WO2025224155A1 - Compositions et procédés pour introduire une macromolécule dans une cellule - Google Patents

Compositions et procédés pour introduire une macromolécule dans une cellule

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Publication number
WO2025224155A1
WO2025224155A1 PCT/EP2025/061036 EP2025061036W WO2025224155A1 WO 2025224155 A1 WO2025224155 A1 WO 2025224155A1 EP 2025061036 W EP2025061036 W EP 2025061036W WO 2025224155 A1 WO2025224155 A1 WO 2025224155A1
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WIPO (PCT)
Prior art keywords
cell
alkyl
oligonucleotide
hydro
compound
Prior art date
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Pending
Application number
PCT/EP2025/061036
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English (en)
Inventor
Anders DAHLÈN
Tom BALADI
Carl-Johan CARLING
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AstraZeneca AB
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AstraZeneca AB
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Filing date
Publication date
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Publication of WO2025224155A1 publication Critical patent/WO2025224155A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01008—Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • compositions and methods for delivering a macromolecule e.g., an oligonucleotide, a polypeptide, or a combination thereof, to a cell
  • a macromolecule e.g., an oligonucleotide, a polypeptide, or a combination thereof
  • compositions and methods for delivering a macromolecule e.g., an oligonucleotide, a polypeptide, or a combination thereof, to the cytosol and/or nucleus of a cell.
  • Antisense oligonucleotides are short, exogenous, single stranded DNA or RNA with a sequence that is complementary to a nucleotide sequence of a target nucleic acid.
  • Classic single stranded antisense oligonucleotides can act in the nucleus to cleave mRNAs via an RNase H dependent mechanism.
  • Many therapeutic ASOs are ‘gapmers’, which have a central DNA region that supports RNase H activity that is flanked by chemically modified ends to increase affinity and reduce susceptibility to nucleases. (Bennett et al. (2010) Annu. Rev. Pharmacol. Toxicol. 50:259-293).
  • Splice switching oligonucleotides are another form of antisense oligonucleotides that hybridize with pre-mRNA to disrupt transcript splicing by blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery (Havens and Hastings (2016) Nucleic Acids Res. 44(14);6549-63). SSOs can be designed to induce intron and exon inclusion or exclusion, ultimately restoring or inhibiting protein function or re-directing splicing to produce alternative protein isoforms. Additionally, SSOs can be used to mask aberrant splice sites, thereby restoring normal alternative splicing to produce functional proteins.
  • any pre-mRNA sequence could be targeted with SSOs, but to date, only four SSOs have been FDA approved.
  • SSOs See e.g., Neil and Bisaccia (2019) J. Pediatr. Pharmacol. Ther. 24(3): 194-203; Kim et al. (2019) N. Engl. J. Med. 381:1644-1652). It is estimated that up to 70% of human genes undergo alternative splicing, and 50% of human genetic diseases arise from mutations that affect splicing. (Bauman et al., (2009) Oligonucleotides.
  • SMA Spinal Muscular Atrophy
  • DMD Duchenne Muscular Dystrophy
  • RNA interference is an endogenous regulatory pathway for control of gene expression in which short (approx. 15 - 22 bp) double-stranded RNA fragments, known as small interfering RNAs (siRNAs), are loaded into an RNA-induced silencing complex (RISC) to cleave target mRNA in a sequence-dependent manner.
  • siRNAs small interfering RNAs
  • RISC RNA-induced silencing complex
  • nucleic acid-based therapeutics are gaining attention as a promising approach for treatment of a variety of diseases and disorders, many have failed to meet therapeutic end points, often due to challenges with effective methods for in vivo delivery.
  • One hindrance to the widespread use of oligonucleotide therapeutics is the inability of the oligonucleotide to escape endosomal compartments and reach the cytosol or nucleus in sufficient concentrations. (Juliano et al. (2008) Nucleic Acids Res. 36(12):4158-4171).
  • Endosomolytic small molecule compounds are compounds that facilitate the release of gymnotically delivered oligonucleotides that might otherwise accumulate in endosomes or lysosomes.
  • Some endosomolytic SMCs induce endosomal membrane destabilization by buffering the lumen of endosomes as the luminal pH decreases with endosomal maturation. The increase in luminal pH occurs quickly and can be reversible with proper dosing. (Maxfield, F.R. (1982) J. Cell Biol. 95(2):676-681). This buffering leads to an increase of luminal osmotic pressure, engorging the endosome and triggering membrane rupture, ultimately allowing the endosomal cargo to leak into the cytosol.
  • Chloroquine and derivatives thereof have been widely used to enhance activity of oligonucleotide-containing nanoparticles by promoting endosomal release. Although these compounds display great potency in vitro, high micromolar concentration ranges are typically required and there is a narrow window between effective and toxic concentrations.
  • chloroquine induces leakage between 40-100 ⁇ M (Ldnn et al. (2016) Sci. Rep. 6:32301; Heath et al. (2019) Nanomedicine. 14(21):2799-2814).
  • compositions and methods for delivering a macromolecule e.g., an oligonucleotide, a polypeptide, or a combination thereof, to a cell are provided.
  • a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula I is provided.
  • Formula I one of Zi and Z2 is N, and the other is C;
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl;
  • Re is hydro or C1-C4 alkyl
  • R7, R8, R9, Rio, R11 are each independently CHR12, CR12R17 or NR13;
  • R12 is hydro, C1-C4 alkyl, -OR14, or -CO2R15;
  • R14 is hydro or C1-C4 alkyl
  • R15 is hydro or C1-C4 alkyl
  • R16 is hydro or C1-C4 alkyl
  • R17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.
  • a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula I is provided.
  • Formula I one of Zi and Z2 is N, and the other is C;
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl;
  • Re is hydro or C1-C4 alkyl
  • R7, R8, R9, Rio, R11 are each independently CHR12, CR12R17 or NR13;
  • R12 is hydro, C1-C4 alkyl, -OR14, or -CO2R15;
  • R14 is hydro or C1-C4 alkyl
  • R15 is hydro or C1-C4 alkyl
  • R16 is hydro or C1-C4 alkyl
  • R17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; and wherein one or more of the alkyl are optionally substituted with one or more halo, and wherein the compound of Formula I is conjugated via R7, R8, R9, Rio or Rn, or a pharmaceutically acceptable salt thereof.
  • a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula la is provided.
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • R14 is hydro or C1-C4 alkyl
  • R15 is hydro or C1-C4 alkyl
  • R16 is hydro or C1-C4 alkyl
  • lb denotes the conjugation point; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.
  • EEE endosomal escape enhancer
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • R14 is hydro or C1-C4 alkyl
  • R15 is hydro or C1-C4 alkyl
  • R16 is hydro or C1-C4 alkyl
  • a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula Ic, Formula Id, Formula le or Formula If is provided.
  • EEE endosomal escape enhancer
  • a macromolecule conjugated to one or more endosomal escape enhancer (EEE) compounds of Formula I, la, lb, Ic, Id, le or If is provided, wherein R1 is hydro, halo, C1-C4 alkyl, -OR4, -C( O)NR5R5, -CO2R6, or cyano; X is hydro, C1-C4 alkyl, or -OR2,
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl
  • R14 is hydro or C1-C4 alkyl
  • R15 is hydro or C1-C4 alkyl
  • R16 is hydro or C1-C4 alkyl; wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.
  • R1 is cyano, halo, or C1-C4 alkyl optionally substituted with one or more chloro or fluoro.
  • R1 is cyano, methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, fluoro, chloro, or bromo.
  • R1 is cyano, bromo, chloro, fluoro, or trifluoromethyl.
  • R1 is fluoro.
  • X is -OR2, and R2 is methyl, ethyl, or isopropyl. In one aspect, X is hydro.
  • R3 is hydro; cyano; C1-C4 alkyl optionally substituted with one or more chloro, fluoro, or hydroxy; or -OR4, wherein R4 is C1-C4 alkyl.
  • R7, R8, Rio, and R11 are each independently CHR12 or CR12R17, and R9 is NR13.
  • each R12 is hydro.
  • R13 is -(CH2)3OH.
  • R13 is methyl.
  • R1 is halo
  • X is -OR2
  • R2 is ethyl or methyl
  • R3 is cyano
  • R1 is haloalkyl
  • X is -OR2
  • R2 is ethyl or methyl
  • R3 is cyano
  • R13 is hydro or methyl.
  • R1 is halo
  • X is - OR2
  • R2 is ethyl or methyl
  • R3 is methyl or methoxy
  • R13 is hydro.
  • R1 is halo
  • X is -OR2
  • R2 is C1-C4 alkyl
  • R3 is hydro
  • R13 is hydro.
  • R1 is haloalkyl
  • X is -OR2
  • R2 is ethyl or methyl
  • R3 is hydro
  • R13 is hydro or methyl.
  • R1 is halo, X is hydro, R3 is cyano, and R13 is hydro.
  • Ri is cyano, X is -OR2, R2 is ethyl or methyl, R3 is haloalkyl, and R13 is hydro.
  • Ri is ethyl or methyl
  • X is -OR2, R2 is ethyl or methyl
  • R3 is hydro
  • R13 is hydro.
  • R1 is halo, X is -OR2, R2 is ethyl or methyl, R3 is -(CH2)yOH a,nd R13 is hydro, wherein y is 0, 1, 2, or 3.
  • EEE Exemplified endosomal escape enhancer
  • composition in one aspect, includes a peptide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof.
  • a pharmaceutical composition in one aspect, includes an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof; and a pharmaceutically acceptable diluent or carrier.
  • composition in one aspect, includes an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof.
  • a pharmaceutical composition in one aspect, includes an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof; and a pharmaceutically acceptable diluent or carrier.
  • the oligonucleotide is single stranded. In one aspect, the oligonucleotide is double stranded. In one aspect, the oligonucleotide includes DNA. In one aspect, the oligonucleotide includes RNA. In one aspect, the oligonucleotide includes from about 8 to about 30 nucleotides.
  • the oligonucleotide is an antisense oligonucleotide (ASO), a splice switching oligonucleotide (SSO), interfering RNA (RNAi), small interfering RNA (siRNA), micro RNA (miRNA), an antagomir, a decoy oligonucleotide, or a combination thereof.
  • ASO antisense oligonucleotide
  • SSO splice switching oligonucleotide
  • RNAi interfering RNA
  • siRNA small interfering RNA
  • miRNA micro RNA
  • antagomir antagomir
  • decoy oligonucleotide or a combination thereof.
  • the oligonucleotide includes one or more modified nucleotides.
  • the one or more modified nucleotides comprise: phosphodiester (PO); phosphorothioate (PS); 2’0-methyl (2’OMe); 2’0-methoxyethyl (MOE); peptide nucleic acid (PNA); phosphoroamidate morpholino (PMO); locked nucleic acid (LNA); 2’-deoxy-2’- fluoro (2’-F); any other 2’ modified oligonucleotide; or a combination thereof.
  • the compound of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof is attached to the macromolecule via a linker.
  • the compound of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof is attached to the oligonucleotide via a linker.
  • the linker is cleavable.
  • a method of introducing an oligonucleotide into a nucleus and/or cytosol of a cell is provided.
  • the method includes: contacting the cell with an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof.
  • the conjugated compound(s) facilitates entry of the oligonucleotide into the nucleus and/or cytosol of the cell.
  • the oligonucleotide is internalized by the cell through endocytosis and encapsulated within an endosome and the compound facilitates release of the oligonucleotide from the endosome. In one aspect, the oligonucleotide is internalized by transient pore formation induced by the conjugated compound(s).
  • contacting the cell with the oligonucleotide conjugated to one or more compounds of Formulae I, la, lb, Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, Ic, Id, le, If or of Table 1, or a combination thereof is performed in a composition, wherein the composition includes about 0.025 ⁇ M to about 20 ⁇ M of the oligonucleotide. In one aspect, the composition includes about 0.1 ⁇ M to about 10 ⁇ M, about 0.1 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M of the oligonucleotide.
  • the oligonucleotide hybridizes to a target nucleic acid in the cell.
  • the target nucleic acid is in the nucleus of the cell. In one aspect, the target nucleic acid is in the cytosol of the cell.
  • the oligonucleotide alters activity of a gene expressed by the cell. In one aspect, the oligonucleotide increases activity of a gene expressed by the cell. In one aspect, the activity of the gene expressed by the cell is increased at least about lOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s).
