Cas9 protein.
  • a Cas9 protein domain comprises an H839A amino acid substitution compared to a naturally occurring S. pyogenes Cas9 protein lacking a N-terminal methionine.
  • a variant of a protein or enzyme for example a variant reverse transcriptase, comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein.
  • a protein comprises one or more protein domains or subdomains.
  • polypeptide domain when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function.
  • a protein comprises multiple protein domains.
  • a protein comprises multiple protein domains that are naturally occurring.
  • a protein comprises multiple protein domains from different naturally occurring proteins.
  • a prime editor can be a fusion protein comprising a Cas9 protein domain of S.
  • a protein comprises a functional variant or functional fragment of a full-length wild type protein.
  • a “functional fragment” or “functional portion”, as used herein, refers to any portion of a reference protein (e.g., a wild type protein) that encompasses less than the entire amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions.
  • a functional fragment of a reverse transcriptase can encompass less than the entire amino acid sequence of a wild type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide.
  • a functional fragment thereof can retain one or more of the functions of at least one of the functional domains.
  • a functional fragment of a Cas9 can encompass less than the entire amino acid sequence of a wild type Cas9, but retains its DNA binding ability and lacks its nuclease activity partially or completely.
  • a “functional variant” or “functional mutant”, as used herein, refers to any variant or mutant of a reference protein (e.g., a wild type protein) that encompasses one or more alterations to the amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions.
  • the one or more alterations to the amino acid sequence comprises amino acid substitutions, insertions or deletions, or any combination thereof.
  • the one or more alterations to the amino acid sequence comprises amino acid substitutions.
  • a functional variant of a reverse transcriptase can comprise one or more amino acid substitutions compared to the amino acid sequence of a wild type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide.
  • the reference protein is a fusion of multiple functional domains
  • a functional variant thereof can retain one or more of the functions of at least one of the functional domains.
  • a functional fragment of a Cas9 can comprise one or more amino acid substitutions in a nuclease domain, e.g., a H840A amino acid substitution, compared to the amino acid sequence of a wild type Cas9, but retains the DNA binding ability and lacks the nuclease activity partially or completely.
  • the term “function” and its grammatical equivalents as used herein refer to a capability of operating, having, or serving an intended purpose. Functional can comprise any percent from baseline to 100% of an intended purpose.
  • functional can comprise or comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of an intended purpose.
  • the term functional can mean over or over about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose.
  • a protein or polypeptides includes naturally occurring amino acids (e.g., one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).
  • a protein or polypeptides includes non-naturally occurring amino acids (e.g., amino acids which is not one of the twenty amino acids commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics).
  • a protein or polypeptide is modified.
  • a protein comprises an isolated polypeptide.
  • isolated means free or removed to varying degrees from components which normally accompany it as found in the natural state or environment. For example, a polypeptide naturally present in a living animal is not isolated, and the same polypeptide partially or completely separated from the coexisting materials of its natural state is isolated.
  • a protein is present within a cell, a tissue, an organ, or a virus particle.
  • a protein is present within a cell or a part of a cell (e.g., a bacteria cell, a plant cell, or an animal cell).
  • the cell is in a tissue, in a subject, or in a cell culture.
  • the cell is a microorganism (e.g., a bacterium, fungus, protozoan, or virus).
  • a protein is present in a mixture of analytes (e.g., a lysate).
  • the protein is present in a lysate from a plurality of cells or from a lysate of a single cell.
  • the terms “homologous,” “homology,” or “percent homology” as used herein refer to the degree of sequence identity between an amino acid and a corresponding reference amino acid sequence, or a polynucleotide sequence and a corresponding reference polynucleotide sequence.
  • Homology can refer to polymeric sequences, e.g., polypeptide or DNA sequences that are similar. Homology can mean, for example, nucleic acid sequences with at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In other embodiments, a “homologous sequence” of nucleic acid sequences can exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid sequence.
  • a “region of homology to a genomic region” can be a region of DNA that has a similar sequence to a given genomic region in the genome.
  • a region of homology can be of any length that is sufficient to promote binding of a spacer, a primer binding site, or a protospacer sequence to the genomic region.
  • the region of homology can comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 or more bases in length such that the region of homology has sufficient homology to undergo binding with the corresponding genomic region.
  • sequence homology or identity when a percentage of sequence homology or identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of two or more sequences across a portion of their length when compared and aligned for maximum correspondence. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. Unless stated otherwise, sequence homology or identity is assessed over the specified length of the nucleic acid, polypeptide or portion thereof. In some embodiments, the homology or identity is assessed over a functional portion or specified portion of the length.
  • Alignment of sequences for assessment of sequence homology can be conducted by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, which is described in Altschul et al, J. Mol. Biol.215:403- 410, 1990.
  • BLAST Basic Local Alignment Search Tool
  • a publicly available, internet interface, for performing BLAST analyses is accessible through the National Center for Biotechnology Information. Additional known algorithms include those published in: Smith & Waterman, “Comparison of Biosequences”, Adv. Appl. Math.2:482, 1981; Needleman & Wunsch, “A general method applicable to the search for similarities in the amino acid sequence of two proteins” J. Mol.
  • Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/) which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000; 16: 276-277), and the GGSEARCH program https://fasta.bioch.virginia.edu/fasta_www2/, which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length.
  • amino acid (or nucleotide) positions can be determined in homologous sequences based on alignment, for example, “H840” in a reference SpCas9 sequence can correspond to “H839” where a variant SpCas9 sequence omits the N- terminal Methionine, or another corresponding position in a Cas9 homolog when the Cas9 homolog is aligned against the reference SpCas9 sequence.
  • the term “homolog” as used herein refers to a gene or a protein that is related to another gene or protein by a common ancestral DNA sequence. A homolog can be an ortholog or a paralog.
  • An ortholog refers to a gene or protein that is related to another gene or protein by a speciation event.
  • a paralog refers to a gene or protein that is related to another gene or protein by a duplication event within a genome.
  • a paralog may be within the same species of the gene or protein it is related to.
  • a paralog may also be in a different species of the gene or protein it is related to.
  • an ortholog may retain the same function.
  • a paralog may evolve a new function.
  • the term “polynucleotide” or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules.
  • a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA.
  • a polynucleotide is double-stranded, e.g., a double- stranded DNA in a gene.
  • a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA.
  • a polynucleotide is a cell-free nucleic acid molecule.
  • a polynucleotide circulates in blood.
  • a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.
  • the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions.
  • Polynucleotides can have any three-dimensional structure.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, single guide RNA (sgRNA), guide RNA, a nucleic acid probe, a primer, a small nuclear RNA (snRNA), a long non-coding RNA, a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a tRNA- derived small RNA (tsRNA), an antisense RNA, a small hairpin RNA (
  • a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof.
  • a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • the polynucleotide can comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G).
  • a polynucleotide can be modified.
  • the terms “modified”, or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides.
  • modifications can be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide.
  • the modification can be on the internucleoside linkage (e.g., phosphate backbone).
  • multiple modifications are included in the modified nucleic acid molecule.
  • a single modification is included in the modified nucleic acid molecule.
  • the term “gene” refers to a nucleic acid sequence that encodes a gene editing agent or phenotype-altering protein.
  • Gene may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites.
  • Nucleic acid sequences encoding the gene editing agent or phenotype-altering protein can be RNA, e.g., mRNA that directs the expression of the gene editing agent or phenotype-altering protein.
  • nucleic acid sequences include RNA sequences, e.g., mRNA sequences that are translated, in some embodiments, into protein.
  • the nucleic acid sequences include both the full-length nucleic acid sequences as well as non- full-length sequences derived from the full-length protein.
  • the sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific lymphocyte.
  • Gene sequences to encode gene editing agents or phenotype-altering proteins disclosed herein are available in publicly available databases and publications.
  • complementary refers to the ability of two polynucleotide molecules to base pair with each other.
  • Complementary polynucleotides can base pair via hydrogen bonding, which can be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • hydrogen bonding can be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • an adenine on one polynucleotide molecule will base pair to a thymine or uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to a guanine on a second polynucleotide molecule.
  • Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence.
  • the two DNA molecules 5’-ATGC-3’ and 5’-GCAT-3’ are complementary, and the complement of the DNA molecule 5’-ATGC-3’ is 5’-GCAT-3’.
  • a percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).
  • Perfectly complementary means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule. “Substantially complementary” as used herein refers to a degree of complementarity that can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules. In some embodiments, the portion of complementarity can be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides.
  • “Substantial complementary” can also refer to a 100% complementarity over a portion or region of two polynucleotide molecules.
  • the portion or region of complementarity between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which polynucleotides, e.g., the transcribed mRNA, translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of the protein encoded by the gene after transcription and translation of the gene.
  • expression of a polynucleotide is determined by the amount of a functional form of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a gene is determined by the amount of the mRNA, or transcript that is encoded by the gene after transcription the gene. In some embodiments, expression of a polynucleotide, e.g., an mRNA, is determined by the amount of the protein encoded by the mRNA after translation of the mRNA.
  • expression of a polynucleotide is determined by the amount of a functional form of the protein encoded by the polypeptide after translation of the polynucleotide.
  • sampling can comprise capillary sequencing, bisulfite- free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE- sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, or any combination thereof.
  • encode refers to a polynucleotide which is said to “encode” another polynucleotide, a polypeptide, or an amino acid if, in its native state or when manipulated by methods well known to those skilled in the art, it can be used as polynucleotide synthesis template, e.g., transcribed into an RNA, reverse transcribed into a DNA or cDNA, and/or translated to produce an amino acid, or a polypeptide or fragment thereof.
  • a polynucleotide comprising three contiguous nucleotides form a codon that encodes a specific amino acid.
  • a polynucleotide comprises one or more codons that encode a polypeptide.
  • a polynucleotide comprising one or more codons comprises a mutation in a codon compared to a wild-type reference polynucleotide.
  • the mutation in the codon encodes an amino acid substitution in a polypeptide encoded by the polynucleotide as compared to a wild-type reference polypeptide.
  • mutation refers to a change and/or alteration in an amino acid sequence of a protein or a nucleic acid sequence of a polynucleotide. Such changes and/or alterations can comprise the substitution, insertion, deletion and/or truncation of one or more amino acids, in the case of an amino acid sequence, and/or nucleotides, in the case of nucleic acid sequence, compared to a reference amino acid or a reference nucleic acid sequence.
  • the reference sequence is a wild-type sequence.
  • a mutation in a nucleic acid sequence of a polynucleotide encodes a mutation in the amino acid sequence of a polypeptide.
  • the mutation in the amino acid sequence of the polypeptide or the mutation in the nucleic acid sequence of the polynucleotide is a mutation associated with a disease state.
  • the term “subject” and its grammatical equivalents as used herein can refer to a human or a non-human.
  • a subject can be a mammal.
  • a human subject can be male or female.
  • a human subject can be of any age.
  • a subject can be a human embryo.
  • a human subject can be a newborn, an infant, a child, an adolescent, or an adult.
  • a human subject can be in need of treatment for a genetic disease or disorder.
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • treatment or “treating” and their grammatical equivalents refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder.
  • Treatment can include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder.
  • Treatment can include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder.
  • this treatment can include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder.
  • Treatment can include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder.
  • a condition can be pathological.
  • a treatment may not completely cure or prevent a disease, condition, or disorder.
  • a treatment ameliorates a disease, condition, or disorder.
  • a subject can be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of the subject.
  • the term “ameliorate” and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • the terms “prevent” or “preventing” means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevent also means reducing risk of developing a disease, disorder, or condition. Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder.
  • a composition prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of a subject.
  • effective amount or “therapeutically effective amount” refers to a quantity of a composition, for example, a prime editing composition comprising a construct that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein.
  • An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is ex vivo or in vivo.
  • An effective amount can be the amount to induce, for example, at least about a 2-fold change (increase or decrease) or more in the amount of target nucleic acid modulation (e.g., expression of a gene to produce functional a protein) observed relative to a negative control.
  • An effective amount or dose can induce, for example, about 2-fold increase, about 3-fold increase, about 4-fold increase, about 5-fold increase, about 6-fold increase, about 7-fold increase, about 8-fold increase, about 9-fold increase, about 10-fold increase, about 25-fold increase, about 50-fold increase, about 100-fold increase, about 200-fold increase, about 500-fold increase, about 700-fold increase, about 1000-fold increase, about 5000-fold increase, or about 10,000-fold increase in target gene modulation (e.g., expression of a target gene to produce a functional protein).
  • target gene modulation e.g., expression of a target gene to produce a functional protein.
  • the “effective amount” or “therapeutically effective amount” is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient. In some embodiments, an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). [0087] An effective amount can be the amount to induce, when administered to a population of cells, a certain percentage of the population of cells to have a correction a mutation.
  • an effective amount can be the amount to induce, when administered to or introduced to a population of cells, installation of one or more intended nucleotide edits that correct a mutation in the target gene, in at least about 1%, 2%, 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the population of cells.
  • RT reverse transcriptase
  • RT refers to a class of enzymes that synthesize a DNA molecule from an RNA template.
  • An RT may require the primer molecule with an exposed 3’ hydroxyl group.
  • the primer molecule of an RT may be a DNA molecule.
  • the primer molecule of an RT may be an RNA molecule.
  • an RT may comprise both DNA polymerase activity and RNase H activity. The two activities may reside in two separate domains in an RT.
  • the term “linker” as used herein refers to a bond, a chemical group, or a molecule linking two molecules or moieties, e.g., two p domains to form a fusion protein.
  • a linker can be a peptide linker.
  • a linker can also be a polynucleotide or oligonucleotide linker.
  • a RNA-binding protein recruitment sequence such as a MS2 polynucleotide sequence, can be used to connect a Cas9 domain and a DNA polymerase domain of a prime editor, wherein one of the Cas9 domain and the DNA polymerase domain is fused to a MS2 coat protein.
  • a peptide linker may have various lengths, depending on the application of a linker or the sequences or molecules being linked by a linker.
  • solvate refers to a physical association of a compound with one or more solvent molecules.
  • solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
  • solvate is intended to encompass both solution-phase and isolatable solvates.
  • suitable solvates include the compound in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, tetrahydrofuran, or ethanolamine, and the like.
  • Solvates may be used in the processes for the preparation of a substance.
  • Solvates are often formed as part of the isolation or purification stages of a chemical synthesis.
  • TGA thermogravimetric analysis
  • DSC differential scanning calorimetry
  • X-ray crystallography e.g., single crystal X-ray crystallography or X-ray powder diffraction
  • SS-NMR Solid State NMR
  • Magic Angle Spinning NMR or MAS-NMR Solid State NMR
  • Alkyl refers to a straight or branched hydrocarbon chain monovalent radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, and preferably having from one to twelve carbon atoms (i.e., C 1 -C 12 alkyl). The alkyl is attached to the remainder of the molecule through a single bond. In certain embodiments, an alkyl comprises one to twelve carbon atoms (i.e., C1-C12 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (i.e., C 1 -C 8 alkyl). In other embodiments, an alkyl comprises one to six carbon atoms (i.e., C 1 -C 6 alkyl).
  • an alkyl comprises one to five carbon atoms (i.e., C1-C6 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (i.e., C 1 -C 4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (i.e., C 1 - C 6 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (i.e., C 1 -C 2 alkyl). In other embodiments, an alkyl comprises one carbon atom (i.e., C 1 alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (i.e., C 5 -C 15 alkyl).
  • an alkyl comprises five to eight carbon atoms (i.e., C5-C8 alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (i.e., C2-C6 alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (i.e., C 3 -C 5 alkyl).
  • the alkyl group may be attached to the rest of the molecule by a single bind, such as, methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2- methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl), and the like.
  • a single bind such as, methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2- methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl), and the like.
  • Alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and preferably having from two to twelve carbon atoms (i.e., C 2 -C 12 alkenyl). In certain embodiments, an alkenyl comprises two to eight carbon atoms (i.e., C 2 -C 8 alkenyl). In certain embodiments, an alkenyl comprises two to six carbon atoms (i.e., C2-C6 alkenyl). In other embodiments, an alkenyl comprises two to four carbon atoms (i.e., C2-C4 alkenyl).
  • alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.
  • Alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and preferably having from two to twelve carbon atoms (i.e., C2-C12 alkynyl).
  • an alkynyl comprises two to eight carbon atoms (i.e., C2-C8 alkynyl). In other embodiments, an alkynyl comprises two to six carbon atoms (i.e., C 2 -C 6 alkynyl). In other embodiments, an alkynyl comprises two to four carbon atoms (i.e., C 2 -C 4 alkynyl).
  • the alkynyl is attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • Alkylene refers to a straight divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation, and preferably having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, butylene, and the like.
  • the alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond.
  • the points of attachment of the alkylene chain to the rest of the molecule and to the radical group are through the terminal carbons respectively.
  • Alkylene chain may be optionally substituted by one or more substituents such as those substituents described herein.
  • an alkylene comprises one to ten carbon atoms (i.e., C1-C10 alkylene). In certain embodiments, an alkylene comprises one to eight carbon atoms (i.e., C 1 -C 8 alkylene). In other embodiments, an alkylene comprises one to five carbon atoms (i.e., C 1 -C 6 alkylene). In other embodiments, an alkylene comprises one to four carbon atoms (i.e., C 1 -C 4 alkylene). In other embodiments, an alkylene comprises one to three carbon atoms (i.e., C 1 -C 6 alkylene).
  • an alkylene comprises one to two carbon atoms (i.e., C1-C2 alkylene). In other embodiments, an alkylene comprises one carbon atom (i.e., C1 alkylene). In other embodiments, an alkylene comprises five to eight carbon atoms (i.e., C 5 -C 8 alkylene). In other embodiments, an alkylene comprises two to five carbon atoms (i.e., C2-C6 alkylene). In other embodiments, an alkylene comprises three to five carbon atoms (i.e., C3-C5 alkylene).
  • Alkenylene refers to a straight divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon double bond, and preferably having from two to twelve carbon atoms.
  • the alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond.
  • the points of attachment of the alkenylene chain to the rest of the molecule and to the radical group are through the terminal carbons respectively.
  • Alkenylene chain may be optionally substituted by one or more substituents such as those substituents described herein.
  • an alkenylene comprises two to ten carbon atoms (i.e., C2-C10 alkenylene).
  • an alkenylene comprises two to eight carbon atoms (i.e., C2-C8 alkenylene). In other embodiments, an alkenylene comprises two to five carbon atoms (i.e., C 2 -C 6 alkenylene). In other embodiments, an alkenylene comprises two to four carbon atoms (i.e., C2-C4 alkenylene). In other embodiments, an alkenylene comprises two to three carbon atoms (i.e., C2-C3 alkenylene). In other embodiments, an alkenylene comprises two carbon atom (i.e., C 2 alkenylene).
  • an alkenylene comprises five to eight carbon atoms (i.e., C 5 -C 8 alkenylene). In other embodiments, an alkenylene comprises three to five carbon atoms (i.e., C3-C5 alkenylene).
  • Alkynylene refers to a straight divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon triple bond, and preferably having from two to twelve carbon atoms. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond.
  • Alkynylene chain may be optionally substituted by one or more substituents such as those substituents described herein.
  • an alkynylene comprises two to ten carbon atoms (i.e., C 2 -C 10 alkynylene).
  • an alkynylene comprises two to eight carbon atoms (i.e., C2-C8 alkynylene).
  • an alkynylene comprises two to five carbon atoms (i.e., C 2 -C 6 alkynylene).
  • an alkynylene comprises two to four carbon atoms (i.e., C 2 -C 4 alkynylene). In other embodiments, an alkynylene comprises two to three carbon atoms (i.e., C 2 -C 3 alkynylene). In other embodiments, an alkynylene comprises two carbon atoms (i.e., C 2 alkynylene). In other embodiments, an alkynylene comprises five to eight carbon atoms (i.e., C5-C8 alkynylene). In other embodiments, an alkynylene comprises three to five carbon atoms (i.e., C3-C5 alkynylene).
  • C x-y when used in conjunction with a chemical moiety, such as alkyl, alkenyl, or alkynyl is meant to include groups that contain from x to y carbons in the chain.
  • C1-6 alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from 1 to 6 carbons.
  • -C x-y alkylene- refers to a substituted or unsubstituted alkylene chain with from x to y carbons in the alkylene chain.
  • -C1-6 alkylene- may be selected from methylene, ethylene, propylene, butylene, pentylene, and hexylene, any one of which is optionally substituted.
  • Cx-y alkenyl and “Cx-y alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively.
  • -C x-y alkenylene- refers to a substituted or unsubstituted alkenylene chain with from x to y carbons in the alkenylene chain.
  • - C2-6 alkenylene- may be selected from ethenylene, propenylene, butenylene, pentenylene, and hexenylene, any one of which is optionally substituted.
  • An alkenylene chain may have one double bond or more than one double bond in the alkenylene chain.
  • the term -Cx-yalkynylene- refers to a substituted or unsubstituted alkynylene chain with from x to y carbons in the alkynylene chain.
  • -C 2-6 alkynylene- may be selected from ethynylene, propynylene, butynylene, pentynylene, and hexynylene, any one of which is optionally substituted.
  • An alkynylene chain may have one triple bond or more than one triple bond in the alkynylene chain.
  • the term “carbocycle” as used herein refers to a saturated, unsaturated or aromatic ring in which each atom of the ring is carbon. Carbocycle include 3- to 10-membered monocyclic rings and 6- to 12-membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings.
  • Bicyclic carbocycles may be fused, bridged or spiro-ring systems.
  • the carbocycle is an aryl.
  • the carbocycle is a cycloalkyl.
  • the carbocycle is a cycloalkenyl.
  • an aromatic ring e.g., phenyl
  • Carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, and naphthyl. Carbocycle may be optionally substituted by one or more substituents such as those substituents described herein.
  • Cycloalkyl refers to a stable fully saturated monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which includes fused or bridged ring systems, and preferably having from three to twelve carbon atoms (i.e., C 3-12 cycloalkyl).
  • a cycloalkyl comprises three to ten carbon atoms (i.e., C3-10 cycloalkyl). In other embodiments, a cycloalkyl comprises five to seven carbon atoms (i.e., C5-7 cycloalkyl).
  • the cycloalkyl may be attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Cycloalkyl may be optionally substituted by one or more substituents such as those substituents described herein.
  • Cycloalkenyl refers to a stable unsaturated non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which includes fused or bridged ring systems, preferably having from three to twelve carbon atoms and comprising at least one double bond (i.e., C3-12 cycloalkenyl).
  • a cycloalkenyl comprises three to ten carbon atoms (i.e., C 3-10 cycloalkenyl).
  • a cycloalkenyl comprises five to seven carbon atoms (i.e., C5-7 cycloalkenyl).
  • the cycloalkenyl may be attached to the rest of the molecule by a single bond.
  • monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.
  • Cycloalkenyl may be optionally substituted by one or more substituents such as those substituents described herein.
  • Aryl refers to a radical derived from an aromatic monocyclic or aromatic multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or aromatic multicyclic hydrocarbon ring system contains only hydrogen and carbon and from five to eighteen carbon atoms, where at least one of the rings in the ring system is aromatic, i.e., it contains a cyclic, delocalized (4n+2) p–electron system in accordance with the Hückel theory.