  • the activity of the gene expressed by the cell is increased at least about lOOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s).
  • the oligonucleotide decreases activity of a gene expressed by the cell.
  • the activity of the gene expressed by the cell is decreased at least about lOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s).
  • the activity of the gene expressed by the cell is decreased at least about lOOx when the cell is contacted with the oligonucleotide conjugated to the compound(s) as compared to a cell that is contacted with the oligonucleotide not conjugated to the compound(s).
  • the method includes in vitro delivery of the oligonucleotide to the cell. In one aspect, the method includes in vivo delivery of the oligonucleotide to the cell.
  • the cell is a cultured cell. In one aspect, the cell is an isolated cell. In one aspect, the cell is an isolated cell from a subject in need of treatment. In one aspect, the cell is part of a tissue or organ. In one aspect, the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, adipose, and/or muscle (e.g., skeletal muscle).
  • CNS central nervous system
  • PNS peripheral nervous system
  • the cell is a brain cell, a CNS cell, a PNS cell, a heart cell, a liver cell, a kidney cell, a spleen cell, a pancreas cell, a lung cell, a muscle cell, an adipose cell, an immune cell, or combination thereof.
  • the cell is a mammalian cell. In another aspect, the cell is a eukaryotic cell and/or a prokaryotic cell.
  • a method of releasing an oligonucleotide from an endosome includes: contacting the cell with the oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, or a combination thereof, wherein the oligonucleotide is internalized by the cell through endocytosis and encapsulated within the endosome, and wherein the compound(s) facilitates release of the oligonucleotide from the endosome.
  • a method for the treatment and/or prevention of a disorder in a subject includes: administering to the subject a therapeutically effective amount of an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, or a combination thereof.
  • administration includes parenteral administration.
  • administration includes intravenous or subcutaneous administration.
  • the subject is a mammal. In one aspect, the subject is a human.
  • a method for the treatment and/or prevention of a disorder in a subject includes: isolating a cell from the subject; contacting the isolated cell with a therapeutically effective amount of an oligonucleotide conjugated to one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, a pharmaceutically acceptable salt of one or more compounds of any one of Formulae I, la, lb, or Ic, Id, le, If or of Table 1, or a combination thereof to produce an engineered cell; and transplanting the engineered cell in the subject.
  • the isolated cell is contacted with a composition that includes about 0.025 ⁇ M to about 20 ⁇ M of the oligonucleotide. In one aspect, the composition includes about 0.1 ⁇ M to about 10 ⁇ M, about 0.1 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M of the oligonucleotide.
  • the subject is a mammal. In one aspect, the subject is a human.
  • FIG. 1 is a general schematic of the synthesis of the preparation of Compounds 2-7, 19-31.
  • FIG. 2 is a breakdown of the starting material “X” identified in FIG. 29 and the required intermediate for each of the Compounds 2-7, 19-31.
  • FIG. 3 is a schematic of the synthesis of Compound 2.
  • FIG. 4 is a schematic of the synthesis of Compound 3.
  • FIG. 5 is a schematic of the synthesis of Compound 4.
  • FIG. 6 is a general schematic of the synthesis of the preparation of Compounds 8-17 with the additional alkylation step.
  • FIG. 7 is a general schematic of the synthesis of the preparation of Compound 18.
  • FIG. 8 is a schematic of the synthesis of Compound 7.
  • FIG. 9a % knockdown (normalized to untreated) in HEK293T wt after 24 hours.
  • FIG. 9b % knockdown (normalized to untreatedjin HEK293 GAL9-mCherry cells after 24 hours.
  • “Lower alkyl” refers to an alkyl group having from 1 and up to about 8 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one aspect, the alkyl group includes from 1 to 4 carbon atoms (C1-C4 alkyl). Lower alkyls include, but are not limited to, methyl, ethyl, propyl, isopropyl, and butyl, including n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • the alkyl group may be substituted or unsubstituted.
  • the alkyl group is substituted with one or more halo groups, e.g., F, Cl, Br, I, At, etc.
  • the alkyl group is substituted with one or more F or Cl, e.g., a mono-, di- or tri- fluoro or chloro alkyl.
  • An alkyl group in which one or more of the hydrogen atoms are replaced by halogen can be referred to as “halo alkyl”, e.g., “halo C1-C4 alkyl” refers to a Ci- C4 alkyl substituted by one or more of the same or different halogen atoms.
  • Examples of C1- C4 alkyls substituted with one or more halo groups include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.
  • Alkoxy also represented as “-OR4” where R4 is an alkyl group, refers to a saturated or unsaturated branched or straight-chain hydrocarbon group attached to a parent molecule through an oxygen atom.
  • the alkoxy group includes from 1 to 4 carbon atoms (C1-C4 alkoxy). Examples of C1-C4 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, including n-butoxy, sec-butoxy iso-butoxy and t-butoxy.
  • the alkoxy group is substituted with one or more halo groups, e.g., F, Cl, Br, I, At, etc.
  • the alkoxy group is substituted with one or more F or Cl, e.g., a mono-, di-, or tri- fluoro or chloro alkoxy.
  • Halogen or “halo” can be used interchangeably to refer to a fluoro, chloro, bromo, or iodo group. In one aspect, halo refers to fluoro, chloro, or bromo. In one aspect, halo refers to fluoro or chloro.
  • Amide “ refers to the group “-C(O)NR5R5” (also represented as “-C( O)NR5R5”) where each R5 is independently hydro or alkyl (optionally substituted and/or interrupted) and includes primary, secondary, and tertiary amides.
  • the alkyl in the amide includes a Ci to Cs alkyl.
  • the alkyl substituent includes a Ci to C4 alkyl.
  • at least one R5 in the formula “-C(O)NR5R5” is a hydro.
  • the ester is a “short chain ester,” wherein Re is C1-C4 alkyl.
  • Re is methyl or ethyl.
  • Re is propyl or isopropyl.
  • Hydro refers to a hydrogen substituent and is also represented by “-H ”
  • Endosomolytic refers to one or more compounds that facilitates release of an oligonucleotide from an endosome/lysosome/autophagosome/multivesicular body or other endosomal vesicle into the cytosol of a cell.
  • the endosomolytic agent is one or more small molecule compounds (SMC).
  • SMC small molecule compounds
  • the endosomolytic agent has a structure represented by any of Formulae I, la, or of Table 1, or a pharmaceutically acceptable salt thereof.
  • “Small molecule compound” or “SMC” refers to an organic molecule with a molecular weight of less than 1000 g/Mol and includes compounds having a structure represented by any of Formulae I, la, or of Table 1.
  • Nucleic acid refers to an oligomer or polymer of nucleotides and includes naturally occurring or synthetically produced single stranded or double stranded deoxyribonucleotides (DNA) or ribonucleotides (RNA).
  • a nucleic acid can include naturally occurring nucleic acid nucleobases such as adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), as well base analogs or modified nucleobases that do not occur in nature.
  • Target nucleic acid refers to a nucleic acid to which an antisense oligonucleotide hybridizes.
  • hybridization of an antisense oligonucleotide to a target nucleic acid in a cell alters activity of a gene expressed by the cell.
  • hybridization of the antisense oligonucleotide to the target nucleic acid increases activity of a gene expressed by the cell.
  • hybridization of the antisense oligonucleotide to the target nucleic acid decreases activity of a gene expressed by the cell.
  • Oligonucleotide refers to an exogeneous, naturally occurring, or non-naturally occurring single- stranded or double- stranded polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA). In one aspect, the oligonucleotide is about 2 to about 50 nucleotides in length.
  • the oligonucleotide includes one or more nucleotide analogs or modified backbone residues or linkages, including, but not limited to, phosphodiester (PO); phosphorothioate (PS); 2’0-methyl (2’0Me); 2’0-methoxyethyl (MOE); peptide nucleic acid (PNA); phosphoroamidate morpholino (PMO); locked nucleic acid (ENA); 2’-deoxy-2’- fluoro (2’-F); or a combination thereof.
  • “Locked nucleic acid nucleoside” or “LNA” refers a nucleoside that includes a bicyclic sugar moiety with a 4’-CH2-O-2’bridge.
  • Phosphorothioate refers to an internucleotide linkage in which one of the non-bridging oxygens is replaced by sulfur.
  • modified oligonucleotide refers to an oligonucleotide that includes at least one modified nucleoside and/or at least one modified intemucleoside linkage.
  • an “antisense oligonucleotide” or “ASO” is an oligonucleotide that includes at least a portion of which is complementary to a target nucleic acid such that the ASO can hybridize to the target nucleic acid.
  • An antisense oligonucleotide can increase or decrease expression of a target nucleic acid.
  • a “splice switching oligonucleotide” or “SSO” is a short, synthetic, antisense oligonucleotide that can hybridize to a pre-mRNA and disrupt splicing of the transcript, for example, by blocking the RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA.
  • Pre-mRNA refers to an RNA transcript that includes one or more introns and has not been fully processed into mRNA.
  • siRNA is a class of double-stranded RNA that is non-coding, typically between about 20 to about 25 base pairs, with hydroxylated 3’ and phosphorylated 5’ ends.
  • siRNA is part of the RNA interference pathway and interferes with expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation.
  • siRNA may be conjugated e.g., to sugars such as GalNAc or lipids such as cholesterol, to enhance delivery to a target cell, e.g., with improved pharmacokinetics and/or efficacy. See, e.g., Osborn et al., Nucleic Acid Ther. 28(3): 128-136 (2016).
  • the present disclosure provides a method of enhancing delivery of unconjugated siRNA.
  • Polypeptide used interchangeably herein with “peptide” or “protein,” refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. In some aspects, a polypeptide comprises about 2 to about 5000 amino acids. In some aspects, the polypeptide is capable of providing a site- specific modification in a target nucleic acid. In some embodiments, the polypeptide is a therapeutic polypeptide.
  • a “macromolecule” includes proteins, nucleic acids, carbohydrates, lipids, nanogels, and macrocycles.
  • a macromolecule of the present disclosure comprises an oligonucleotide.
  • a macromolecule of the present disclosure comprises a polypeptide.
  • a macromolecule of the present disclosure comprises one or more components of a site-specific modification (SSM) system described herein, e.g., a CRISPR system, a Cre-Lox system, and/or a FLP-FRT system.
  • SSM site-specific modification
  • references to a SSM system can mean any one or more components of the system.
  • a SSM system comprises a CRISPR system, a Cre-Lox system, a FLP-FRT system, any component thereof, or any combination thereof.
  • a “CRISPR” system is a SSM system capable of performing an SSM at a target nucleic acid.
  • a CRISPR system includes (a) a protein capable of providing the SSM, e.g., a Cas protein; and (b) a guide RNA (also referred to herein as “gRNA”), which includes (i) a “crRNA” or “spacer” region that hybridizes to the target nucleic acid, and (ii) a “tracrRNA” or “scaffold” region that associates with the protein.
  • the SSM comprises single-stranded cleavage of the target nucleic acid.
  • the SSM comprises double- stranded cleavage of the target nucleic acid. In some aspects, the SSM comprises a deletion. In some aspects, the SSM comprises an insertion. In some aspects, the SSM comprises a mutation. In some aspects, the SSM comprises a base edit, e.g., conversion of a C-G base pair to a T-A base pair.
  • a “Cre-Lox” system is a SSM system capable of performing an SSM at a target nucleic acid.
  • a Cre/Lox system includes a Cre recombinase, which recognizes a pair of Lox (also called LoxP) sequences flanking the target nucleic acid and catalyzes site- specific recombination at the target nucleic acid.
  • LoxP also called LoxP
  • An analogous system to the Cre-Lox system is the “FLP-FRT” system.
  • a FLP-FRT system is a SSM system capable of performing an SSM at a target nucleic acid.
  • a FLP-FRT system includes the FLP recombinase, which recognizes a pair of FRT sequences flanking the target nucleic acid and catalyzes site-specific recombination at the target nucleic acid.
  • the SSM comprises an inversion.
  • the SSM comprises an insertion.
  • the SSM comprises a deletion.
  • the SSM comprises a translocation.
  • the location and orientation of the Lox sequences (or the FRT sequences) determines the type of SSM (e.g., inversion, deletion, or translocation) performed by the Cre recombinase (or the FLP recombinase).