  • the ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene.
  • Aryl may be optionally substituted by one or more substituents such as those substituents described herein.
  • C x-y carbocycle is meant to include groups that contain from x to y carbons in a ring.
  • C3-6 carbocycle can be a saturated, unsaturated or aromatic ring system that contains from 3 to 6 carbon atoms ⁇ any of which is optionally substituted as provided herein.
  • heterocycle refers to a saturated, unsaturated, non-aromatic or aromatic ring comprising one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3- to 10-membered monocyclic rings and 6- to 12- membered bicyclic rings.
  • Each ring of a bicyclic heterocycle may be selected from saturated, unsaturated, and aromatic rings.
  • the heterocycle comprises at least one heteroatom selected from oxygen, nitrogen, sulfur, or any combination thereof.
  • the heterocycle comprises at least one heteroatom selected from oxygen, nitrogen, or any combination thereof.
  • the heterocycle comprises at least one heteroatom selected from oxygen, sulfur, or any combination thereof.
  • the heterocycle comprises at least one heteroatom selected from nitrogen, sulfur, or any combination thereof.
  • the heterocycle may be attached to the rest of the molecule through any atom of the heterocycle, valence permitting, such as a carbon or nitrogen atom of the heterocycle.
  • the heterocycle is a heteroaryl.
  • the heterocycle is a heterocycloalkyl.
  • exemplary heterocycles include pyrrolidinyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, piperidinyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiophenyl, oxazolyl, thiazolyl, morpholinyl, indazolyl, indolyl, and quinolinyl.
  • Heterocycle may be optionally substituted by one or more substituents such as those substituents described herein.
  • Bicyclic heterocycles may be fused, bridged or spiro-ring systems.
  • a heterocycle e.g., pyridyl
  • a saturated or unsaturated ring e.g., cyclohexane, cyclopentane, or cyclohexene.
  • Heterocycle may be optionally substituted by one or more substituents such as those substituents described herein.
  • “Heterocycloalkyl” refers to a stable 3- to 12-membered non-aromatic ring radical that comprises two to twelve carbon atoms and at least one heteroatom wherein each heteroatom may be selected from N, O, Si, P, B, and S atoms.
  • the heterocycloalkyl comprises at least one heteroatom selected from oxygen, nitrogen, sulfur, or any combination thereof. In some embodiments, the heterocycloalkyl comprises at least one heteroatom selected from oxygen, nitrogen, or any combination thereof. In some embodiments, the heterocycloalkyl comprises at least one heteroatom selected from oxygen, sulfur, or any combination thereof. In some embodiments, the heterocycloalkyl comprises at least one heteroatom selected from nitrogen, sulfur, or any combination thereof.
  • the heterocycloalkyl may be selected from monocyclic or bicyclic, and fused or bridged ring systems. The heteroatoms in the heterocycloalkyl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized.
  • the heterocycloalkyl radical is partially or fully saturated.
  • the heterocycloalkyl is attached to the rest of the molecule through any atom of the heterocycloalkyl, valence permitting, such as any carbon or nitrogen atoms of the heterocycloalkyl.
  • heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2- oxopiperazinyl, 2oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4- piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothio
  • Heterocycloalkyl may be optionally substituted by one or more substituents such as those substituents described herein.
  • the term “heteroaryl” refers to a radical derived from a 3- to 12-membered aromatic ring radical that comprises one to eleven carbon atoms and at least one heteroatom wherein each heteroatom may be selected from N, O, and S.
  • the heteroaryl comprises at least one heteroatom selected from oxygen, nitrogen, sulfur, or any combination thereof.
  • the heteroaryl comprises at least one heteroatom selected from oxygen, nitrogen, or any combination thereof.
  • the heteroaryl comprises at least one heteroatom selected from oxygen, sulfur, or any combination thereof.
  • the heteroaryl comprises at least one heteroatom selected from nitrogen, sulfur, or any combination thereof.
  • the heteroaryl ring may be selected from monocyclic or bicyclic and fused or bridged ring systems rings wherein at least one of the rings in the ring system is aromatic, i.e., it contains a cyclic, delocalized (4n+2) p–electron system in accordance with the Hückel theory.
  • the heteroatom(s) in the heteroaryl radical may be optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • heteroaryl may be attached to the rest of the molecule through any atom of the heteroaryl, valence permitting, such as a carbon or nitrogen atom of the heteroaryl.
  • Heteroaryl includes aromatic single ring structures, preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • Heteroaryl may be optionally substituted by one or more substituents such as those substituents described herein.
  • Heteroaryl also includes polycyclic ring systems having two or more rings in which two or more atoms are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other rings can be aromatic or non-aromatic carbocyclic, or heterocyclic.
  • Heteroaryl may be optionally substituted by one or more substituents such as those substituents described herein.
  • An “X-membered heterocycle” refers to the number of endocylic atoms, i.e., X, in the ring.
  • a 5-membered heteroaryl ring or 5-membered aromatic heterocycle has 5 endocyclic atoms, e.g., triazole, oxazole, thiophene, etc.
  • Alkoxy refers to a radical bonded through an oxygen atom of the formula –O-alkyl, where alkyl is an alkyl chain as defined above.
  • Halo or “halogen” refers to halogen substituents such as bromo, chloro, fluoro and iodo substituents.
  • haloalkyl or “haloalkane” refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, for example, trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.
  • the alkyl part of the fluoroalkyl radical is optionally further substituted.
  • haloalkanes examples include halomethane (e.g., chloromethane, bromomethane, fluoromethane, iodomethane), di-and trihalomethane (e.g., trichloromethane, tribromomethane, trifluoromethane, triiodomethane), 1-haloethane, 2- haloethane, 1,2-dihaloethane, 1-halopropane, 2-halopropane, 3-halopropane, 1,2-dihalopropane, 1,3-dihalopropane, 2,3-dihalopropane, 1,2,3-trihalopropane, and any other suitable combinations of alkanes (or substituted alkanes) and halogens (e.g., Cl, Br, F, and I).
  • halomethane e.g., chloromethane, bromomethane, fluoromethane, iodomethane
  • each halogen may be independently selected for example, 1-chloro, 2-fluoroethane.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., an NH or NH 2 of a compound. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group.
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • salts or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, 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 or “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • a subject in need thereof refers to a subject, as described infra, that suffers from, or is at risk for, a pathology to be prophylactically or therapeutically treated with a compound or salt described herein.
  • the terms “administer”, “administered”, “administers” and “administering” are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration.
  • oral routes of administering a composition can be used.
  • the terms “administer”, “administered”, “administers” and “administering” a compound should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to the individual in need.
  • a “therapeutic effect,” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above.
  • a prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • Compounds [0120] In some aspects, the present disclosure provides compounds of Formula (I): or a stereoisomer, tautomer, solvate, or salt thereof, wherein:
  • R 1 is H, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-
  • the present disclosure provides compounds of Formula (I): or a stereoisomer, tautomer, solvate, or salt thereof, wherein: R 1 is H, C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently selected at each instance from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, modified uracil, , , , , ,
  • R 1 is hydrogen or methyl while R 2 is methyl and B 2 is is C 2 -C 6 alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 - CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • the present disclosure provides compounds of Formula (I): ⁇ I) or a stereoisomer, tautomer, solvate, or salt thereof, wherein: R 1 is H, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently selected at each instance from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one C1-C3 alkoxy or 1-3 groups of fluoro, C2-C6 alkenyl, or C2-C6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified , modified , with the proviso that when R 1 is hydrogen or methyl while R 2 is methyl
  • R 1 is H, C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle;
  • the present disclosure provides compounds of Formula (I): or a stereoisomer, tautomer, solvate, or salt thereof, wherein: R 1 is H, C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted
  • R 2 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo
  • R 2 is C2-C6 alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2- CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle,
  • R 2 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C 2 -C 6 alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C1-C6 alkyl, C1-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is C 2 -C 6 alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C1-C6 alkyl, C1-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is C2-C6 alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is C1-C6 alkyl, C1-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl- CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is C 2 -C 6 alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 - CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro. In some embodiments, R 2 is C 1 -C 6 alkyl. In some embodiments, R 2 is H3C , some embodiments, some embodiments, R 2 is C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro. In some embodiments, R 2 is C1-C6 alkyl substituted with one hydroxyl. In some embodiments, R 2 is . In some embodiments, R 2 is C1-C6 alkyl substituted with 1-3 fluoro.
  • R 2 is C 1 -C 6 alkyl substituted with 3 fluoro. In some embodiments, R 2 is [0128] In some embodiments, R 2 is C 2 -C 6 alkyl or C 2 -C 6 alkyl substituted with one hydroxyl H 3 C or 1-3 fluoro. In some embodiments, R 2 is C 2 -C 6 alkyl. In some embodiments, R 2 is , substituted with one hydroxyl or 1-3 fluoro. In s stituted with one hydroxyl. In some embodiments, R 2 is . , . In some embodiments, R 2 is C2-C6 alkyl substituted with 1-3 fluoro.
  • R 2 is C2-C6 alkyl substituted with 3 fluoro. In some embodiments, R 2 is . In some embodiments, R 2 is . [0129] In some embodiments, R 2 is C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl- CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, or C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 2 is C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl, or C 3 -C 6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 2 is C 3 -C 6 cycloalkyl-CH 2 -CH 2 - or C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 2 is C3-C6 cycloalkyl-CH2-CH2-. In some embodiments, R 2 is . In some embodiments, R 2 is C3- C 6 cycloalkyl-CH 2 - or C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro. In some embodiments, R 2 is C 3 -C 6 cycloalkyl-CH 2 -. In some embodiments, R 2 is . In some embodiments, R 2 is C3-C6 cycloalkyl- or C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 2 is C3-C6 cycloalkyl-. In some embodiments, . [0130] In some embodiments, R 2 is arylalkyl or arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl or arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl or arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl.
  • R 2 is arylalkyl. In some embodiments, some embodiments, R 2 is arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl. In some [0131] In some embodiments, R 2 is heteroarylalkyl. In some embodiments, .
  • R 2 is heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl or heterocycloalkyl substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl.
  • R 2 is heterocycloalkyl substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl-CH2-CH2- or heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl-CH 2 -CH 2 -.
  • R 2 is heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl-CH2- or heterocycloalkyl-CH2- substituted with methyl on the heterocycle. In some embodiments, R 2 is heterocycloalkyl-CH 2 -. In some embodiments, R 2 is heterocycloalkyl-CH 2 - substituted with
  • B 2 A 2 wherein indicates a site of connected with B 2 .
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 . In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, fluoro, or -OR 20 .
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen or halogen. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen or fluoro. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen or -OR 20 .
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence halogen or -OR 20 . In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence fluoro or -OR 20 . In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen.
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence halogen. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence fluoro. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence -OR 20 . [0141] In some embodiments, R 3 is independently at each occurrence hydrogen, halogen, or - OR 20 .
  • R 3 is independently at each occurrence hydrogen, fluoro, or -OR 20 . In some embodiments, R 3 is independently at each occurrence hydrogen or halogen. In some embodiments, R 3 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 3 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 3 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 3 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 3 is independently at each occurrence hydrogen. In some embodiments, R 3 is independently at each occurrence halogen. In some embodiments, R 3 is independently at each occurrence fluoro.
  • R 3 is independently at each occurrence -OR 20 . In some embodiments, R 3 is independently at each occurrence -OCH3, -OCH2CH2OCH3, or -F. In some embodiments, R 3 is independently at each occurrence -OCH2CH2OCH3 or -F. In some embodiments, R 3 is -OCH3. In some embodiments, R 3 is -OCH 2 CH 2 OCH 3 . In some embodiments, R 3 is -F. [0142] In some embodiments, R 3′ is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 3′ is independently at each occurrence hydrogen, fluoro, or -OR 20 .
  • R 3′ is independently at each occurrence hydrogen or halogen. In some embodiments, R 3′ is independently at each occurrence hydrogen or fluoro. In some embodiments, R 3′ is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 3′ is independently at each occurrence halogen or -OR 20 . In some embodiments, R 3′ is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 3′ is independently at each occurrence hydrogen. In some embodiments, R 3′ is independently at each occurrence halogen. In some embodiments, R 3′ is independently at each occurrence fluoro. In some embodiments, R 3′ is independently at each occurrence -OR 20 .
  • R 3′′ is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 3′′ is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 3′′ is independently at each occurrence hydrogen or halogen. In some embodiments, R 3′′ is independently at each occurrence hydrogen or fluoro. In some embodiments, R 3′′ is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 3′′ is independently at each occurrence halogen or -OR 20 . In some embodiments, R 3′′ is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 3′′ is independently at each occurrence hydrogen.
  • R 3′′ is independently at each occurrence halogen. In some embodiments, R 3′′ is independently at each occurrence fluoro. In some embodiments, R 3′′ is independently at each occurrence -OR 20 .
  • R 4 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 4 is independently at each occurrence hydrogen, fluoro, or -OR 20 . In some embodiments, R 4 is independently at each occurrence hydrogen or halogen. In some embodiments, R 4 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 4 is independently at each occurrence hydrogen or -OR 20 .
  • R 4 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 4 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 4 is independently at each occurrence hydrogen. In some embodiments, R 4 is independently at each occurrence halogen. In some embodiments, R 4 is independently at each occurrence fluoro. In some embodiments, R 4 is independently at each occurrence -OR 20 . In some embodiments, R 4 is -OH, -OCH3, -F, or - OCH 2 CH 2 OCH 3 . In some embodiments, R 4 is -OH, -OCH 3 , or -F. In some embodiments, R 4 is - OH.
  • R 4 is -OCH 3 . In some embodiments, R 4 is -F.
  • R 11 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 11 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 11 is independently at each occurrence hydrogen or halogen. In some embodiments, R 11 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 11 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 11 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 11 is independently at each occurrence fluoro or -OR 20 .
  • R 11 is independently at each occurrence hydrogen. In some embodiments, R 11 is independently at each occurrence halogen. In some embodiments, R 11 is independently at each occurrence fluoro. In some embodiments, R 11 is independently at each occurrence -OR 20 . [0146] In some embodiments, R 12 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 12 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 12 is independently at each occurrence hydrogen or halogen. In some embodiments, R 12 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 12 is independently at each occurrence hydrogen or -OR 20 .
  • R 12 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 12 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 12 is independently at each occurrence hydrogen. In some embodiments, R 12 is independently at each occurrence halogen. In some embodiments, R 12 is independently at each occurrence fluoro. In some embodiments, R 12 is independently at each occurrence -OR 20 . [0147] In some embodiments, R 13 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 13 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 13 is independently at each occurrence hydrogen or halogen.
  • R 13 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 13 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 13 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 13 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 13 is independently at each occurrence hydrogen. In some embodiments, R 13 is independently at each occurrence halogen. In some embodiments, R 13 is independently at each occurrence fluoro. In some embodiments, R 13 is independently at each occurrence -OR 20 . [0148] In some embodiments, R 14 is independently at each occurrence hydrogen, halogen, or - OR 20 .
  • R 14 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 14 is independently at each occurrence hydrogen or halogen. In some embodiments, R 14 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 14 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 14 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 14 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 14 is independently at each occurrence hydrogen. In some embodiments, R 14 is independently at each occurrence halogen. In some embodiments, R 14 is independently at each occurrence fluoro.
  • R 14 is independently at each occurrence -OR 20 . In some embodiments, R 14 is independently at each occurrence -OH or -OCH 3 . In some embodiments, R 14 is -OH. In some embodiments, R 14 is -OCH 3 . In some embodiments, R 14 is -OCH 2 CH 2 OCH 3 . [0149] In some embodiments, R 20 is independently at each instance hydrogen, C1-C6 alkyl, C1- C 6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl.
  • R 20 is independently at each instance hydrogen, C 1 -C 6 alkyl, C 1 - C 6 alkyl substituted with one C 1 -C 3 alkoxy or 3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl. In some embodiments, R 20 is independently at each instance hydrogen or C 1 -C 6 alkyl, C1-C6 alkyl substituted with one methoxy or 3 groups of fluoro, C2-C6 alkenyl, or C2-C6 alkynyl. In some embodiments, R 20 is independently at each instance hydrogen. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl.
  • R 20 is independently at each instance methyl, ethyl, propyl, or isopropyl. In some embodiments, R 20 is independently at each instance C1-C6 alkyl substituted with one C1-C3 alkoxy or 1-3 groups of fluoro. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl substituted with one C 1 -C 3 alkoxy or 3 groups of fluoro. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl substituted with one methoxy or 3 groups of fluoro. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl substituted with one methoxy.
  • R 20 is independently at each instance (2-methoxy)ethyl. In some embodiments, R 20 is independently at each instance C1-C6 alkyl substituted with 3 groups of fluoro. In some embodiments, R 20 is independently at each instance trifluoromethyl. In some embodiments, R 20 is independently at each instance C 2 -C 6 alkenyl. In some embodiments, R 20 is independently at each instance allyl. In some embodiments, R 20 is independently at each instance C2-C6 alkynyl. In some embodiments, R 20 is independently at each instance propargyl.
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, with the proviso that when R 1 is hydrogen or methyl while R 2 is methyl alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl- CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, hetero
  • R 1 is hydrogen or methyl while R 2 is methyl alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl- CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified t some embodiments, when alkyl, C3-C6 cycloalkyl-CH 2 -CH 2 -,C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, t some embodiments, when alkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • alkyl when alkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • alkyl when alkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH2- CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl- CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, [0157] In some embodiments, B 2 is adenine, cytosine, uracil, modified adenine, modified t some embodiments, when alkyl, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, or heterocyclo
  • B 2 is adenine, cytosine, uracil, modified adenine, modified ot alkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 - C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • alkyl when alkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH2- CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl- CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • the compound is of Formula (IIa), Formula (IIb), Formula (IIc), Formula (IId), Formula (IIe), or Formula (IIf): (IId), (IIf).
  • the compound is of Formula (IIa). In some embodiments, the compound is of Formula (IIb). In some embodiments, the compound is of Formula (IIc). In some embodiments, the compound is of Formula (IId). In some embodiments, the compound is of Formula (IIe). In some embodiments, the compound is of Formula (IIf).
  • R 1 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle,
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl-CH2- CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2- CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl- CH 2 - substituted with methyl on the heterocycle.
  • R 1 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 2 or 3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl- CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 3 groups of fluoro, C3-C6 cycloalkyl, C3- C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, arylalkyl, arylalkyl substituted with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 1 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 3 groups of fluoro, C 3 -C 6 cycloalkyl, C 3 - C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, arylalkyl, arylalkyl substituted with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 1 is C1-C6 alkyl or C1-C6 alkyl substituted with hydroxyl or 1- 3 groups of fluoro. In some embodiments, R 1 is C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted with hydroxyl or 2 or 3 groups of fluoro. In some embodiments, R 1 is C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted with hydroxyl or 3 groups of fluoro. In some embodiments, R 1 is C1-C6 alkyl. In some embodiments, . In som 1 e embodiments, R is C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro.
  • R 1 is C1-C6 alkyl substituted with hydroxyl or 2 or 3 groups of fluoro. In some embodiments, R 1 is C 1 -C 6 alkyl substituted with hydroxyl or 1 or 3 groups of fluoro. In some embodiments, R 1 is C 1 -C 6 alkyl substituted with hydroxyl or one group of fluoro. In some embodiments, R 1 is C1-C6 alkyl substituted with hydroxyl or 3 groups of fluoro. In some embodiments, R 1 is C1-C6 alkyl substituted with hydroxyl. In some embodiments, R 1 is . In some embodiments, R 1 is C 1 -C 6 alkyl substituted with 3 groups of fluoro.
  • R 1 is methyl substituted with hydroxyl or 1-3 groups of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or 2 or 3 groups of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or 1 or 3 groups of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or one group of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or 3 groups of fluoro. In some HO F 3 C embodiments, R 1 is or .
  • R 1 is C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, or C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 1 is C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, or C 3 -C 6 cycloalkyl-CH 2 -. In some embodiments, R 1 is C 3 -C 6 cycloalkyl. In some embodiments, R 1 is . In some embodiments, R 1 is C3-C6 cycloalkyl-CH2-CH2-. In some embodiments, R 1 is . In some embodiments, R 1 is C3-C6 cycloalkyl-CH2-. In some embodiments, R 1 is .
  • R 1 is arylalkyl or arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl or arylalkyl substituted with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl or arylalkyl substituted with group of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl.
  • R 1 is some embodiments, R 1 is arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl substituted with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl substituted with group of methoxy, fluoro, chloro, or bromo on the . [0169] In some embodiments, R 1 is heteroarylalkyl. In some embodiments, .
  • R 1 is heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl or heterocycloalkyl substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl.
  • R 1 is heterocycloalkyl substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH2-CH2- or heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH 2 -CH 2 -.
  • R 1 is heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH2- or heterocycloalkyl-CH2- substituted with methyl on the heterocycle. In some embodiments, R 1 is heterocycloalkyl-CH 2 -. In some embodiments, R 1 is heterocycloalkyl-CH 2 - substituted with
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, or uracil. In some embodiments, each of B 3 , B 4 , B 5 and B 6 is independently modified adenine, modified cytosine, modified guanine, or modified uracil.
  • B 3 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 3 is adenine, cytosine, guanine, or uracil. In some embodiments, B 3 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 3 is adenine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is guanine. In some embodiments, B 3 is uracil. In some embodiments, B 3 is modified adenine.
  • B 3 is modified cytosine. In some embodiments, B 3 is modified guanine. In some embodiments, B 3 is modified uracil.
  • B 4 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 4 is adenine, cytosine, guanine, or uracil. In some embodiments, B 4 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 4 is adenine. In some embodiments, B 4 is cytosine. In some embodiments, B 4 is cytosine.
  • B 4 is guanine. In some embodiments, B 4 is uracil. In some embodiments, B 4 is modified adenine. In some embodiments, B 4 is modified cytosine. In some embodiments, B 4 is modified guanine. In some embodiments, B 4 is modified uracil. [0175] In some embodiments, B 5 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 5 is adenine, cytosine, guanine, or uracil.
  • B 5 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 5 is adenine. In some embodiments, B 5 is cytosine. In some embodiments, B 5 is guanine. In some embodiments, B 5 is uracil. In some embodiments, B 5 is modified adenine. In some embodiments, B 5 is modified cytosine. In some embodiments, B 5 is modified guanine. In some embodiments, B 5 is modified uracil.
  • B 6 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 6 is adenine, cytosine, guanine, or uracil. In some embodiments, B 6 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 6 is adenine. In some embodiments, B 6 is cytosine. In some embodiments, B 6 is guanine. In some embodiments, B 6 is uracil. In some embodiments, B 6 is modified adenine.
  • B 6 is modified cytosine. In some embodiments, B 6 is modified guanine. In some embodiments, B 6 is modified uracil.
  • R 3 is methyl, ethyl, propyl, isopropyl, -CH2CH2OCH3 or -CF3. In some embodiments, R 3 is methyl, ethyl, propyl, or isopropyl. In some embodiments, R 3 is methyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is propyl. In some embodiments, R 3 is isopropyl. In some embodiments, R 3 is -CH 2 CH 2 OCH 3 .