  • Hybridize refers to the pairing of complementary oligomeric compounds, for example, pairing between an antisense oligonucleotide and its corresponding target nucleic acid. While not limited to any mechanism, the most common mechanism of pairing involves hydrogen bonding between complementary nucleobases, including, for example, Watson- Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, in Watson-Crick base pairing, guanine (G) is complementary to cytosine (C), adenine (A) is complementary to thymine (T) in DNA, and adenine (A) is complementary to uracil (U) in RNA.
  • G guanine
  • C cytosine
  • A adenine
  • T thymine
  • U uracil
  • Hybridization can occur between two complementary DNA molecules (DNA-DNA hybridization), two RNA molecules (RNA-RNA hybridization), or between complementary DNA and RNA molecules (DNA-RNA hybridization). Hybridization can occur between a short nucleotide sequence that is complementary to a portion of a longer nucleotide sequence. Hybridization can occur between sequences that do not have 100% “sequence complementarity,” i.e., complementary sequences need not have nucleobase complementarity at each nucleoside, although sequences having less sequence complementarity are less stable and less likely hybridize than sequences having greater sequence complementarity.
  • Specifically hybridizes refers to the ability of an oligonucleotide to hybridize to a target nucleic acid with greater affinity than to a different nucleic acid.
  • the antisense oligonucleotide specifically hybridizes to a target nucleic acid sequence under physiological conditions, for example, for in vivo or therapeutic use.
  • Targeting or “targeted to,” in the context of antisense oligonucleotides, refers to the association of an antisense oligonucleotide with a particular target nucleic acid or region of a target nucleic acid.
  • An antisense oligonucleotide targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
  • Targeting refers to the association of the protein of the SSM system (e.g., Cas protein, Cre recombinase, or FLP recombinase) with a particular target nucleic acid or region of a target nucleic acid.
  • the Cas protein of a CRISPR system targets a target nucleic acid upon hybridization of the guide RNA with the target nucleic acid.
  • the Cre recombinase of a Cre-Lox system targets a target nucleic acid upon recognition of the Lox sequences flanking the target nucleic acid.
  • the FLP recombinase of a FLP-FRT system targets a target nucleic acid upon recognition of the FRT sequences flanking the target nucleic acid.
  • “Alter” or “modulate” refer a change in an amount, function, or activity of a molecule, e.g., a macromolecule described herein, when compared to the amount, function, or activity prior to treatment.
  • one or more compounds described herein increases or decreases an amount, function or activity of a gene expressed by a target nucleic acid sequence.
  • the compound(s) increases the activity of an antisense oligonucleotide (ASO) that acts on pre-mRNA via RNase H in the nucleus.
  • ASO antisense oligonucleotide
  • the compound(s) increases the activity of an siRNA that acts via the RISC complex in the cytosol.
  • the compound(s) increases the alteration of pre-mRNA splicing by a splice switching oligonucleotide (SSO), as reflected by an increase in the desired splice variant. In one aspect, the compound(s) results in reduced levels of the corresponding target mRNA and/or protein as compared to treatment with the ASO in the absence of the compound. In some aspects, the compound(s) increases the activity of a Cas protein in a CRISPR system. In some aspects, the compound(s) increases the activity of a Cre recombinase. In some aspects, the compound(s) increases the activity of a FLP recombinase.
  • SSO splice switching oligonucleotide
  • the compound(s) increases the frequency of the SSM at the target nucleic acid. In some aspects, the compound(s) increases the editing efficiency of a SSM system (e.g., the CRISPR, Cre-Lox, and/or FLP-FRT systems).
  • a SSM system e.g., the CRISPR, Cre-Lox, and/or FLP-FRT systems.
  • the compound(s) described herein “enhances the delivery” of a macromolecule provided herein, e.g., an oligonucleotide and/or polypeptide. In one aspect, the compound described herein “enhances the delivery” of an antisense oligonucleotide to increase cytosolic and/or nuclear concentration, accumulation, and/or half-life of the oligonucleotide as compared to that found without administration of the compound(s).
  • the compound(s) described herein “enhances the delivery” of one or more components of a SSM system to increase cytosolic and/or nuclear concentration, accumulation, and/or half-life of the SSM system as compared to that found without administration of the compound.
  • the SSM system comprises a CRISPR system, a Cre-Lox system, a FLP-FRT system, any component thereof, or any combination thereof.
  • “Expression” refers to a process by which a protein is produced in a host cell from a nucleic acid and includes, but is not limited to, transcription, translation, post-translational modification, and secretion. “Increased” expression is in the context of a comparison between a treated cell and an untreated control, for example, a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, versus an untreated cell, or a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas
  • “decreased” expression is in the context of a comparison between a treated cell and an untreated control, for example, a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, versus an untreated cell, or a cell treated with a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, and conjugated to one or more compounds described herein versus a cell treated with only the macromolecule, e.g., the antisense oligonucleotide, re
  • Disease refers to any disease, disorder, condition, symptom, or indication.
  • Treating” or “treatment” refer to curative, symptomatic, preventive and prophylactic treatment and include, but are not limited to, arresting or ameliorating a disease or at least one clinical symptoms of a disease, reducing the risk of acquiring a disease or at least one clinical symptoms of a disease, reducing the development of a disease or at least one clinical symptoms of the disease, reducing the risk of developing a disease or at least one clinical symptoms of a disease, or delaying the onset of the disease or at least one clinical symptoms of a disease.
  • a “subject” and “patient” can be used interchangeably to refer to any animal subjects, for example, mammalian subjects such as humans, primates, cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In one aspect, the subject is human.
  • the term “cell” can include a single cell, a plurality of cells or a population of cells where context permits, unless otherwise specified.
  • the cell is in vitro, for example, a cell explanted from a subject.
  • the cell is a cell grown in batch culture or in tissue culture.
  • the cell is in vivo, for example, located in a subject in need of treatment.
  • the subject is a human subject.
  • “Pharmaceutically acceptable” as used herein means approved by a regulatory agency of a Federal or state government, or listed in the U.S. Pharmacopeia, European Pharmacopia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “pharmaceutical composition” includes one or more active agents, including, for example, a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, conjugated to one or more compounds described herein, and a pharmaceutically acceptable carrier or diluent.
  • the carrier or diluent is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.
  • “Pharmaceutically acceptable salt” refers to a salt of one or more compounds that is physiologically and pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound and includes a salt prepared from pharmaceutically acceptable non-toxic acid or base, including inorganic or organic acids and bases. “Pharmaceutically acceptable salts” of the compounds described herein may be prepared by methods well-known in the art. For a review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002).
  • an “effective amount” of a macromolecule conjugated to one or more compounds refers to an amount sufficient to increase the efficacy of a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA.
  • a macromolecule e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA.
  • an “effective” amount of a macromolecule conjugated to one or more compounds refers to an amount sufficient to facilitate entry of a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, into the nucleus and/or cytosol of a cell.
  • a macromolecule e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA
  • an “effective” amount of a macromolecule conjugated to one or more compounds refers to an amount that facilitates the release of a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, from an endosome into the cytosol of a cell.
  • a macromolecule e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA
  • a “therapeutically effective amount” of a macromolecule refers to an amount sufficient to provide a therapeutic benefit in the treatment of a disease, or to delay or reduce one or more symptoms associated with the disease.
  • a “therapeutically effective amount” can vary depending on many factors, including, but not limited to, the macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA, being administered, the disease, the severity of the disease, the age of the subject being treated, and/or the weight of the subject being treated.
  • the macromolecule e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA
  • Dose refers to a specified quantity of an active agent (for example, a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA conjugated to one or more compounds) provided in a single administration, or in a specified time -period.
  • a dose can be administered in one, two, or more boluses or injections.
  • the active agent is administered by infusion over an extended period of time or continuously.
  • Doses can be stated as the amount of pharmaceutical agent per unit time (e.g., hour, day, week, or month). Doses can also be stated as the amount per unit weight of the subject (e.g., mg/kg or g/kg).
  • Dosage unit refers to a form in which an active agent, for example, a macromolecule, e.g., an oligonucleotide such as an antisense oligonucleotide, a polypeptide such as a recombinase (e.g., Cre or FLP), or a combination thereof such as a Cas protein and a guide RNA conjugated to one or more compounds, is provided.
  • the dosage unit is a vial containing lyophilized active agent.
  • a dosage unit is a vial containing reconstituted active agent.
  • the active agent is a macromolecule.
  • the active agent is an oligonucleotide.
  • the active agent comprises a polypeptide. In one aspect, the active agent comprises an antisense oligonucleotide. In one aspect, the active agent comprises a Cre recombinase. In one aspect, the active agent comprises a FLP recombinase. In one aspect, the active agent comprises Cas protein. In one aspect, the active agent comprises Cas protein and a guide RNA. In one aspect, the active agent comprises a CRISPR system, a Cre-Lox system, a FLP-FRT system, any component thereof, or any combination thereof.
  • compositions described herein can be used in vitro on a sample (for example, on isolated cells, organs, or tissues) or in vivo in a subject (for example, in a living organism, such as a patient).
  • compositions described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and the area to be treated.
  • the composition is administered parenterally.
  • Parenteral administration includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Administration can be continuous, chronic, short, or intermittent.
  • oligonucleotides or proteins Delivering macromolecules such as oligonucleotides or proteins into cells requires that the macromolecule, e.g., oligonucleotide or protein, traverse cellular membranes, including the plasma membrane and/or endosomal membranes.
  • the macromolecule e.g., oligonucleotide or protein
  • traverse cellular membranes including the plasma membrane and/or endosomal membranes.
  • Use of macromolecules such as oligonucleotides or proteins can be hindered by their inability to effectively reach the cytosolic and/or nucleus of the cell, for example, due to their inability to cross the cell membrane or to escape from endosomal compartments following endocytosis.
  • a macromolecule that can increase the activity of a macromolecule, e.g., an oligonucleotide, such as an antisense oligonucleotide or siRNA; a polypeptide, such as recombinase (e.g., Cre or FLP); or a combination thereof, such as a Cas protein and a guide RNA.
  • the compound is a small molecule compound (SMC).
  • the compound is an endosomolytic compound that facilitates the release of the macromolecule, e.g., oligonucleotide and/or polypeptide from an endosome into the cytosol of a cell.
  • the compound disclosed herein increase transfection efficiency of a macromolecule, e.g., an oligonucleotide and/or polypeptide.
  • a macromolecule e.g., an oligonucleotide and/or polypeptide.
  • the compounds described herein interfere with the normal cell trafficking machinery to a minimal extent, i.e., until leakage is induced and do not induce damage or toxicity which irreversibly impedes cell proliferation or results in cell death.
  • compositions and methods are provided for delivering a macromolecule, e.g., an oligonucleotide, to the cytosol and/or nucleus of a cell.
  • a macromolecule e.g., an oligonucleotide
  • the oligonucleotide is single stranded.
  • the oligonucleotide is double stranded.
  • the oligonucleotide includes deoxyribonucleic acid (DNA).
  • the oligonucleotide includes ribonucleic acid (RNA).
  • the oligonucleotide is about 5 nucleotides to about 100 nucleotides, about 5 nucleotides to about 50 nucleotides, about 8 nucleotides to about 30 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 30 nucleotides, or about 18 to about 30 nucleotides in length. In one aspect, the oligonucleotide has a molecular weight from about 5 kDa to about 15 kDa.
  • the oligonucleotide reduces expression of a target nucleic acid, which can be referred to as “gene silencing.” In one aspect, the oligonucleotide increases expression of a target nucleic acid, which can be referred to as “gene activation.” In one aspect, the oligonucleotide alters the splicing of a target nucleic acid, which can be referred to as “splice switching.” In one aspect, the oligonucleotide interacts with a target protein. In one aspect, the oligonucleotide is an agonist or antagonist of a target protein.
  • the oligonucleotide is an antisense oligonucleotide (ASO), a small (-18-30 nucleotides), synthetic, single-stranded nucleic acid polymer which modulates gene expression via various mechanisms.
  • ASO antisense oligonucleotide
  • the ASO includes DNA and forms an RNA-DNA heteroduplex that is recognized by endogenous RNase H enzyme which catalyzes the degradation of RNA, thereby decreasing expression of the gene.
  • the ASO binds to a target nucleic acid but does not induce degradation.