  • R 3 is -CF 3 .
  • each phosphorus atom between independently has a configuration of S p or R p .
  • each phosphorus atom between has a configuration of Sp.
  • each phosphorus atom between has a configuration of R p .
  • the present disclosure provides compounds of the formula: ,
  • mRNA sequences comprising a 5’-end motif of Formula (III): or a stereoisomer, tautomer, solvate, or salt thereof, wherein:
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, hetero
  • R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently selected at each instance from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified adenine, modified
  • the mRNA sequence comprises a 5’-end motif of Formula (III): or a stereoisomer, tautomer, solvate, or salt thereof, wherein: R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl- CH 2 -CH
  • R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently selected at each instance from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified adenine, modified
  • alkyl when alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl- CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • the mRNA sequence comprises a 5’-end motif of Formula (III): (III) or a stereoisomer, tautomer, solvate, or salt thereof, wherein: R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH 2
  • R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently selected at each instance from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytos
  • the mRNA sequence comprises a 5’-end motif of Formula (III):
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl with the heterocycle,
  • the mRNA sequence comprises a 5’-end motif of Formula (III): or a stereoisomer, tautomer, solvate, or salt thereof, wherein:
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl- CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle;
  • R 2 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3
  • B is . In some embodiments, B is . In some embodiments, B 1 is embodiments, some embodiments, B 1 is
  • R 2 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with
  • R 2 is C2-C6 alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2- CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle,
  • R 2 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C2-C6 alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C 2 -C 6 alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C1-C6 alkyl, C1-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is C2-C6 alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is C1-C6 alkyl, C1-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl- CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 2 is C 2 -C 6 alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 - CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is C1-C6 alkyl or C1-C6 alkyl substituted with one hydroxyl or 1-3 fluoro. In some embodiments, R 2 is C -C alkyl. In some embodiment 2 H3C 1 6 s, R is , embodiments, R 2 is C1-C6 alkyl substituted with one hydroxyl or 1-3 fluoro. In some embodiments, R 2 is C1-C6 alkyl substituted with one hydroxyl. In some embodiments, R 2 is . In some embodiments, R 2 is C1-C6 alkyl substituted with 1-3 fluoro. In some embodiments, R 2 is C1-C6 alkyl substituted with 3 fluoro.
  • R 2 is [0188] In some embodiments, R 2 is C 2 -C 6 alkyl or C 2 -C 6 alkyl substituted with one hydroxyl H 3C or 1-3 fluoro. In some embodiments, R 2 is C2-C6 alkyl. In some embodiments, R 2 is , some embodiments, R 2 is C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro. In some embodiments, R 2 is C2-C6 alkyl substituted HO with one hydroxyl. In some embodiments, R 2 is , , , or . , . In some embodiments, R 2 is C2-C6 alkyl substituted with 1-3 fluoro.
  • R 2 is C2-C6 alkyl substituted with 3 fluoro. In some embodiments, R 2 is . , . [0189] In some embodiments, R 2 is C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl- CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, or C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 2 is C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, or C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 2 is C 3 -C 6 cycloalkyl-CH 2 -CH 2 - or C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 2 is C 3 -C 6 cycloalkyl-CH2-CH2-.
  • R 2 is . In some embodiments, R 2 is C3- C 6 cycloalkyl-CH 2 - or C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro. In some embodiments, R 2 is C3-C6 cycloalkyl-CH2-. In some embodiments, R 2 is . In some embodiments, R 2 is C 3 -C 6 cycloalkyl- or C 3 -C 6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro. In some embodiments, R 2 is C3-C6 cycloalkyl-.
  • R 2 is arylalkyl or arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl or arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl or arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl.
  • R 2 is arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl substituted independently with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 2 is arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl. In some
  • R 2 is heteroarylalkyl. In some embodiments, . [0192] In some embodiments, R 2 is heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle. In some embodiments, R 2 is heterocycloalkyl or heterocycloalkyl substituted with methyl on the heterocycle. In some embodiments, R 2 is heterocycloalkyl.
  • R 2 is heterocycloalkyl substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl-CH2-CH2- or heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle.
  • R 2 is heterocycloalkyl-CH2-CH2-.
  • R 2 is heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle. In some embodiments, R 2 is heterocycloalkyl-CH2- or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle. In some embodiments, R 2 is heterocycloalkyl-CH 2 -. In some embodiments, R 2 is heterocycloalkyl-CH 2 - substituted with
  • RNA ribonucleic acid
  • M is a bond linked to the RNA.
  • RNA ribonucleic acid
  • M in connected with a ribonucleic acid (RNA), or M is a bond linked to the RNA.
  • M in connected with a ribonucleic acid (RNA), or M is a bond linked to the RNA.
  • RNA ribonucleic acid
  • M in is the bond linked to the RNA.
  • M in [0199] indicates a site of connected with a ribonucleic acid (RNA), or M is a bond linked to the RNA; and R 14 is -OH, -OCH 3 , -OCH 2 CH 2 OCH 3 , or -F.
  • M in is , wherein indicates a site of connected with a ribonucleic acid (RNA), or M is a bond linked to the RNA; and R 14 is -OH or -OCH 3 .
  • M connected with a ribonucleic acid (RNA), or M is a bond linked to the RNA; and R 14 is -OCH3.
  • R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 .
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, fluoro, or -OR 20 .
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen or halogen. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen or fluoro. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen or -OR 20 .
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence halogen or -OR 20 . In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence fluoro or -OR 20 . In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen.
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence halogen. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence fluoro. In some embodiments, each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence -OR 20 . [0201] In some embodiments, R 3 is independently at each occurrence hydrogen, halogen, or - OR 20 .
  • R 3 is independently at each occurrence hydrogen, fluoro, or -OR 20 . In some embodiments, R 3 is independently at each occurrence hydrogen or halogen. In some embodiments, R 3 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 3 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 3 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 3 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 3 is independently at each occurrence hydrogen. In some embodiments, R 3 is independently at each occurrence halogen. In some embodiments, R 3 is independently at each occurrence fluoro.
  • R 3 is independently at each occurrence -OR 20 . In some embodiments, R 3 is independently at each occurrence -OCH3, -OCH2CH2OCH3, or -F. In some embodiments, R 3 is independently at each occurrence -OCH 2 CH 2 OCH 3 or -F. In some embodiments, R 3 is -OCH 3 . In some embodiments, R 3 is -OCH2CH2OCH3. In some embodiments, R 3 is -F. [0202] In some embodiments, R 3′ is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 3′ is independently at each occurrence hydrogen, fluoro, or -OR 20 .
  • R 3′ is independently at each occurrence hydrogen or halogen. In some embodiments, R 3′ is independently at each occurrence hydrogen or fluoro. In some embodiments, R 3′ is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 3′ is independently at each occurrence halogen or -OR 20 . In some embodiments, R 3′ is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 3′ is independently at each occurrence hydrogen. In some embodiments, R 3′ is independently at each occurrence halogen. In some embodiments, R 3′ is independently at each occurrence fluoro. In some embodiments, R 3′ is independently at each occurrence -OR 20 .
  • R 3′′ is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 3′′ is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 3′′ is independently at each occurrence hydrogen or halogen. In some embodiments, R 3′′ is independently at each occurrence hydrogen or fluoro. In some embodiments, R 3′′ is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 3′′ is independently at each occurrence halogen or -OR 20 . In some embodiments, R 3′′ is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 3′′ is independently at each occurrence hydrogen.
  • R 3′′ is independently at each occurrence halogen. In some embodiments, R 3′′ is independently at each occurrence fluoro. In some embodiments, R 3′′ is independently at each occurrence -OR 20 .
  • R 4 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 4 is independently at each occurrence hydrogen, fluoro, or -OR 20 . In some embodiments, R 4 is independently at each occurrence hydrogen or halogen. In some embodiments, R 4 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 4 is independently at each occurrence hydrogen or -OR 20 .
  • R 4 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 4 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 4 is independently at each occurrence hydrogen. In some embodiments, R 4 is independently at each occurrence halogen. In some embodiments, R 4 is independently at each occurrence fluoro. In some embodiments, R 4 is independently at each occurrence -OR 20 . In some embodiments, R 4 is -OH, -OCH3, -F, or - OCH 2 CH 2 OCH 3 . In some embodiments, R 4 is -OH, -OCH 3 , or -F. In some embodiments, R 4 is - OH.
  • R 4 is -OCH3. In some embodiments, R 4 is -F.
  • R 11 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 11 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 11 is independently at each occurrence hydrogen or halogen. In some embodiments, R 11 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 11 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 11 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 11 is independently at each occurrence fluoro or -OR 20 .
  • R 11 is independently at each occurrence hydrogen. In some embodiments, R 11 is independently at each occurrence halogen. In some embodiments, R 11 is independently at each occurrence fluoro. In some embodiments, R 11 is independently at each occurrence -OR 20 . [0206] In some embodiments, R 12 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 12 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 12 is independently at each occurrence hydrogen or halogen. In some embodiments, R 12 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 12 is independently at each occurrence hydrogen or -OR 20 .
  • R 12 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 12 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 12 is independently at each occurrence hydrogen. In some embodiments, R 12 is independently at each occurrence halogen. In some embodiments, R 12 is independently at each occurrence fluoro. In some embodiments, R 12 is independently at each occurrence -OR 20 . [0207] In some embodiments, R 13 is independently at each occurrence hydrogen, halogen, or - OR 20 . In some embodiments, R 13 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 13 is independently at each occurrence hydrogen or halogen.
  • R 13 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 13 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 13 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 13 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 13 is independently at each occurrence hydrogen. In some embodiments, R 13 is independently at each occurrence halogen. In some embodiments, R 13 is independently at each occurrence fluoro. In some embodiments, R 13 is independently at each occurrence -OR 20 . [0208] In some embodiments, R 14 is independently at each occurrence hydrogen, halogen, or - OR 20 .
  • R 14 is independently at each occurrence hydrogen, fluoro, or - OR 20 . In some embodiments, R 14 is independently at each occurrence hydrogen or halogen. In some embodiments, R 14 is independently at each occurrence hydrogen or fluoro. In some embodiments, R 14 is independently at each occurrence hydrogen or -OR 20 . In some embodiments, R 14 is independently at each occurrence halogen or -OR 20 . In some embodiments, R 14 is independently at each occurrence fluoro or -OR 20 . In some embodiments, R 14 is independently at each occurrence hydrogen. In some embodiments, R 14 is independently at each occurrence halogen. In some embodiments, R 14 is independently at each occurrence fluoro.
  • R 14 is independently at each occurrence -OR 20 . In some embodiments, R 14 is independently at each occurrence -OH or -OCH3. In some embodiments, R 14 is -OH. In some embodiments, R 14 is -OCH 3 . In some embodiments, R 14 is -OCH 2 CH 2 OCH 3 . [0209] In some embodiments, R 20 is independently at each instance hydrogen, C 1 -C 6 alkyl, C 1 - C6 alkyl substituted with one C1-C3 alkoxy or 1-3 groups of fluoro, C2-C6 alkenyl, or C2-C6 alkynyl.
  • R 20 is independently at each instance hydrogen, C1-C6 alkyl, C1- C 6 alkyl substituted with one C 1 -C 3 alkoxy or 3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl. In some embodiments, R 20 is independently at each instance hydrogen or C1-C6 alkyl, C1-C6 alkyl substituted with one methoxy or 3 groups of fluoro, C2-C6 alkenyl, or C2-C6 alkynyl. In some embodiments, R 20 is independently at each instance hydrogen. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl.