  • the oligonucleotide is a splice switching oligonucleotide (SSO) that masks sequences within a target nucleic acid and thereby interferes with transcript RNA-RNA and/or RNA-protein interactions.
  • the oligonucleotide is a small-interfering RNA (siRNA), which has a characteristic 19 + 2mer structure (e.g., a duplex of two 21 -nucleotide RNA molecules with 19 complementary bases and terminal 2-nucleotide 3' overhangs).
  • the oligonucleotide is a microRNA (miRNA).
  • the oligonucleotide targets a non- coding RNA sequence associated with transcriptional repression to reverse the effects of this negative regulation thereby activating gene expression.
  • Other oligonucleotides include, but are not limited to, interfering RNA (RNAi) and decoy oligonucleotides.
  • RNAi interfering RNA
  • decoy oligonucleotides include, but are not limited to, interfering RNA (RNAi) and decoy oligonucleotides.
  • the oligonucleotide is a gapmer.
  • the oligonucleotide is an aptamer.
  • the oligonucleotide is part of a site-specific modification (SSM) system, e.g., a CRISPR system, described herein.
  • the oligonucleotide is a guide RNA.
  • the guide RNA comprises one or both of: (i) a scaffold region or tracrRNA capable of associating with a Cas protein (e.g., Cas nuclease); and (ii) a spacer region or crRNA capable of hybridizing to a specific target nucleic acid sequence, thereby directing the Cas protein to make a site-specific modification at the target nucleic acid.
  • the spacer region is about 15 to about 25 nucleotides in length.
  • the guide RNA is a single guide RNA (sgRNA) comprising both the tracrRNA and the crRNA.
  • the guide RNA comprises a tracrRNA and a crRNA as two separate oligonucleotides that, together with the Cas protein, are capable of forming a complex.
  • the oligonucleotide includes one or more modified nucleotides. In one aspect, the oligonucleotide includes one or more modifications to the oligonucleotide phosphate linkages. In one aspect, the oligonucleotide includes one or more modifications to the ribose sugar. In one aspect, the oligonucleotide includes one or more nucleotides that are covalently modified to limit conformation, i.e., locked nucleic acids (LNA). In one aspect, the oligonucleotide includes a peptide nucleic acid (PNA). In one aspect, the oligonucleotide includes a methylated cystosine at the 5’ position.
  • LNA locked nucleic acids
  • the oligonucleotide includes one or more modified nucleotides selected from: phosphodiester (PO); phosphorothioate (PS); 2’0-methyl (2’0Me); 2’0-methoxyethyl (MOE); peptide nucleic acid (PNA); phosphoroamidate morpholino (PMO); locked nucleic acid (LNA); 2’-deoxy-2’-fluoro (2’-F); or a combination thereof.
  • the oligonucleotide is an antisense oligonucleotide targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1).
  • the oligonucleotide is an antisense oligonucleotide targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) comprising at least one nucleic acid with an LNA.
  • MALAT1 metastasis-associated lung adenocarcinoma transcript 1
  • the antisense oligonucleotide targets MALAT1 and has the sequence: GM5CAttm5ctaatagm5cAGM5C, where m5c is 5- methylcytidine and capital letters are LNA nucleosides (SEQ ID NO:1).
  • the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1% to about 100%. In embodiments, the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.
  • compositions and methods are provided for delivering a macromolecule, e.g., a polypeptide, to the cytosol and/or nucleus of a cell.
  • the polypeptide comprises a therapeutic protein, which may be an antibody or a non-antibody protein.
  • the polypeptide is a therapeutic peptide, e.g., as described in Wang et al., Sig Transduct Target Ther. 7:48 (2022).
  • the polypeptide is capable of providing a SSM at a target nucleic acid.
  • the target nucleic acid is DNA.
  • the target nucleic acid is RNA.
  • the polypeptide is a nuclease.
  • the polypeptide is a recombinase.
  • the polypeptide is part of a SSM system described herein, e.g., a CRISPR system, a Cre-Lox system, or a FLP-FRT system.
  • the polypeptide is a Cas protein.
  • the polypeptide is Cas9.
  • the polypeptide is Casl2a.
  • the polypeptide is a recombinase.
  • the polypeptide is Cre.
  • the polypeptide is FEP.
  • the polypeptide comprises a modified Cas protein.
  • the modified Cas protein is a Cas nickase, e.g., Cas9 nickase or Cas 12a nickase, which cleaves only one strand of a double- stranded target nucleic acid.
  • the modified Cas protein is a catalytically inactivated Cas protein (dead Cas), e.g., dCas9 or dCasl2a, which does not comprise nuclease activity.
  • the dead Cas is capable of binding to a target nucleic acid and preventing other enzymes such as transcription factors from binding to the target nucleic acid.
  • Cas nickase and dead Cas proteins are further described, e.g., in Xu et al., J Mol Biol. 431(l):34-47 (2019); Qi et al., Cell 152(5): 1173-1183 (2013); and Liu et al., Microbial Cell Factories 19:172 (2020).
  • the polypeptide comprises a Cas fusion protein.
  • the Cas fusion protein comprises a modified Cas protein, e.g., a Cas nickase or a dead Cas, fused to an effector domain.
  • the polypeptide comprises a Cas nickase or a dead Cas, e.g., dCas9 or dCasl2, fused to a nucleotide deaminase, e.g., cytidine deaminase or adenosine deaminase, and optionally further fused to a DNA glycosylase inhibitor.
  • the polypeptide comprises a Cas nickase, e.g., Cas9 nickase or Casl2 nickase, fused to a reverse transcriptase.
  • Cas fusion proteins are further described, e.g., in Rees et al., Nat Rev Genet. 19(12):770-788 (2016); Anzalone et al., Nature 576(7785): 149- 157 (2019); and Liu et al., Microbial Cell Factories 19:172 (2020).
  • the polypeptide comprises a recombinase. In some aspects, the polypeptide comprises Cre. In some aspects, the polypeptide comprises FLP. In some aspects, the recombinase is a modified recombinase. In some aspects, the recombinase is an inducible recombinase. Modified (e.g., inducible) recombinases are further described, e.g., in Kaczmarcyk et al., Nucleic Acids Res. 29(12): e56 (2001); Badea et al., PLOS One 4(11): e7859 (2009); and Akbudak et al., Mol Biotechnol. 49(l):82-89 (2011).
  • Modified (e.g., inducible) recombinases are further described, e.g., in Kaczmarcyk et al., Nucleic Acids Res. 29(12):
  • the macromolecule is delivered to a target cell.
  • the target cell is a cultured cell.
  • the target cell is an isolated cell.
  • the target cell is an isolated cell from a subject in need of treatment.
  • the target cell is a mammalian cell.
  • the target cell is a eukaryotic cell.
  • the target cell is a prokaryotic cell.
  • the target cell is part of a tissue or organ.
  • the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, adipose, and/or muscle (e.g., skeletal muscle).
  • the target cell is a brain cell, a CNS cell, a PNS cell, a heart cell, a liver cell, a kidney cell, a spleen cell, a pancreas cell, a lung cell, a muscle cell, an adipose cell, an immune cell, or combination thereof.
  • the target cell is a CNS cell.
  • the CNS cell comprises a glial cell and/or a neuron.
  • Glial cells of the CNS include, e.g., astrocytes, oligodendrocytes, microglia, and ependymal cells.
  • Neurons include, e.g., afferent neurons, efferent neurons, and interneurons.
  • the target cell is a liver cell, e.g., a hepatocyte or a non-parenchymal cell.
  • the target cell comprises a plateable metabolism qualified human hepatocyte, a plateable induction qualified human hepatocyte, plateable human hepatocyte, suspension qualified human hepatocyte (including 10-donor and 20-donor pooled hepatocytes), human hepatic kupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled Beagle hepatocytes), mouse hepatocytes (including CD-I and C57BI/6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus monkey hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes).
  • a plateable metabolism qualified human hepatocyte including 10-donor and 20-donor pooled hepatocytes
  • the target cell is an immune cell.
  • immune cells include T cells, B cells, dendritic cells, NK cells, T helper cells, cytotoxic T cells, regulatory T cells, gamma delta T cells, neutrophils, mast cells, monocytes, antigen- presenting cells, lymphocytes, basophils, and phagocytes.
  • the macromolecule to be delivered to a target cell is an oligonucleotide.
  • the oligonucleotide is an antisense oligonucleotide.
  • the oligonucleotide is a siRNA.
  • the siRNA is unconjugated, i.e., not conjugated to a lipid or sugar.
  • the siRNA is conjugated to a lipid and/or a sugar.
  • the oligonucleotide is a guide RNA of a CRISPR system.
  • the macromolecule to be delivered to a target cell is an polypeptide.
  • the polypeptide is a Cas protein, e.g., Cas9 or Casl2a.
  • the Cas protein is a modified Cas protein or a Cas fusion protein as described herein.
  • the polypeptide is a recombinase.
  • the polypeptide is Cre.
  • macromolecules such as oligonucleotides and polypeptides must traverse the plasma membrane and enter the cytosol and/or nucleus of the cell.
  • the macromolecule e.g., oligonucleotide and/or polypeptide
  • the macromolecule is taken up by endocytosis, in which the macromolecule, e.g., oligonucleotide and/or polypeptide, is surrounded by the plasma membrane, which then buds off inside the cell to form a vesicle containing the ingested macromolecule, e.g., oligonucleotide and/or polypeptide.
  • the macromolecule e.g., oligonucleotide and/or polypeptide
  • the endocytic pathway of mammalian cells includes distinct membrane compartments which include: early endosomes, late endosomes and lysosomes.
  • Ees Early endosomes (EE) are the first compartment of the endocytic pathway and are the main sorting station in the endocytic pathway (Huotari and Helenius (2011) EMBO J. 30(17):3481-3500). EEs recycle the majority of cargo internalized by endocytosis. Ees are heterogenous in terms of morphology, localization, composition, and function. Most Ees are relatively small and remain close to the plasma membrane, although the overall distribution of Ees is cell-type dependent.
  • Late endosomes receive endocytosed material, usually from early endosomes in the endocytic pathway and include proteins characteristic of nucleosomes, mitochondria and mRNAs including lysosomal membrane glycoproteins and acid hydrolases. They are acidic (35pprox.. pH 5.5) and are thought to mediate a final sorting of the internalized cargo prior to delivery of the cargo to the lysosomes.
  • Lysosomes are compartment of the endocytic pathway which sequester cargo for arrest or degradation. Their chief function is to break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules and return them to the cytoplasm as new cell-building materials. To accomplish this, lysosomes include a variety of hydrolytic enzymes that function in an acidic environment (35 approx.. pH 4.8).
  • macromolecules When taken up by endocytosis, macromolecules, e.g., oligonucleotides and/or polypeptides, often accumulate in endosomes, in particular, late endosomes or lysosomes, where they are pharmacologically inert.
  • the macromolecule e.g., oligonucleotide and/or polypeptide
  • the endosomal compartment to access their cytosolic or nuclear targets before degradation or exportation via exocytosis.
  • a small molecule compound that facilitates entry of the macromolecule, e.g., oligonucleotide and/or polypeptide, into the cytosol and/or nucleus of a cell.
  • SMC small molecule compound
  • the compound facilitates entry of the macromolecule, e.g., oligonucleotide and/or polypeptide, into the cytosol by forming pores in the plasma membrane of the cell.
  • the compound facilitates release of the macromolecule, e.g., oligonucleotide and/or polypeptide, from an endosomal compartment within the cell.
  • the macromolecule is an oligonucleotide
  • the compound facilitates the endosomal escape of a gymnotically delivered oligonucleotide.
  • the compound engorges the endosomal compartment, physically inducing membrane rupture and concurrent macromolecule, e.g., oligonucleotide and/or polypeptide, escape into the cytosol during the early-to-late endosomal transition and/or late endosomal transition to lysosome.
  • the endosomal escape enhancer (EEE) compound has a structure represented by Formula I:
  • Formula I one of Zi and Z2 is N, and the other is C;
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl
  • Rs is hydro or C1-C4 alkyl
  • Re is hydro or C1-C4 alkyl
  • R7, R8, R9, Rio, R11 are each independently CHR12, CR12R17 or NR13;
  • R12 is hydro, C1-C4 alkyl, -OR14, or -CO2R15;
  • R14 is hydro or C1-C4 alkyl;
  • R15 is hydro or C1-C4 alkyl
  • R16 is hydro or C1-C4 alkyl
  • R17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; and wherein one or more of the alkyl are optionally substituted with one or more halo, or a pharmaceutically acceptable salt thereof.
  • R1 is hydro. In one aspect, R1 is halo, including, for example, chloro, fluoro, bromo or iodo. In one aspect, R1 is bromo, chloro, or fluoro. In one aspect, R1 is fluoro. In one aspect, R1 is alkyl. In one aspect, R1 is a saturated alkyl. In one aspect, R1 is an unsaturated alkyl, e.g., an alkenyl or alkynyl. In some aspects, R1 can be polyunsaturated. In one aspect, R1 is a straight chain alkyl. In one aspect, R1 is a branched alkyl. In one aspect, Ri is C1-C4 alkyl.
  • R1 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R1 is a substituted alkyl, e.g., substituted with one or more halo. In some embodiments, R1 is a C1-C4 alkyl, wherein at least one carbon of the alkyl is substituted with one or more chloro or fluoro. In some aspects, R1 is substituted with a hydroxyl or a ketone. In one aspect, R1 is an unsubstituted alkyl.
  • R1 is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl.
  • R1 is a halo C1-C4 alkyl.
  • R1 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.
  • R1 is alkoxy, as represented by -OR4. In one aspect, R1 is a saturated alkoxy. In one aspect, R1 is an unsaturated alkoxy. In some embodiments, R4 is C1-C4 alkyl, e.g., R4 is methyl, ethyl, propyl, isopropyl, or butyl, including n-butyl, sec -butyl, isobutyl, and tert-butyl. In some embodiments, the alkoxy can be optionally substituted with one or more halo. In some aspects, R1 is alkoxy substituted with a hydroxyl or a ketone.
  • R1 is alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a mono-haloalkoxy, a di-haloalkoxy, or a tri-haloalkoxy.
  • R1 is a straight chain alkoxy.
  • R1 is a branched alkoxy.
  • R1 is C1-C4 alkoxy (-OR4), where R4 is C1-C4 alkyl, e.g., methyl or ethyl.
  • R1 is an alkoxy that includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, or butoxy, including for example, n-butoxy, sec- butoxy iso-butoxy, and t-butoxy.
  • R5 is C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.
  • R1 is a primary amide.
  • R1 is a secondary amide.
  • R1 is a tertiary amide.
  • R1 is a short chain ester, which can be represented by the group “-C(O)OR6” or “-CO2R6” where R6 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.
  • R1 is cyano
  • X is hydro. In some embodiments, X is an alkyl. In one aspect, X is a saturated alkyl. In one aspect, Xis an unsaturated alkyl. In one aspect, Xis a straight chain alkyl. In one aspect, X is a branched alkyl. In one aspect, X is C1-C4 alkyl. In one aspect, X is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, X is an unsubstituted alkyl.
  • X is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri- haloalkyl.
  • a halo group i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri- haloalkyl.
  • X is a halo C1-C4 alkyl.
  • X is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.
  • X is -OR2, where R2 is defined below.
  • R2 is hydro. In some embodiments, R2is an alkyl. In one aspect, R2 is a saturated alkyl. In one aspect, R2 is an unsaturated alkyl. In one aspect, R2is a straight chain alkyl. In one aspect, R2 is a branched alkyl. In one aspect, R2 is C1-C4 alkyl. In one aspect, R2 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, R2 is methyl. In one aspect, R2 is ethyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • R2 is an unsubstituted alkyl.
  • R2 is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl.
  • R2 is a halo C1-C4 alkyl.
  • R2 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.
  • R3 is hydro. In some aspect, R3 is a halo, e.g., a chloro, fluoro, or bromo. In one aspect, R3 is alkyl. In one aspect, R3 is a saturated alkyl. In one aspect, R3 is an unsaturated alkyl. In one aspect, R3 is a straight chain alkyl. In one aspect, R3 is a branched alkyl. In one aspect, R3 is C1-C4 alkyl. In one aspect, R3 is methyl, ethyl, propyl, isopropyl, or butyl.
  • butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • R3 is a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl.
  • R3 is a C1-C4 alkyl optionally substituted with one or more halo.
  • R3 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, or chloromethyl.
  • R3 is amide, which can be represented by the group “-C(O)NR5R5,” in which each R5 is independently hydro or alkyl, e.g., methyl or ethyl.
  • the alkyl substituent includes from 1 and up to about 8 carbon atoms. In one aspect, the alkyl substituent includes from about 1 and up to about 4 carbon atoms.
  • R3 is a primary amide. In one aspect, R3 is a secondary amide. In one aspect, R3 is a tertiary amide.
  • R3 is a short chain ester, which can be represented by the group “-C(0)0R6” or “-CO2R6” where Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl.
  • R3 is cyano.
  • R3 is alkyl substituted with one or more hydroxyl, as represented by “-(CH0H) n CH20H” wherein n is 0, 1, 2, or 3.
  • R3 is methylhydroxyl, as represented by “-CH2OH.”
  • R3 is alkoxy, as represented by “-OR4.” In one aspect, R3 is a saturated alkoxy. In one aspect, R3 is an unsaturated alkoxy. In some embodiments, R4 is C1-C4 alkyl, e.g., R4 is methyl, ethyl, propyl, or butyl. In some embodiments, the alkoxy can be optionally substituted with one or more halo. In some aspects, R3 is alkoxy substituted with a hydroxyl or a ketone.
  • R3 is alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a mono-haloalkoxy, a di-haloalkoxy, or a tri-haloalkoxy.
  • R3 is a straight chain alkoxy.
  • R3 is a branched alkoxy.
  • R3 is C1-C4 alkoxy (-OR4), where R4 is C1-C4 alkyl, e.g., methyl or ethyl.
  • R3 is an alkoxy that includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, including for example, n- butoxy, sec -butoxy iso-butoxy and t-butoxy. In one aspect, R3 is methoxy.
  • R7, R8, R9, Rio, or Rn are each independently CHR12, CR12R17 or NR13.
  • all of R7, R8, R9, Rio, and Rn are CHR12, where R12 for each atom is independently chosen from hydro, C1-C4 alkyl, -OR14, or -CO2R15.
  • all of R7, R8, R9, Rio, and Rn are CHR12, where each R12 is hydro.
  • all of R7, R8, R9, Rio, and Rn are CHR12, where R12 is each independently chosen from hydro or an unsubstituted alkyl.
  • R12 may be substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri- haloalkyl.
  • R12 may be a halo C1-C4 alkyl, for instance trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.
  • R7, R8, R9, Rio, and Rn are CHR12, where each R12 is independently chosen from hydro, C1-C4 alkyl, or -OR14, where R14 is further chosen from hydro or C1-C4 alkyl.
  • R12 is alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, including for example, n-butoxy, sec -butoxy iso-butoxy and t-butoxy.
  • all of R7, R8, R9, Rio, and Rn are CHR12, where each R12 is independently chosen from hydro, C1-C4 alkyl, -OR14, or -CO2R15, where each of R14 and R15 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.
  • At least one of R7, R8, R9, Rio, and Rn is CR12R17, where R12 and R17 are each independently chosen from hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.
  • at least two of R7, R8, R9, Rio, and Rn are CR12R17, where R12 and R17 are each independently chosen from hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.
  • R7 is CR12R17, where R12 is methyl and R17 is methyl, and R8, R9, Rio, and Rn are each independently chosen from CHR12 or NR13.
  • Rs is CR12R17, where R12 is methyl and R17 is methyl, and R7, R9, Rio, and Rn are each independently chosen from CHR12 or NR13.
  • R9 is CR12R17, where R12 is methyl and R17 is methyl, and R7, R8, Rio, and Rn are each independently chosen from CHR12 or NR13.
  • Rio is CR12R17, where R12 is methyl and R17 is methyl, and R7, R8, R9, and Rn are each independently chosen from CHR12 or NR13.
  • Rn is CR12R17, where R12 is methyl and R17 is methyl, and R7, R8, R9, and Rio are each independently chosen from CHR12 or NR13.
  • At least one of R7, R8, R9, Rio, and Rn is NR13, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.
  • at least two of R7, R8, R9, Rio, and Rn are NR13, where each of the two R13 groups are independently hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl.
  • R7 is NR13 and R8, R9, Rio, and Rn are CHR12, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro.
  • Rs is NR13 and R7, R9, Rio, and Rn are CHR12, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro.
  • R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro.
  • Rio is NR13 and R7, R8, R9, and Rn are CHR12 where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro.
  • Rn is NR13 and R7, R8, R9, and Rio are CHR12 where R13 is hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl and R12 is hydro.
  • y is chosen from 0, 1, 2, or 3
  • R14, R15, and Ri6 are each independently chosen from hydro and C1-C4 alkyl.
  • R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where each R12 and R13 are hydro. In one aspect, R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where each R12 is hydro, and R13 is C1-C4 alkyl. In one aspect, R9 is NR13 and R7, R8, Rio, and Rn are CHR12, where each R12 is hydro, and R13 is methyl.
  • R9 is NR13, where R13 is hydro; at least one of R7, R8, Rio, and Rn is CR12R17; where R12 and R17 are each C1-C4 alkyl; and the remaining of R7, R8, Rio, and Rn are CHR12, where each R12 is hydro, and R13 is hydro or C1-C4 alkyl.
  • R9 is NR13, where R13 is hydro; R7, Rio, and Rn are CHR12, where R12 is hydro; and Rs is CR12R17, where each of R12 and R17 is methyl.
  • R9 is NR13, where R13 is hydro; R7, R8, and Rn are CHR12; where R12 is hydro; and Rio is CR12R17, where each of R12 and R17 is methyl.
  • R9 is NR13, where R13 is hydro; R7 and Rn are CHR12, where R12 is hydro; and Rs and Rio are CR12R17, where each of R12 and R17 is methyl.
  • EEE endosomal escape enhancer
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl;
  • R4 is C1-C4 alkyl
  • R5 is hydro or C1-C4 alkyl, e.g., methyl or ethyl
  • R6 is hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • R16 is hydro or C1-C4 alkyl
  • the endosomal escape enhancer (EEE) compound has a structure represented by Formula lb:
  • Formula lb wherein one of Z1 and Z2 is N, and the other is C;
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl
  • Rs is hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • R16 is hydro or C1-C4 alkyl
  • the endosomal escape enhancer (EEE) compound has a structure represented by Formula Ic, Formula Id, Formula le or Formula If:
  • X is hydro, C1-C4 alkyl, or -OR2;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl
  • Rs is hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl;
  • R16 is hydro or C1-C4 alkyl
  • a compound of Formula la, Formula lb, Ic, Id, le or If is provided, wherein R1 is hydro, halo, C1-C4 alkyl, -OR4, -C( O)NR5R5, -CO2R6, or cyano;
  • X is -OR 2 ;
  • R2 is hydro or C1-C4 alkyl
  • R4 is C1-C4 alkyl
  • Rs is hydro, methyl or ethyl
  • Re is hydro, methyl or ethyl
  • R1 is hydro. In one aspect, R1 is halo, including, for example, chloro, fluoro, bromo, or iodo. In one aspect, R1 is bromo, chloro, or fluoro. In one aspect, R1 is fluoro. In one aspect, R1 is alkyl. In one aspect, R1 is a saturated alkyl. In one aspect, R1 is an unsaturated alkyl, e.g., an alkenyl or alkynyl. In some aspects, R1 can be polyunsaturated. In one aspect, R1 is a straight chain alkyl. In one aspect, R1 is a branched alkyl. In one aspect, Ri is C1-C4 alkyl.
  • R1 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R1 is a substituted alkyl, e.g., substituted with one or more halo. In some embodiments, R1 is a C1-C4 alkyl, wherein at least one carbon of the alkyl is substituted with one or more chloro or fluoro. In some aspects, R1 is substituted with a hydroxyl or a ketone. In one aspect, R1 is an unsubstituted alkyl.
  • R1 is substituted with a halo group, i.e., a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl.
  • R1 is a halo C1-C4 alkyl.
  • R1 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.
  • R1 is alkoxy, as represented by “-OR4”. In one aspect, R1 is a saturated alkoxy. In one aspect, R1 is an unsaturated alkoxy. In some embodiments, R4 is C1-C4 alkyl, e.g., R4 is methyl, ethyl, propyl, isopropyl, or butyl, including n-butyl, sec -butyl, isobutyl, and tert-butyl. In some embodiments, the alkoxy can be optionally substituted with one or more halo. In some aspects, R1 is alkoxy substituted with a hydroxyl, or a ketone.