  • R 20 is independently at each instance methyl, ethyl, propyl, or isopropyl. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl substituted with one C 1 -C 3 alkoxy or 3 groups of fluoro. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl substituted with one methoxy or 3 groups of fluoro. In some embodiments, R 20 is independently at each instance C1-C6 alkyl substituted with one methoxy.
  • R 20 is independently at each instance (2-methoxy)ethyl. In some embodiments, R 20 is independently at each instance C 1 -C 6 alkyl substituted with 3 groups of fluoro. In some embodiments, R 20 is independently at each instance trifluoromethyl. In some embodiments, R 20 is independently at each instance C2-C6 alkenyl. In some embodiments, R 20 is independently at each instance allyl. In some embodiments, R 20 is independently at each instance C 2 -C 6 alkynyl. In some embodiments, R 20 is independently at each instance propargyl.
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, the same time when some embodiments, when alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl- CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, with the proviso that when R 1 is hydrogen or methyl while R 2 is methyl alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl- CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH 2 - substituted with methyl
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, modified uracil, , , ,
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, modified uracil, , , ,
  • B 2 is adenine, cytosine, uracil, modified adenine, modified t the same time when some embodiments, when alkyl, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • B 2 is adenine, cytosine,
  • R 1 is hydrogen or methyl while R 2 is methyl
  • alkyl when alkyl, C3-C6 cycloalkyl-CH2-CH2-, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl, arylalkyl, arylalkyl substituted independently with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH2- CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl- CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • the mRNA sequence comprises a 5’-end motif of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), Formula (IVe), or Formula (IVf):
  • the mRNA sequence comprises a 5’-end motif of Formula (IVa). In some embodiments, the mRNA sequence comprises a 5’-end motif of Formula (IVb). In some embodiments, the mRNA sequence comprises a 5’-end motif of Formula (IVc). In some embodiments, the mRNA sequence comprises a 5’-end motif of Formula (IVd). In some embodiments, the mRNA sequence comprises a 5’-end motif of Formula (IVe). In some embodiments, the mRNA sequence comprises a 5’-end motif of Formula (IVf).
  • R 1 is C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl, heterocycloalkyl,
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl-CH 2 - CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH 2 - CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl- CH2- substituted with methyl on the heterocycle.
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 2 or 3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl- CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 3 groups of fluoro, C3-C6 cycloalkyl, C3- C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -, arylalkyl, arylalkyl substituted with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH 2 -CH 2 - substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 1 is C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 3 groups of fluoro, C3-C6 cycloalkyl, C3- C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-, arylalkyl, arylalkyl substituted with one group of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl-CH 2 -CH 2 -, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 1 is C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted with hydroxyl or 1- 3 groups of fluoro. In some embodiments, R 1 is C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted with hydroxyl or 2 or 3 groups of fluoro. In some embodiments, R 1 is C1-C6 alkyl or C1-C6 alkyl substituted with hydroxyl or 3 groups of fluoro. In some embodiments, R 1 is C 1 -C 6 alkyl. In some embodiments, . In some embodiments, R 1 is C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro.
  • R 1 is C 1 -C 6 alkyl substituted with hydroxyl or 2 or 3 groups of fluoro. In some embodiments, R 1 is C1-C6 alkyl substituted with hydroxyl or 1 or 3 groups of fluoro. In some embodiments, R 1 is C1-C6 alkyl substituted with hydroxyl or one group of fluoro. In some embodiments, R 1 is C 1 -C 6 alkyl substituted with hydroxyl or 3 groups of fluoro. In som hydroxyl. In some embodiments, R 1 is some embodiments, R 1 is C1-C6 alkyl substituted with 3 groups of fluoro. In some embodiments, .
  • R 1 is methyl substituted with hydroxyl or 1-3 groups of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or 2 or 3 groups of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or 1 or 3 groups of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or one group of fluoro. In some embodiments, R 1 is methyl substituted with hydroxyl or 3 groups of fluoro. In some embodiments, .
  • R 1 is C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, or C3-C6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro.
  • R 1 is C3-C6 cycloalkyl, C3-C6 cycloalkyl-CH2-CH2-, or C3-C6 cycloalkyl-CH2-. In some embodiments, R 1 is C3-C6 cycloalkyl. In some embodiments, R 1 is . In some embodiments, R 1 is C3-C6 cycloalkyl-CH2-CH2-. In some embodiments, R 1 is . In some embodiments, R 1 is C3-C6 cycloalkyl-CH2-. In some embodiments, R 1 is .
  • R 1 is arylalkyl or arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl or arylalkyl substituted with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl or arylalkyl substituted with group of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl.
  • R 1 is some embodiments, R 1 is arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl substituted with 1 or 2 groups of methoxy, fluoro, chloro, or bromo on the aryl. In some embodiments, R 1 is arylalkyl substituted with group of methoxy, fluoro, chloro, or bromo on the . [0229] In some embodiments, R 1 is heteroarylalkyl. In some embodiments, .
  • R 1 is heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH 2 -, or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl or heterocycloalkyl substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl.
  • R 1 is heterocycloalkyl substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH2-CH2-, heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle, heterocycloalkyl-CH2-, or heterocycloalkyl-CH2- substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH 2 -CH 2 - or heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH2-CH2-.
  • R 1 is heterocycloalkyl-CH2-CH2- substituted with methyl on the heterocycle.
  • R 1 is heterocycloalkyl-CH 2 - or heterocycloalkyl-CH 2 - substituted with methyl on the heterocycle. In some embodiments, R 1 is heterocycloalkyl-CH2-. In some embodiments, R 1 is heterocycloalkyl-CH2- substituted with methyl on the heterocycle. In some embodiments, ,
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, or uracil. In some embodiments, each of B 3 , B 4 , B 5 and B 6 is independently modified adenine, modified cytosine, modified guanine, or modified uracil.
  • B 3 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 3 is adenine, cytosine, guanine, or uracil. In some embodiments, B 3 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 3 is adenine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is guanine. In some embodiments, B 3 is uracil. In some embodiments, B 3 is modified adenine.
  • B 3 is modified cytosine. In some embodiments, B 3 is modified guanine. In some embodiments, B 3 is modified uracil.
  • B 4 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 4 is adenine, cytosine, guanine, or uracil. In some embodiments, B 4 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 4 is adenine. In some embodiments, B 4 is cytosine. In some embodiments, B 4 is cytosine.
  • B 4 is guanine. In some embodiments, B 4 is uracil. In some embodiments, B 4 is modified adenine. In some embodiments, B 4 is modified cytosine. In some embodiments, B 4 is modified guanine. In some embodiments, B 4 is modified uracil. [0235] In some embodiments, B 5 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 5 is adenine, cytosine, guanine, or uracil.
  • B 5 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 5 is adenine. In some embodiments, B 5 is cytosine. In some embodiments, B 5 is guanine. In some embodiments, B 5 is uracil. In some embodiments, B 5 is modified adenine. In some embodiments, B 5 is modified cytosine. In some embodiments, B 5 is modified guanine. In some embodiments, B 5 is modified uracil.
  • B 6 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 6 is adenine, cytosine, guanine, or uracil. In some embodiments, B 6 is modified adenine, modified cytosine, modified guanine, or modified uracil. In some embodiments, B 6 is adenine. In some embodiments, B 6 is cytosine. In some embodiments, B 6 is guanine. In some embodiments, B 6 is uracil. In some embodiments, B 6 is modified adenine.
  • B 6 is modified cytosine. In some embodiments, B 6 is modified guanine. In some embodiments, B 6 is modified uracil.
  • R 3 is methyl, ethyl, propyl, isopropyl, -CH2CH2OCH3 or -CF3. In some embodiments, R 3 is methyl, ethyl, propyl, or isopropyl. In some embodiments, R 3 is methyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is propyl. In some embodiments, R 3 is isopropyl. In some embodiments, R 3 is -CH2CH2OCH3.
  • R 3 is -CF 3 .
  • each phosphorus atom between independently has a configuration of Sp or Rp. In some embodiments, each phosphorus atom between has a configuration of S p . In some embodiments, each phosphorus atom between has a configuration of Rp. [0239] In some aspects, the present disclosure provides analogs of 5’-cap mRNA that display a plurality of biological activities, including but not limited to, RNA splicing, RNA transport, prevention of degradation by exonucleases, and promotion of translation by ribosome. In some embodiments. In some embodiments, the analogs of 5’-cap mRNA disclosed herein can provide therapies and vaccines to treat various diseases.
  • the present disclosure provides one or more cells comprising a ribonucleic acid (RNA) molecule comprising an mRNA sequence provided herein.
  • RNA ribonucleic acid
  • the present disclosure provides one or more cells comprising a polypeptide or protein translated from an RNA molecule comprising an mRNA sequence provided herein.
  • the present disclosure provides pharmaceutical compositions comprising an RNA molecule comprising an mRNA sequence provided herein.
  • the compositions further comprise one or more single guide RNA designed to target one or more specific locations of one or more genes of interest to elicit pharmacological effect upon administration into a mammal.
  • the present disclosure provides methods for synthesizing a ribonucleic acid (RNA) molecule comprising: (a) introducing an mRNA sequence provided herein into a mixture comprising an RNA polymerase, and (b) incubating the mixture for a time sufficient to allow for transcription of the RNA molecule.
  • the mixture further comprises a DNA template and nucleoside triphosphates.
  • the present disclosure provides methods of gene editing comprising introducing into a cell a ribonucleic acid (RNA) molecule comprising an mRNA sequence provided herein, or a pharmaceutical composition provided herein, wherein the RNA molecule encodes a therapeutic protein, wherein the RNA molecule is translated in the cell.
  • RNA ribonucleic acid
  • the present disclosure provides vectors comprising an mRNA sequence provided herein.
  • the vector is an adeno-associated viral vector (AAV) particle comprising a capsid and a viral genome, and wherein the viral genome comprises the mRNA sequence.
  • AAV adeno-associated viral vector
  • the vector is a lipid nanoparticle (LNP) comprising the mRNA sequence, wherein the mRNA sequence is encapsulated in or associated with the LNP.
  • the vector is a virus-like particle (VLP) comprising the mRNA sequence, wherein the mRNA sequence is encapsulated in or associated with the VLP.
  • VLP virus-like particle
  • the present disclosure provides ribonucleic acid (RNA) molecules comprising an mRNA sequence provided herein, wherein the RNA molecule has a half-life that is at least 1.2 times of that of a corresponding natural RNA molecule in a cellular environment.
  • the RNA molecules further comprise an RNA sequence encoding a therapeutic protein.
  • the present disclosure provides methods of producing a protein in a cell, comprising: (a) introducing a modified messenger ribonucleic acid (mRNA) molecule into a cell; and (b) translating the mRNA molecule in the cell to make a protein; wherein the modified mRNA molecule comprises an mRNA sequence provided herein, and wherein: (i) the mRNA molecule has a translation efficiency of at least 1.2-fold of that of a corresponding unmodified mRNA molecule, or; (ii) the mRNA molecule persists in the cell at least 20% longer than the corresponding unmodified mRNA molecule, or; (iii) the mRNA molecule produces the protein at least 20% more than the corresponding unmodified mRNA molecule, or; (iv) the mRNA molecule induces an innate immune response at a frequency of at least 20% less than the corresponding unmodified mRNA molecule, or; (v) the mRNA molecule has a
  • the RNA e.g., mRNA may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g.
  • 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%).
  • a modified RNA e.g., modified mRNA may exhibit enhanced translation efficiency relative to that of a corresponding unmodified mRNA molecule.
  • the mRNA molecule has a translation efficiency of at least 1-fold, at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, or at least 10-fold of that of a corresponding unmodified mRNA molecule.
  • the mRNA molecule has a translation efficiency of at least 1.2-fold of that of a corresponding unmodified mRNA molecule.