  • R1 is alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a mono-haloalkoxy, a di-haloalkoxy, or a tri-haloalkoxy.
  • R1 is a straight chain alkoxy.
  • R1 is a branched alkoxy.
  • R1 is C1-C4 alkoxy (-OR4), where R4 is C1-C4 alkyl, e.g., methyl or ethyl.
  • R1 is an alkoxy that includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, or butoxy, including for example, n-butoxy, sec- butoxy iso-butoxy and t-butoxy.
  • R1 is a short chain ester, which can be represented by the group “-C(O)OR6” or “-CO2R6” where Re is hydro or C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R1 is cyano.
  • X is hydro. In one aspect, X is C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, X is -OR2.
  • R2 is hydro. In some embodiments, R2is an alkyl. In one aspect, R2 is a saturated alkyl. In one aspect, R2 is an unsaturated alkyl. In one aspect, R2is a straight chain alkyl. In one aspect, R2 is a branched alkyl. In one aspect, R2 is C1-C4 alkyl. In one aspect, R2 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, R2 is methyl. In one aspect, R2 is ethyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • R3 is hydro. In some aspects, R3 is a halo, e.g., a chloro, fluoro or bromo. In one aspect, R3 is alkyl. In one aspect, R3 is a saturated alkyl. In one aspect, R3 is an unsaturated alkyl. In one aspect, R3 is a straight chain alkyl. In one aspect, R3 is a branched alkyl. In one aspect, R3 is C1-C4 alkyl. In one aspect, R3 is methyl, ethyl, propyl, isopropyl, or butyl.
  • butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • R3 is a haloalkyl, e.g., a mono-haloalkyl, a di-haloalkyl, or a tri-haloalkyl.
  • R3 is a C1-C4 alkyl optionally substituted with one or more halo.
  • R3 is a halo C1-C4 alkyl such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, or chloromethyl.
  • R3 is amide, which can be represented by the group “-C(O)NR5R5,” in which each R5 is independently hydro or alkyl, e.g., methyl or ethyl.
  • the alkyl substituent includes from 1 and up to about 8 carbon atoms. In one aspect, the alkyl substituent includes from about 1 and up to about 4 carbon atoms.
  • R3 is a primary amide. In one aspect, R3 is a secondary amide. In one aspect, R3 is a tertiary amide.
  • R3 is a short chain ester, which can be represented by the group “-C(O)OR6” or “-CO2R6” where R6 is hydro or C1-C4 alkyl, e.g., methyl or ethyl.
  • R3 is cyano.
  • R3 is alkyl substituted with one or more hydroxyl, as represented by “-(CH0H) 11 CH20H” wherein n is 0, 1, 2, or 3.
  • R3 is methylhydroxyl, as represented by “-CH2OH.”
  • the endosomal escape enhancer (EEE) compounds of Formula la, lb, Ic, Id, le and If as described herein can include, by way of non-limiting example, any of the following as found in the Table 2A below:
  • the endosomal escape enhancer (EEE) compound has a structure represented by Formula la, lb, Ic, Id, le or If, and wherein Ri, X and R3 are as found in Table 2B.
  • the compound has a structure represented by Formula I, wherein Ri is halo, X is -OR2, Riis methyl, R3 is cyano, R7, R8, R10, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is C1-C4 alkyl or -(CFDyOH, where y is 1-3.
  • the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2is methyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is C1-C4 alkyl.
  • the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, Riis methyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where Rn is hydro, and R9 is NR13, where R13 is methyl.
  • the compound has a structure represented by Formula I wherein Zi is N, Z2 is C, R1 is fluoro, X is -OR2, R2 is methyl, R3 is cyano, R7, R8, R10, and R11 are CHR12, where R12 is hydro, and R9 is NR13, where R13 is hydro.
  • the compound has a structure represented by Formula I wherein Zi is N, Z2 is C, R1 is fluoro, X is -OR2, Riis ethyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is methyl.
  • the compound has a structure represented by Formula I wherein Zi is N, Z2 is C, R1 is fluoro, X is -OR2, Riis methyl, R3 is cyano, R7, Rio, and Rn are CHR12, where R12 is hydro, Rs is CR12R17, where R12 and R17 are each methyl, and R9 is NR13, where R13 is methyl.
  • the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2is methyl, R3 is cyano, R7, R8, R10, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is (CH2) y OH, where y is 1.
  • the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2is methyl, R3 is cyano, R7, R8, R10, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is (CH2) y OH, where y is 2.
  • the compound has a structure represented by Formula I wherein Ri is fluoro, X is -OR2, R2 is methyl, R3 is cyano, R7, R8, Rio, and Rn are CHR12, where R12 is hydro, and R9 is NR13, where R13 is (CH2) y OH, where y is 3.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl and R3 is cyano. In some embodiments, R1 is fluoro and R2 is ethyl. In some embodiments, R1 is fluoro and R2 is methyl. In some embodiments, R1 is chloro and R2 is ethyl. In some embodiments, Ri is chloro and R2 is methyl.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl and R3 is cyano. In some embodiments, R1 is haloalkyl, e.g., trifluoromethyl, R2 is ethyl and R3 is cyano.
  • R1 is haloalkyl, e.g., trifluoromethyl, R2 is methyl and R3 is cyano
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl and R3 is methyl or methoxy.
  • R1 is fluoro, R2 is ethyl and R3 is methyl.
  • R1 is fluoro, R2 is methyl and R3 is methoxy.
  • R1 is fluoro, R2 is methyl and R3 is methyl.
  • R1 is fluoro, R2 is ethyl and R3 is methoxy. In some embodiments, R1 is chloro, R2 is ethyl and R3 is methyl. In some embodiments, R1 is chloro, R2 is methyl and R3 is methoxy. In some embodiments, R1 is chloro, R2 is methyl and R3 is methyl. In some embodiments, R1 is chloro, R2 is ethyl and R3 is methoxy.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is C1-C4 alkyl and R3 is hydro.
  • Ri is fluoro, R2 is methyl and R3 is hydro.
  • R1 is chloro, R2 is methyl and R3 is hydro.
  • R1 is bromo, R2 is methyl and R3 is hydro.
  • R1 is fluoro, R2 is ethyl and R3 is hydro.
  • R1 is chloro, R2 is ethyl and R3 is hydro.
  • R1 is bromo, R2 is ethyl and R3 is hydro.
  • R1 is fluoro, R2 is propyl, e.g., isopropyl and R3 is hydro.
  • R1 is chloro, R2 is propyl, e.g., isopropyl and R3 is hydro.
  • R1 is bromo, R2 is propyl, e.g., isopropyl and R3 is hydro.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl and R3 is hydro. In some embodiments, R1 is trifluoromethyl, R2 is methyl and R3 is hydro. In some embodiments, Ri is trifluoromethyl, R2 is ethyl and R3 is hydro. In some embodiments, R1 is trifluoromethyl, R2 is methyl and R3 is hydro. In some embodiments, R1 is trifluoromethyl, R2 is ethyl and R3 is hydro.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is hydro and R3 is cyano. In some embodiments, R1 is fluoro. In some embodiments, R1 is chloro. In some embodiments, R1 is bromo.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is cyano, X is -OR2, R2 is ethyl or methyl and R3 is haloalkyl. In some embodiments, R1 is cyano, R2 is methyl and R3 is trifluoromethyl. In some embodiments, Ri is cyano, R2 is ethyl and R3 is trifluoromethyl.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is ethyl or methyl, X is -OR2, R2 is ethyl or methyl and R3 is hydro. In some embodiments, R1 is methyl, R2 is methyl and R3 is hydro. In some embodiments, R1 is methyl, R2 is ethyl and R3 is hydro. In some embodiments, R1 is ethyl, R2 is methyl and R3 is hydro. In some embodiments, R1 is ethyl, R2 is ethyl and R3 is hydro.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl and R3 is -(CFDyOH, wherein y is 0, 1, 2, or 3.
  • R1 is fluoro, R2 is methyl and R3 is -OH.
  • R1 is fluoro, R2 is methyl and R3 is -CH2OH.
  • Ri is fluoro, R2 is methyl and R3 is -(CH2)2OH.
  • R1 is chloro, R2 is methyl and R3 is -OH.
  • R1 is chloro, R2 is methyl and R3 is -CH2OH. In some embodiments, R1 is chloro, R2 is methyl and R3 is -(CH2)2OH. In some embodiments, Ri is chloro, R2 is methyl and R3 is -(CH2)3OH. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is -OH. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is -CH2OH. In some embodiments, R1 is fluoro, R2 is ethyl and R3 is -(CH2)2OH.
  • Ri is fluoro, R2 is ethyl and R3 is -(CH2)3OH.
  • R1 is chloro, R2 is ethyl and R3 is -OH.
  • R1 is chloro, R2 is ethyl and R3 is -CH2OH.
  • R1 is chloro, R2 is ethyland R3 is -(CH2)2OH.
  • Ri is chloro, R2 is ethyl and R3 is -(CH2)3OH.
  • the compound has a structure represented by Formula la, lb, Ic, Id, le or If, wherein R1 is fluoro, X is -OR2, R2is methyl and R3 is cyano.
  • the compound comprises a structure as shown in Table 1.
  • the compound is any one of Compounds 2-7 as shown in Table 1.
  • the compound is Compound 4 as shown in Table 1.
  • the compound is Compound 8 as shown in Table 1.
  • the compound is Compound 32 as shown in Table 1.
  • the compound is any one of Compounds 32a or 32b as shown below.
  • the compounds represented by Formula I, la, lb, Ic, Id, le or If may have one or more stereocenters and may exist as racemates or racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • Stereoisomers may be separated using conventional techniques, e.g., chromatography or fractional crystallization, or the stereoisomers may be made by stereoselective synthesis.
  • Compounds 32-35 contain at least one stereocenter and their individual isomers (further depicted below) are all disclosed herein.
  • Compound 32a
  • a composition that includes a macromolecule conjugated to one or more compounds described herein.
  • the macromolecule comprises an oligonucleotide.
  • the macromolecule is an antisense oligonucleotide.
  • the macromolecule is a siRNA.
  • the siRNA is conjugated to a lipid and/or a sugar.
  • the macromolecule comprises a polypeptide.
  • the macromolecule comprises one or more components of a SSM system described herein.
  • the SSM system is a CRISPR system.
  • the SSM system is a Cre-Lox system.
  • the SSM system is a FLP-FRT system.
  • the macromolecule comprises a Cas protein and/or a guide RNA.
  • the macromolecule comprises Cre.
  • the macromolecule comprises FLP.
  • a composition that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds described herein. In one aspect, a composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by Formula I, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by Formula la, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by Formula lb, or a pharmaceutically acceptable salt thereof.
  • a composition that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by Formula Ic, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by Formula Id, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by Formula le, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by Formula If, or a pharmaceutically acceptable salt thereof.
  • a composition that includes an oligonucleotide and/or polypeptide conjugated to one or more of Compounds 2-35, or a pharmaceutically acceptable salt thereof.
  • a composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more of Compounds 4, 32, and 33, or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition is provided that includes an oligonucleotide and/or polypeptide conjugated to one or more compounds described herein, and a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutical composition in one aspect, includes an oligonucleotide and/or polypeptide conjugated to one or more compounds represented by any one of Formulae I, la, lb, Id, le, andlf, or one or more compounds listed in Table 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutical composition in one aspect, includes an oligonucleotide and/or polypeptide conjugated to one or more of Compounds 2-35, a pharmaceutically acceptable salt thereof, or combinations thereof; and a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutical composition in one aspect, includes an oligonucleotide and/or polypeptide conjugated to one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof; and a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition can be formulated to be compatible with the intended route of administration, including, but not limited to, parenteral administration, such as intravenous (IV), or subcutaneous (SC or SQ) administration; intraperitoneal, intramuscular, oral, transdermal, or transmucosal administration.
  • a composition that includes an oligonucleotide. In one aspect, a composition is provided that includes an antisense oligonucleotide (ASO). In one aspect, a composition is provided that includes a splice switching oligonucleotide (SSO). In one aspect, a composition is provided that includes siRNA. In some aspects, the siRNA is conjugated to a lipid and/or a sugar. In one aspect, a composition is provided that includes a guide RNA. In one aspect, a composition is provided that includes about 0.025 ⁇ M to about 20 ⁇ M oligonucleotide.
  • the composition includes at least about 0.1 ⁇ M and up to about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, or about 20 ⁇ M oligonucleotide. In one aspect, the composition includes from about 0.1 ⁇ M to about 10 ⁇ M, about 0.1 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M oligonucleotide.
  • a composition that includes a polypeptide.
  • a composition is provided that includes a Cas protein, e.g., a Cas9 or Casl2a protein.
  • the Cas protein is a modified Cas protein or a Cas fusion protein as described herein.
  • a composition is provided that includes Cre.
  • a composition is provided that includes FLP.
  • a composition is provided that includes about 0.001 ⁇ M to about 20 ⁇ M polypeptide.
  • the composition includes at least about 0.01 ⁇ M and up to about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, or about 20 ⁇ M polypeptide. In one aspect, the composition includes from about 0.01 ⁇ M to about 10 ⁇ M, about 0.05 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M polypeptide.
  • a composition that includes an oligonucleotide and a polypeptide.
  • a composition is provided that includes a Cas protein and a guide RNA.
  • the Cas protein is Cas9.
  • the Cas protein is Cas 12a.
  • the Cas protein is a modified Cas protein or a Cas fusion protein as described herein.
  • a composition is provided that includes about 0.001 ⁇ M to about 20 ⁇ M of each of the oligonucleotide and the polypeptide.
  • the composition includes at least about 0.1 ⁇ M and up to about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, or about 20 ⁇ M of each of the oligonucleotide and the polypeptide.
  • the composition includes from about 0.01 ⁇ M to about 10 ⁇ M, about 0.05 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M of each of the oligonucleotide and the polypeptide.
  • a composition that includes one or more compounds described herein. In one aspect, a composition is provided that includes a small molecule compound described herein. In one aspect, a composition is provided that includes one or more compounds represented by Formulae I, la, lb, Ic, Id, le or If, or the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes one or more compounds represented by any one of Formulae I, la, lb, Ic, Id, le or If, or the compounds listed in Table 1, a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, a composition is provided that includes one or more of Compounds 4, 32, and 33, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided that includes one or more compounds of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.
  • compositions comprising the oligonucleotides and/or polypeptides and/or the “compositions comprising the compounds of Formulae I, la, lb, Ic, Id, le or If, or the compounds listed in Table 1” can include a biological cell, e.g., an in vitro cell.
  • the compositions comprising the oligonucleotides, polypeptides, and/or the compounds of Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1 can include an in vitro cell culture, e.g., a mammalian cell culture.
  • compositions comprising the oligonucleotides, polypeptides, and/or the compounds of Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1 can include an in vitro isolated cell from a subject, e.g., a mammalian subject, e.g., a human subject.
  • a method for introducing a macromolecule into a cell, e.g., a target cell described herein.
  • the cell is a cultured cell.
  • the cell is an isolated cell.
  • the cell is an isolated cell from a subject in need of treatment.
  • the cell is a mammalian cell.
  • the cell is a eukaryotic cell and/or a prokaryotic cell.
  • the cell is part of a tissue or organ of a mammal, e.g., a human.
  • the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, adipose, and/or muscle (e.g., skeletal muscle).
  • the cell is a brain cell, a CNS cell, a PNS cell, a heart cell, a liver cell, a kidney cell, a spleen cell, a pancreas cell, a lung cell, a muscle cell, an adipose cell, an immune cell, or combination thereof.
  • the cell is a CNS cell.
  • the CNS cell comprises a glial cell and/or a neuron.
  • Glial cells of the CNS include, e.g., astrocytes, oligodendrocytes, microglia, and ependymal cells.
  • Neurons include, e.g., afferent neurons, efferent neurons, and interneurons.
  • the cell is a liver cell, e.g., a hepatocyte or a non-parenchymal cell.
  • the cell comprises a plateable metabolism qualified human hepatocyte, a plateable induction qualified human hepatocyte, plateable human hepatocyte, suspension qualified human hepatocyte (including 10-donor and 20-donor pooled hepatocytes), human hepatic Kupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled Beagle hepatocytes), mouse hepatocytes (including CD-I and C57BI/6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus monkey hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White he
  • the cell is a human stem cell.
  • the stem cells can be, for example, pluripotent stem cells, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue specific stem cells (e.g., hematopoietic stem cells) and mesenchymal stem cells (MSCs).
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • tissue specific stem cells e.g., hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • the cell is a differentiated form of any of the cells described herein.
  • the eukaryotic cell is a cell derived from any primary cell in culture.
  • the cell is an immune cell.
  • immune cells include T cells, B cells, dendritic cells, NK cells, T helper cells, cytotoxic T cells, regulatory T cells, gamma delta T cells, neutrophils, mast cells, monocytes, antigen-presenting cells, lymphocytes, basophils, and phagocytes.
  • a method for introducing an oligonucleotide into a cell. In one aspect, a method is provided for introducing a polypeptide into a cell. In one aspect, a method is provided for introducing an oligonucleotide into a nucleus and/or cytosol of a cell. In one aspect, a method is provided for introducing a polypeptide into a nucleus and/or cytosol of a cell. In one aspect, the oligonucleotide is ASO, SSO, and/or siRNA. In one aspect, the oligonucleotide is siRNA, wherein the siRNA is unconjugated.
  • the oligonucleotide is siRNA, wherein the siRNA is conjugated to a lipid and/or a sugar.
  • the oligonucleotide is a guide RNA.
  • the oligonucleotide hybridizes to a target nucleic acid in the cell.
  • the polypeptide is capable of providing a SSM in a target nucleic acid in the cell.
  • the target nucleic acid is in the nucleus of the cell.
  • the target nucleic acid is in cytosol of the cell.
  • the method includes contacting the cell with the oligonucleotide and/or polypeptide conjugated to one or more compounds described herein.
  • the oligonucleotide is an antisense oligonucleotide (ASO).
  • the oligonucleotide is a splice switching oligonucleotide (SSO).
  • the oligonucleotide is siRNA.
  • the siRNA is conjugated to a lipid and/or a sugar.
  • the cell is contacted with an oligonucleotide.
  • the cell is contacted with a polypeptide.
  • the cell is contacted with both an oligonucleotide and a polypeptide.
  • the oligonucleotide is a guide RNA.
  • the polypeptide is a recombinase.
  • the polypeptide is Cre.
  • the polypeptide is FLP.
  • the polypeptide is a Cas protein.
  • the polypeptide is a Cas protein and the oligonucleotide is a guide RNA.
  • the compound is a small molecule compound (SMC).
  • the compound is an endosomolytic compound.
  • the compound has a structure represented by Formula I, or a pharmaceutically acceptable salt thereof.
  • the compound has a structure represented by Formula la, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula lb, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula Ic, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula Id, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula le, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula If, or a pharmaceutically acceptable salt thereof.
  • the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof.
  • the compound is any one of Compounds 4, 32, and 33, or a pharmaceutically acceptable salt thereof.
  • the method includes in vitro delivery of the macromolecule, e.g., oligonucleotide and/or polypeptide conjugated to one or more of the compounds to the cell. In one aspect, the method includes in vivo delivery of the macromolecule, e.g., oligonucleotide and/or polypeptide conjugated to one or more of the compounds to the cell.
  • the cell is a cultured cell. In one aspect, the cell is an isolated cell from a patient in need of treatment. In one aspect, the cell is part of a tissue or organ. In one aspect, the cell is a mammalian cell. In one aspect, the cell is a human cell. In one aspect the cell is a eukaryotic and/or a prokaryotic cell.
  • the macromolecule e.g., oligonucleotide and/or polypeptide
  • the macromolecule is internalized by the cell through endocytosis and encapsulated within an endosome.
  • the conjugated compound(s) facilitates release of the macromolecule, e.g., oligonucleotide and/or polypeptide, from the endosome.
  • the macromolecule e.g., oligonucleotide and/or polypeptide
  • the cell is internalized by the cell through transient pore formation.
  • the conjugated compound facilitates transient pore formation in the plasma membrane.
  • the cell is contacted with a composition that includes an oligonucleotide. In one aspect, the cell is contacted with a composition that includes an antisense oligonucleotide (ASO). In one aspect, the cell is contacted with a composition that includes a splice switching oligonucleotide (SSO). In one aspect, the cell is contacted with a composition that includes a siRNA. In some aspects, the siRNA is conjugated to a lipid and/or a sugar. In some aspects, the siRNA is unconjugated. In one aspect, the cell is contacted with a composition that includes a guide RNA.
  • the cell is contacted with a composition that includes oligonucleotide in an amount sufficient to provide a therapeutic effect. In one aspect, the cell is contacted with a composition that includes oligonucleotide in an amount sufficient to provide a SSM at a target nucleic acid. In one aspect, the cell is contacted with a composition that includes about 0.025 ⁇ M to about 20 ⁇ M oligonucleotide.
  • the cell is contacted with a composition that includes at least about 0.1 ⁇ M and up to about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, or about 20 ⁇ M, oligonucleotide.
  • the cell is contacted with a composition that includes from about 0.1 ⁇ M to about 10 ⁇ M, about 0.1 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M oligonucleotide.
  • the cell is contacted with a composition that includes a polypeptide. In one aspect, the cell is contacted with a composition that includes a Cas protein. In one aspect, the cell is contacted with a composition that includes a recombinase, e.g., Cre or FLP. In one aspect, the cell is contacted with a composition that includes polypeptide in an amount sufficient to provide a therapeutic effect. In one aspect, the cell is contacted with a composition that includes polypeptide in an amount sufficient to provide a SSM at a target nucleic acid. In one aspect, the cell is contacted with a composition that includes about 0.3 ⁇ M to about 20 ⁇ M polypeptide.
  • the cell is contacted with a composition that includes at least about 0.01 ⁇ M and up to about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, or about 20 ⁇ M, polypeptide.
  • the cell is contacted with a composition that includes from about 0.01 ⁇ M to about 10 ⁇ M, about 0.05 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M polypeptide.
  • the cell is contacted with a composition that includes an oligonucleotide and a polypeptide. In one aspect, the cell is contacted with a composition that includes a Cas protein and a guide RNA. In one aspect, the cell is contacted with a composition that includes oligonucleotide and polypeptide in an amount sufficient to provide a therapeutic effect. In one aspect, the cell is contacted with a composition that includes oligonucleotide and polypeptide in an amount sufficient to provide a SSM at a target nucleic acid.
  • the cell is contacted with a composition that includes about 0.001 ⁇ M to about 20 ⁇ M of each of an oligonucleotide (e.g., guide RNA) and a polypeptide (e.g., Cas protein).
  • the cell is contacted with a composition that includes at least about 0.01 ⁇ M and up to about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, or about 20 ⁇ M of each of the oligonucleotide and polypeptide.
  • the cell is contacted with a composition that includes from about 0.01 ⁇ M to about 10 ⁇ M, about 0.05 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M of each of the oligonucleotide and polypeptide.
  • the cell is contacted with a composition that includes one or more compounds described herein.
  • the cell is contacted with a composition that includes one or more compounds represented by Formula I or a pharmaceutically acceptable salt thereof.
  • the cell is contacted with a composition that includes one or more compounds represented by Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1, a pharmaceutically acceptable salt thereof, or combinations thereof.
  • the cell is contacted with a composition that includes any one of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.
  • the cell is contacted with a composition at a concentration sufficient to (i) reduce accumulation of the oligonucleotide in endosomes and/or lysosomes, (ii) induce physical rupture of endosomes, and/or (iii) facilitate transient pore formation in the plasma membrane, thereby facilitating release of a macromolecule, e.g., an oligonucleotide and/or polypeptide, into the cytosol or nucleus.
  • a macromolecule e.g., an oligonucleotide and/or polypeptide
  • contacting the cell with one or more compounds described herein results in endosomal membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds with a structure shown in Formula I, or a pharmaceutically acceptable salt thereof results in endosomal membrane permeabilization as determined by mCherry-GAL9 recruitment assay. In one aspect, contacting the cell with one or more compounds with a structure shown in Formula I, Formula la, lb, Ic, Id, le or If, or the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay.