  • a modified RNA e.g., modified mRNA may persist in the cell longer than a corresponding unmodified mRNA molecule.
  • the mRNA molecule persists in the cell at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% longer than a corresponding unmodified mRNA molecule.
  • the mRNA molecule persists in the cell at least 20% longer than a corresponding unmodified mRNA molecule.
  • a modified RNA e.g., modified mRNA may produce the protein more than a corresponding unmodified mRNA molecule.
  • the mRNA molecule produces the protein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% more than a corresponding unmodified mRNA molecule.
  • the mRNA molecule produces the protein at least 20% more than a corresponding unmodified mRNA molecule.
  • a modified RNA e.g., modified mRNA may induce an innate immune response at a frequency less than a corresponding unmodified mRNA molecule.
  • the mRNA molecule induces an innate immune response at a frequency at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% less than a corresponding unmodified mRNA molecule.
  • the mRNA molecule induces an innate immune response at a frequency at least 20% less than a corresponding unmodified mRNA molecule.
  • a modified RNA e.g., modified mRNA may exhibit enhanced gene transfer rate relative to that of a corresponding unmodified mRNA molecule.
  • the mRNA molecule has a gene transfer rate of at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, or at least 10-fold of that of a corresponding unmodified mRNA molecule.
  • the mRNA molecule has a gene transfer rate of at least 1.2-fold of that of a corresponding unmodified mRNA molecule.
  • a modified RNA e.g., modified mRNA may degrade in the cell at a lower rate than a corresponding unmodified mRNA molecule.
  • the mRNA molecule degrades in the cell at a rate at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than a corresponding unmodified mRNA molecule.
  • the mRNA molecule degrades in the cell at a rate at least 20% less than a corresponding unmodified mRNA molecule.
  • the translation is in vivo, ex vivo, in culture, or in vitro.
  • the translation is in vivo. In some embodiments, the translation is ex vivo, in culture, or in vitro. In some embodiments, the translation is ex vivo. In some embodiments, the translation is in culture. In some embodiments, the translation is in vitro.
  • the protein, e.g., fusion protein is therapeutic. In some embodiments, the protein, e.g., fusion protein provides a functional activity substantially absent in the cell, wherein the functional activity is enzymatic, structural, or gene regulatory. In some embodiments, the functional activity is enzymatic or structural. In some embodiments, the functional activity is enzymatic or gene regulatory. In some embodiments, the functional activity is structural or gene regulatory.
  • the functional activity is enzymatic. In some embodiments, the functional activity is structural. In some embodiments, the functional activity is gene regulatory. [0258] In some embodiments, the modified RNA, e.g., modified mRNA encodes a fusion protein, e.g., a fusion protein comprising a polypeptide domain having DNA binding activity (e.g., a DNA binding domain) and a polypeptide domain (e.g., a DNA polymerase domain) having DNA polymerase activity.
  • a fusion protein e.g., a fusion protein comprising a polypeptide domain having DNA binding activity (e.g., a DNA binding domain) and a polypeptide domain (e.g., a DNA polymerase domain) having DNA polymerase activity.
  • the modified RNA e.g., modified mRNA encodes fusion polypeptide that comprises a DNA binding domain and a DNA polymerase domain linked by a linker, e.g., a peptide linker, e.g., a GS rich peptide linker.
  • the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity.
  • the DNA binding domain comprises a nuclease domain or nuclease activity.
  • the polypeptide domain having the nuclease activity comprises a nickase, or a fully active nuclease.
  • the DNA binding domain comprises a nickase, or a fully active nuclease.
  • nickase refers to a nuclease capable of cleaving only one strand of a double- stranded DNA target.
  • the DNA binding domain comprises a comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease.
  • the DNA binding domain e.g., a nucleic acid guided DNA binding domain
  • Cas protein domain for example, a Cas protein domain.
  • the DNA binding domain is a Cas9 or a variant thereof (e.g., a nickase variant).
  • the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR- Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease.
  • the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
  • the DNA binding domain comprises a template-dependent DNA polymerase for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
  • the DNA polymerase domain comprises a reverse transcriptase domain (RT domain) or a reverse transcriptase (RT).
  • the DNA polymerase domain is a RT domain or a RT.
  • a prime editor comprises a reverse transcriptase (RT) activity.
  • the first polypeptide of the prime editor may have activity for target primed reverse transcription.
  • the polypeptide domain having DNA polymerase activity comprises a reverse transcriptase activity (e.g., activity for target primed reverse transcription).
  • the fusion protein components can be encoded by one or more polynucleotides in whole or in part.
  • modified RNAs e.g., modified mRNAs encoding the fusion protein components, for example, a modified RNA, e.g., modified mRNA encoding a DNA binding domain, and a modified RNA, e.g., modified mRNA encoding a DNA polymerase domain.
  • modified RNA e.g., modified mRNA comprising a modified RNA, e.g., modified mRNA encoding a DNA binding domain, and a modified RNA, e.g., modified mRNA encoding a DNA polymerase domain.
  • the modified RNA e.g., modified mRNA encoding a DNA binding domain
  • the modified RNA e.g., modified mRNA encoding a DNA polymerase domain
  • a linker modified RNA e.g., modified mRNA (e.g., that encodes a peptide linker) to result in a fusion protein that comprises the DNA polymerase domain and DNA binding domain linked by a peptide linker.
  • the modified RNA, e.g., modified mRNA sequence encoding a DNA binding domain, and the modified RNA, e.g., modified mRNA encoding a DNA polymerase domain are linked by a linker modified RNA, e.g., modified mRNA (e.g., that encodes a peptide linker) further comprises one or more modified RNA, e.g., modified mRNA sequences encoding one or more nuclear localization signals (NLS) to result in a fusion protein that comprises the DNA polymerase domain and DNA binding domain linked by a peptide linker and further fused to or linked to one or more NLS.
  • modified RNA e.g., modified mRNA sequence encoding a DNA binding domain
  • modified RNA e.g., modified mRNA encoding a DNA polymerase domain
  • NLS nuclear localization signals
  • a fusion protein encoded by one or more polynucleotides in whole or in part comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) can have the following structure: [NLS]-[Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)].
  • a fusion protein encoded by one or more polynucleotides in whole or in part comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) can have the following structure: [NLS]-[Cas9 (R221K N394K H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)].
  • the modified RNA e.g., modified mRNA encodes a protein
  • a double stranded target DNA e.g., a target gene of interest by editing, e.g., prime editing.
  • the disclosure is not limited to the embodiments described and exemplified above but is capable of variation and modification within the scope of the appended claims.
  • EXAMPLES [0266] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.
  • EXAMPLE 1 General Material and Methods [0267] Chemical synthesis of the cap dinucleotide begins with monophosphorylation of the commercially available 5’-hydroxy group of the nucleoside using POCl 3 and trialkyl phosphate. The corresponding monophosphate can be activated to imidazolide salt using imidazole, triphenylphosphine and aldrithiol. Use of imidazolide as phosphate group activator for adding phosphate or coupling reactions with another nucleotide acting as the nucleophile is a widely adapted strategy (Sawai and Wakai, J Org Chem 1999, 64 (16), 5836–5840, which is incorporated by reference in its entirety).
  • the next step is key reaction of methylating the guanosine using dimethyl sulfate in the aqueous solution as shown in Scheme 1 (Kore and Shanmugasundaram, Lett Org Chem 2009, 6 (2), 141–144, which is incorporated by reference in its entirety).
  • Scheme 1 Synthesis of dinucleotide cap from 3’-OMe-GMP.
  • the coupling reaction between the two nucleotides can be conducted in three ways as shown in Scheme 2. Although different divalent metal catalysts such as MgCl2, MnCl2, CaCl2, CdCl2, and ZnCl2 have been employed in both aqueous and anhydrous conditions, the use of zinc chloride under anhydrous conditions involving polar aprotic solvent, DMF provides high yields of product with shortening of reaction time (Kadokura et al., Tetrahedron Lett 1997, 38 (48), 8359–8362).
  • Non-limiting examples of R 1 groups include: , Scheme 5.
  • N7-G Modifications [0269] 5’-cap analogs having N7-G modifications are prepared analogously to literature reports (e.g., Grudzien et al., Rna 2004, 10 (9), 1479–1487; Kore et al., Bioorgan Med Chem 2013, 21 (15), 4570–4574; Wojcik et al., Pharm 2021, 13 (11), 1941, each of which is incorporated by reference in its entirety).
  • a representative synthetic scheme illustrating the preparation of 5’-cap analogs having N7-G modifications is shown in Scheme 6.
  • Non-limiting examples of such analogs are shown in Scheme 7.
  • Non-limiting examples of R 2 groups include:
  • Triphosphate Bridge Modifications [0270] 5’-Cap analogs possessing modifications of the triphosphate bridge—including but not limited to CX2, phosphorothioate, and triazole modifications—are prepared analogously to literature reports (e.g., Rydzik et al., Nucleic Acids Res 2017, 45 (15), 8661–8675; Wojtczak et al., J Am Chem Soc 2018, 140 (18), 5987–5999; Warminski, ACS Chem Biol 2021, 16 (2), 334– 343; Kowalska et al., RNA 2008, 14(6), 1119–1131; Kowalska et al., ChemBioChem 2009, 10(15), 2469–2473; Warminski et al., Bioorg.
  • Non-limiting example structures of triphosphate bridging oxygen modifications include -O-, -S-, -CH2-, -CCl2-, -CF2-, or -NH-.
  • Non-limiting example structures of triphosphate non-bridging oxygen modifications include -O ⁇ , -S ⁇ (S p isomer), -S ⁇ (R p isomer), -Se ⁇ or -BH3 ⁇ , with the proviso that at least one of Y 1 , Y 2 and Y 3 is -O ⁇ .
  • Scheme 10A General synthetic protocol for further nucleobase modifications. Further Sugar Modifications [0273] 5’-Cap analogs possessing further sugar modifications are prepared analogously to literature reports (e.g., Sikorskiet al., Nucleic Acids Res 2020, 48 (4), 1607–1626; Kore and Shanmugasundaram, Lett Org Chem 2009, 6 (2), 141–144; Shoji et al., J Org Chem 2022, 87 (17), 11743–11750). Non-limiting examples of such analogs are shown in Scheme 11 and 11A.
  • limiting R 3 groups include: methoxy, ethoxy, propoxy, isopropoxy, allyloxy, propargyloxy, - OCH2CH2OCH3, and -OCF3.
  • Scheme 11A A general synthetic protocol for further sugar modifications.
  • nucleotide length of 5’-cap molecule can be from two nucleotides to up to six nucleotides.5’-cap analogs possessing nucleotide chain length modifications are prepared analogously to literature reports (see, e.g., Worch et al., Nucleosides Nucleotides Nucleic Acids 2005, 24 (5–7), 1131–1134). Non-limiting examples of such analogs are shown in Scheme 12 and 12A.
  • nucleotides were eluted using a linear gradient of triethylammonium bicarbonate (TEAB) in deionized water and the collected fractions were tested for the presence of the product by MS and UPLC. Fractions containing the desired product were pooled and evaporated in vacuo with repeated addition of ethanol to decompose TEAB. Compounds were isolated in the form of triethylammonium (TEA + ) salts. These were lyophilized to obtain white fluffy solids.
  • TEAB triethylammonium bicarbonate
  • RNAs attached to the solid support were treated with 0.1 M 1,8-Diazabicyclo(5.4.0)undec-7-ene or 20% Diethylamine in acetonitrile to remove 2-cyanoethyl protecting groups. Finally, the solid support was washed with acetonitrile and flushed with argon. [0285] The product was cleaved from the solid support and de-protected with AMA (methylamine/ammonium hydroxide 1:1; 55 °C, 1 h), evaporated to dryness and redissolved in DMSO.
  • AMA methylamine/ammonium hydroxide 1:1; 55 °C, 1 h
  • the 2’-TBDMS groups were removed using triethylammonium trihydrofluoride (TEA ⁇ 3HF 65 °C, 3 h), and then the mixture was cooled down for 30 mins at 4 °C. The product was precipitated out using cold n-butanol. In cases where the crude purity was not satisfactory, RP-prep-HPLC or ion-exchange chromatography on DEAE Sepharose was used to (0-1 M TEAB) to afford after evaporation triethylammonium salt of pNpG dinucleotide.
  • TEA ⁇ 3HF 65 °C, 3 h triethylammonium trihydrofluoride
  • Product was precipitated by pouring the crude into anhydrous sodium perchlorate solution (10 eq in dry acetone) in a centrifuge tube. The mixture is centrifuged at 3300 RPM, 4 °C for 10 mins. The supernatant is discarded, and additional dry acetone (10 mL) is added and centrifuged. This process is repeated three times. The resulting solid is dried under vacuum to get desired activated phosphate product as white solid and used as such in the next reaction.
  • the washed resin was added to sample in 0.4-1 mL water with 0.4 mL wash of the sample vials. Resin and sample solution were shaken at 190 rpm for 60 minutes. The solution was decanted, and resin was washed further with RNase free water (1 mL), and decanted. Combined decanted solutions were checked on nanodrop to determine concentration and lyophilized to get the sodium salts as dry powder. The final sodium salt was resuspended in water to desired concentrations for in vitro transcription. [0289] The following scheme 13 shows a general synthetic route to prepare an example synthetic route to an example compound mCAP001. 2.5a General Synthesis of mCAP001
  • Reaction mixture was quenched by adding 10 volumes of 0.05M aq. solution of EDTA (20 mg/mL). After adjusting the pH to neutral, the crude product was purified by anion exchange chromatography (DEAE resin and TEAB) and then prep-HPLC (20 mM TEAA and Methanol) to give mCAP005 (0.4 mg, yield: 3.6%) as white solid after repeated evaporations and lyophilization.
  • the compound was characterized by HPLC and MS (FIG.4).
  • reaction mixture was quenched by adding 10 volumes of 0.05M aq. solution of EDTA (20 mg/mL). Adjusted the pH to neutral and the crude was purified by anion exchange chromatography (DEAE resin and TEAB) and then prep-HPLC (20 mM TEAA and Methanol) to give mCAP006 (0.4 mg, yield: 3.6%) as white solid after repeated evaporations and lyophilization.
  • the product was characterized by HPLC, and MS (FIG.8).
  • the final compound obtained as TEA salt was converted to sodium salt form using Amberlite IR- 120 Na+ form (PLUS) ion-exchange resin.
  • LCMS showed the consumption of starting material and a main peak with desired mass corresponding to PM002_1.
  • TEA was added to the reaction mixture to adjust the pH to 8 and the mixture was concentrated under reduced pressure.
  • the crude was purified by prep-HPLC (Column: Phenomenex Titank C18 Bulk 250 ⁇ 70 mm 10 u; mobile phase: [H2O (50 mM TEAB)-acetonitrile]; gradient: 0%-10% B over 18.0 min).
  • mCAP008 was synthesized and purified as described above for other final caps and characterized by HPLC, MS (FIG.10).
  • HPLC: t R 13.678 min, Purity: 95% Yield: 2.4 mg (9.5%).
  • mRNA sequences to be produced were previously cloned into a pDNA vector containing a T7 promoter augmented to enable co-transcriptional capping of “AG” initiating cap analogs, the mRNA sequence of interest, and a unique linearization site adjacent to the poly(A) tail via BspQ1, BbsI, BsaI, or similar DNA restriction endonuclease (All DNA endonucleases provided by New England Biolabs, NEB).
  • IVT In Vitro Transcription
  • N1-Methyl-Pseudouridine modified Prime Editor mRNA for each cap analog was produced using New England BioLab’s HiScribe® T7 High Yield RNA Synthesis Kit (NEB, Cat# E2040S) with minor protocol modifications.
  • in vitro transcription with co- transcriptional capping was performed by transcribing ⁇ 0.04 mg/mL of linear IVT template leveraging 10 mM of each Nucleotide Triphosphate (Adenosine-Triphosphate [ATP], Cytidine- Triphosphate [CTP], Guanosine-Triphosphate [GPT], and N1-Methyl-Pseudourine-Triphosphate [N1MePsU]; ATP, CTP, and GTP provided within HiScribe kit, N1MePsU provided by Trilink Cat# N-1081), 7.5 mM of the cap analog being tested (e.g.
  • ATP Addenosine-Triphosphate
  • CTP Cytidine- Triphosphate
  • GTP Guanosine-Triphosphate
  • N1MePsU N1-Methyl-Pseudourine-Triphosphate
  • mCap001 1x HiScribe T7 Reaction buffer, and 11unit/ ⁇ L HiScribe Enzyme Mix.
  • the reaction was incubated at 37C for 2 hours.
  • To the IVT reaction was then added 1x DNase Buffer and 50 U/mL of Turbo DNAse (ThermoFisher Scientific Cat# AM2238), and the mixture was mixed an additional 30 min. at 37 C.
  • the IVT reaction was quenched by the addition of 0.5M EDTA (InVitrogen, Cat# AM9260G) to a final concentration of 50 mM EDTA in the quenched reaction. Quenched IVT (QIVT) was then stored at 4 °C or -20 °C until further processed.
  • Qiagen RNAEasy purification kit Qiagen, Mini Cat# 74104, or Midi Cat# 75144, or Maxi Cat# 75162
  • FLP Full-Length Product
  • R 1 is H, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently at each instance hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified , modified , with the proviso that when R 1 is hydrogen or methyl while R 2 is
  • R 2 is C 1 -C 6 alkyl, C 2 -C 6 alkyl substituted with one hydroxyl or 1-3 fluoro, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle, heterocycloalkyl-CH2-CH2-
  • R 2 is C1-C6 alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo
  • a messenger RNA (mRNA) sequence comprising a 5’-end motif of Formula (III): or a stereoisomer, tautomer, solvate, or salt thereof, wherein:
  • R 1 is H, C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl, C3-C6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH 2 -CH 2 -, C 3 -C 6 cycloalkyl-CH 2 -CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH 2 -, C 3 -C 6 cycloalkyl-CH 2 - substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkyl substituted with methyl on the heterocycle
  • each of R 3 , R 3′ , R 3′′ , R 4 , R 11 , R 12 , R 13 , and R 14 is independently at each occurrence hydrogen, halogen, or -OR 20 ;
  • R 20 is independently at each instance hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with one C 1 -C 3 alkoxy or 1-3 groups of fluoro, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl;
  • each of B 3 , B 4 , B 5 and B 6 is independently adenine, cytosine, guanine, uracil, modified adenine, modified cytosine, modified guanine, or modified uracil;
  • B 2 is adenine, cytosine, guanine, uracil, modified adenine, modified cyto
  • R 2 is C1-C6 alkyl, C2-C6 alkyl substituted with one hydroxyl or 1-3 fluoro, C3-C6 cycloalkyl, C 3 -C 6 cycloalkyl substituted with hydroxyl or 1-3 groups of fluoro, C 3 -C 6 cycloalkyl-CH2-CH2-, C3-C6 cycloalkyl-CH2-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, C3-C6 cycloalkyl-CH2-, C3-C6 cycloalkyl-CH2- substituted with one hydroxyl or 1-3 groups of fluoro, arylalkyl, arylalkyl substituted independently with 1-3 groups of methoxy, fluoro, chloro, or bromo on the aryl, hetero
  • R 14 is -OH, -OCH 3 , -F, or -OCH 2 CH 2 OCH 3 .
  • 34 The mRNA sequence of any one of Embodiments 24-33, or a stereoisomer, A 2 tautomer, solvate, or salt thereof, wherein M in is the bond linked to the RNA.
  • 35 The mRNA sequence of any one of Embodiments 24-33, or a stereoisomer, tautomer, solvate, or salt thereof, wherein: R 14 is -OH, -OCH 3 , -F, or -OCH 2 CH 2 OCH 3 .
  • 36 36.
  • a cell comprising a ribonucleic acid (RNA) molecule comprising the mRNA sequence according to any one of Embodiments 24-45.
  • RNA ribonucleic acid
  • a cell comprising a polypeptide or protein translated from an RNA molecule comprising the mRNA sequence according to any one of Embodiments 24-45.
  • 48. A pharmaceutical composition comprising an RNA molecule comprising the mRNA sequence according to any one of Embodiments 24-45.
  • Embodiment 48 further comprising one or more single guide RNA designed to target one or more specific locations of one or more genes of interest to elicit pharmacological effect upon administration into a mammal.
  • 50 A method for synthesizing a ribonucleic acid (RNA) molecule comprising: (a) introducing the mRNA sequence according to any one of Embodiments 24-45 into a mixture comprising an RNA polymerase, and (b) incubating the mixture for a time sufficient to allow for transcription of the RNA molecule.
  • the mixture further comprises a DNA template and nucleoside triphosphates.
  • a method of gene editing comprising introducing into a cell a ribonucleic acid (RNA) molecule comprising the mRNA sequence according to any one of Embodiments 24-45, or a pharmaceutical composition of Embodiment 45 or Embodiment 46, wherein the RNA molecule encodes a therapeutic protein, wherein the RNA molecule is translated in the cell.
  • RNA ribonucleic acid
  • AAV adeno-associated viral vector
  • the vector of Embodiment 53 wherein the vector is a lipid nanoparticle (LNP) comprising the mRNA sequence, wherein the mRNA sequence is encapsulated in or associated with the LNP.
  • LNP lipid nanoparticle
  • VLP virus-like particle
  • RNA ribonucleic acid
  • RNA molecule of Embodiment 57 further comprising an RNA sequence encoding a therapeutic protein.
  • a method of producing a protein in a cell comprising: (a) introducing a modified messenger ribonucleic acid (mRNA) molecule into a cell; and (b) translating the mRNA molecule in the cell to make a protein; wherein the modified mRNA molecule comprises the mRNA sequence according to any one of Embodiments 24-45, and wherein: (i) the mRNA molecule has a translation efficiency of at least 1.2 fold of that of a corresponding unmodified mRNA molecule, or; (ii) the mRNA molecule persists in the cell at least 20% longer than the corresponding unmodified mRNA molecule, or; (iii) the mRNA molecule produces the protein at least 20% more than the corresponding unmodified mRNA molecule, or; (iv) the mRNA molecule induces an innate
  • mRNA messenger ribonucleic acid
  • RNA molecule of Embodiment 57 or Embodiment 58 for use as a medicament.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pain & Pain Management (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne une coiffe en 5' d'ARNm chimiquement modifiée, et des procédés de préparation et d'utilisation de celle-ci.
PCT/US2023/082144 2022-12-02 2023-12-01 Coiffe en 5' d'arnm modifiée et ses méthodes d'utilisation WO2024119117A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8519110B2 (en) * 2008-06-06 2013-08-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College mRNA cap analogs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8519110B2 (en) * 2008-06-06 2013-08-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College mRNA cap analogs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GRUDZIEN ET AL.: "Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency", RNA, vol. 10, no. 9, 2004, pages 1479 - 1487, XP002452227, DOI: 10.1261/rna.7380904 *
KLOCKER ET AL.: "Photocaged 5' cap analogues for optical control of mRNA translation in cells", NATURE CHEMISTRY, vol. 14, August 2022 (2022-08-01), pages 905 - 913, XP037928369, DOI: 10.1038/s41557-022-00972-7 *

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