  • contacting the cell with one or more compounds of any one of Compounds 2-35, or a pharmaceutically acceptable salt thereof results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay.
  • contacting the cell with one or more compounds with the structure of Compound 32, or a pharmaceutically acceptable salt thereof results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay.
  • contacting the cell with one or more compounds with the structure of Compound 33, or a pharmaceutically acceptable salt thereof results in membrane permeabilization as determined by mCherry-GAL9 recruitment assay.
  • the macromolecule e.g., oligonucleotide and/or polypeptide, alters activity of a gene expressed by the cell.
  • the gene is an endogenous gene.
  • the gene is an exogenous gene.
  • the macromolecule, e.g., oligonucleotide and/or polypeptide increases activity of a gene expressed by the cell.
  • the activity of the gene expressed by the cell is increased at least about lOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide not conjugated to the compound(s).
  • the activity of the gene expressed by the cell is increased at least about lOx, about 20x, about 30x, about 40x or about 50x and up to about lOOx, 200x, 300x, 400x, 500x or lOOOx.
  • the activity of the gene expressed by the cell is increased from about lOx to about lOOOx, about lOx to about 500x, about lOx to about 400x, about lOx to about 300x, about lOx to about 200x, about lOx to about lOOx, about lOOx to about 300x, or about lOOx to about 200x.
  • the activity of the gene expressed by a cell is increased from about lOx to about lOOOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide not conjugated to the compound(s).
  • the activity of the gene expressed by a cell is increased from about lOOx to about 500x.
  • the activity of the gene expressed by a cell is increased from about lOOx to about 300x.
  • the activity of the gene expressed by a cell is increased from about lOOx to about 200x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 500x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 300x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 200x. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about lOOx. In one aspect, the activity of the gene expressed by a cell is increased from about lOx to about 50x.
  • the macromolecule decreases the activity of a gene expressed by the cell.
  • the activity of the gene expressed by the cell is decreased at least about lOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide not conjugated to the compound(s).
  • the activity of the gene expressed by the cell is decreased at least about lOx, about 20x, about 30x, about 40x or about 50x and up to about lOOx, 200x, 3OOx, 400x, 5OOx or lOOOx. In one aspect, the activity of the gene expressed by the cell is decreased from about lOx to about lOOOx, about lOx to about 500x, about lOx to about 400x, about lOx to about 300x, about lOx to about 200x, about lOx to about lOOx, about lOOx to about 300x, or about lOOx to about 200x.
  • the activity of the gene expressed by a cell is decreased from about lOx to about lOOOx when the cell is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide conjugated to the compound(s) as compared to a cell that is contacted with the macromolecule, e.g., oligonucleotide and/or polypeptide not conjugated to the compound(s).
  • the activity of the gene expressed by a cell is decreased from about lOOx to about 500x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOOx to about 300x.
  • the activity of the gene expressed by a cell is decreased from about lOOx to about 200x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 500x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 300x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 200x. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about lOOx. In one aspect, the activity of the gene expressed by a cell is decreased from about lOx to about 50x.
  • a method for altering expression of a target nucleic acid in a cell.
  • the method includes: contacting the cell with an oligonucleotide that is capable of hybridizing to the target nucleic acid, wherein the oligonucleotide is conjugated to one or more compounds described herein and is internalized by the cell through endocytosis and encapsulated within an endosome, wherein the compound facilitates release of the oligonucleotide from the endosome, wherein the hybridization of the oligonucleotide to the target nucleic acid alters expression of the target nucleic acid.
  • hybridization of the oligonucleotide to the target nucleic acid increases expression of the target nucleic acid. In one aspect, hybridization of the oligonucleotide to the target nucleic acid decreases expression of the target nucleic acid.
  • the oligonucleotide is an ASO. In some embodiments, the oligonucleotide is an SSO. In some embodiments, the oligonucleotide is an siRNA. In some embodiments, the oligonucleotide is conjugated to a lipid and/or a sugar and/or a peptide.
  • the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof.
  • the compound is any one of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.
  • a method is provided for altering expression of a target nucleic acid in a cell.
  • the method includes: contacting the cell with a polypeptide, conjugated to one or more compounds described herein, that is capable of performing a SSM at the target nucleic acid, wherein the polypeptide is internalized by the cell through endocytosis and encapsulated within an endosome, wherein the compound facilitates release of the polypeptide from the endosome, wherein the SSM at the target nucleic acid alters expression of the target nucleic acid.
  • the SSM increases expression of the target nucleic acid.
  • the SSM decreases expression of the target nucleic acid.
  • the polypeptide is a Cas protein.
  • the polypeptide is a recombinase/meganuclease. In some embodiments, the polypeptide is Cre or FLP. In some embodiments, the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or is one or more compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In one aspect, the compound is one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.
  • a method for releasing a macromolecule, e.g., an oligonucleotide and/or a polypeptide, from an endosome.
  • the method includes: contacting the cell with the macromolecule, e.g., oligonucleotide and/or polypeptide conjugated to one or more compounds described herein, wherein the macromolecule, e.g., oligonucleotide and/or polypeptide, is internalized by the cell through endocytosis and encapsulated within the endosome, wherein the compounds facilitates release of the macromolecule, e.g., oligonucleotide and/or polypeptide, from the endosome.
  • the oligonucleotide is an ASO. In some embodiments, the oligonucleotide is an SSO. In some embodiments, the oligonucleotide is a siRNA. In some embodiments, the oligonucleotide is a guide RNA. In some embodiments, the polypeptide is a Cas protein. In some embodiments, the polypeptide is a recombinase. In some embodiments, the polypeptide is Cre. In some embodiments, the polypeptide is FLP. In some embodiments, the polypeptide is a Cas protein, and the polynucleotide is a guide RNA.
  • the one or more compounds has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or are compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof.
  • the compound is one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.
  • a method for the treatment and/or prevention of a disorder in a subject, such as a genetic disorder.
  • administration includes parenteral administration.
  • parenteral administration includes intravenous (IV) or subcutaneous (SC) administration.
  • the subject is a mammal.
  • the subject is a human.
  • the oligonucleotide is an ASO.
  • the oligonucleotide is an SSO.
  • the oligonucleotide is a siRNA.
  • the oligonucleotide is a guide RNA.
  • the polypeptide is a Cas protein.
  • the polypeptide is a recombinase. In some embodiments, the polypeptide is Cre. In some embodiments, the polypeptide is FLP. In some embodiments, the polypeptide is a Cas protein, and the polynucleotide is a guide RNA.
  • the one or more compounds has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or are one or more compounds compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In some embodiments, the compound is one or more of Compounds 4, 32, and 33, a pharmaceutically acceptable salt thereof, or combinations thereof.
  • a method for the treatment and/or prevention of a disorder in a subject.
  • the method includes: isolating a cell from the subject; contacting the isolated cell with a therapeutically effective amount of a macromolecule, e.g., an oligonucleotide and/or polypeptide, conjugated to one or more compounds described herein to produce an engineered cell; and transplanting the engineered cell in the subject.
  • the isolated cell is contacted with a composition comprising about 0.025 ⁇ M to about 20 ⁇ M of the macromolecule, e.g., oligonucleotide and/or polypeptide.
  • the isolated cell is contacted with about 0.1 ⁇ M to about 10 ⁇ M, about 0.1 ⁇ M to about 5 ⁇ M, or about 0.1 ⁇ M to about 1 ⁇ M of the macromolecule, e.g., oligonucleotide and/or polypeptide.
  • the oligonucleotide is an ASO.
  • the oligonucleotide is an SSO.
  • the oligonucleotide is a siRNA.
  • the oligonucleotide is a guide RNA.
  • the polypeptide is a Cas protein.
  • the polypeptide is a recombinase.
  • the polypeptide is Cre. In some embodiments, the polypeptide is FLP. In some embodiments, the polypeptide is a Cas protein, and the polynucleotide is a guide RNA. In some embodiments, the compound has a structure represented by Formula I, Formula la, lb, Ic, Id, le or If, or is one or more compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, or combinations thereof. In some embodiments, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or combinations thereof. Provided herein is a use of one or more compounds described herein in the manufacture of a medicament for gene therapy.
  • Provided herein is a use of one or more compounds of Formula I conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy.
  • Provided herein is a use of one or more compounds of Formula Ic conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy.
  • Provided herein is a use of one or more compounds of Formula Id conjugated to a macromolecule, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for gene therapy.
  • SMC Small-molecule compounds
  • ASO gapmer antisense oligonucleotide
  • FIG. 1 provides the overall generic scheme and includes starting material X shown in step 3.
  • FIG. 2 describes the definition of starting material X and required intermediate for each of the depicted compounds (e.g., Compound II depicted in FIG. 2 is the starting material “X” in the preparation described in FIG. 1 for Compound 2). Further descriptions of exemplified intermediates and Compounds are provided below.
  • the amino pyrazole and amino isopropoxypyrazole derivative intermediates identified in FIG. 2 that are used to make Compounds 19 and 30, respectively, were prepared according to the same procedures described below for intermediate 3 (amino methoxypyrazole) and 7 (amino ethoxypyrazole), respectively.
  • TLC show Cpd.l consumed, a new spot formed.
  • Compounds 32-35 can be further separated into their isomeric parts by supercritical fluid chromatography (SFC).
  • SFC supercritical fluid chromatography
  • Compound 32 was separated into its enantiomers by SFC with use of a mobile phase consisting of ethanol/DEA/CO2 and a YMC SA (IA) column (particle size 5 um).
  • the preparative conditions consisted of a flow of 3.5 ml/min at 40 degC and the compounds were detected at 272 nm.
  • the enantiomeric excess of both enantiomers exceeded 99%ee according to LC/MS analysis.
  • the fractions containing the separated enantiomers were evaporated which yielded the final compounds as solids.
  • MALAT1 ASO 5’-GCATTCTAATAGCAGC-3’ (SEQ ID NO: 2)
  • PPIB siRNA 5’ - CAGCAAAUUCCAUCGUGA-3 ’ (SEQ ID NO: 4), 5’- UCACGAUGGAAUUUGCUGUU-3’ (SEQ ID NO: 5)
  • Conjugate structure F (SEQ ID NO: 9): ( - denotes attachment point to oligonucleotide), peptide as disclosed in
  • Synthesis of VI Synthesis was carried out on OP 10 synthesizer using Polystyrene (90 mg) as solid support using traditional conditions with the following improvements: The trebler was coupled with 3 cycles and the BCN with 4 cycles. The compound was oxidized 3 times after the last BCN coupling. The product was removed from the solid support in ammonium hydroxide at 55°C overnight. The suspension was filtered thorugh a 0.45 pm syringe filter and the solution was concentrated on a speedvac, followed by freeze-drying. Isolated a white solid that was dissolved in water (2 mL) and precipitated with NaOAc/EtOH solution (40 mL). The suspension was kept in freezer for 30 min then centrifuged.
  • PPIB passenger strand
  • Malatl sequences were synthesized on an AKTA OligoPilot 10 using routine methods and starting either 3’ amino functionalized controlled-pore glass (CPG) solid support at 8 pmol scale (for ASO1-4, sense strands for siRNA3-8) or regular PS solid support at 32 pmol scale (ASO5, ASO6, sense strand for siRNAl, siRNA2 and antisense strand for all siRNA).
  • CPG controlled-pore glass
  • DMT dimethoxytrityl
  • Ancillary synthesis reagents and solvents were purchased from Sigma-Aldrich/Merck, TCI Chemicals or other commercial suppliers.
  • Phosphoramidites were prepared as 0.1 M solutions in either dry MeCN or 15% DMF in dry MeCN. Couplings were carried out using 0.25 M 5-[3,5-Bis(trifluoromethyl)phenyl]-lH- tetrazole (Activator 42®) in MeCN for 3x6 min or 2x8min. Thiolation was performed using 0.2 M Xanthane hydride in pyridine for 6 min. Oxidation was carried out with 0.02 M iodine in tetrahydrofuran/water/pyridine 90.54/9.05/0.41 (v/v/v) for 1 min.
  • Trityl groups were removed using 3% dichloroacetic acid (DCA) in toluene. After completed synthesis, final detritylation was not performed. The solid support with oligos attached were dried under high vacuum, and then further functionalized on 3 'or 5 'end.
  • DCA dichloroacetic acid
  • oligonucleotides except ASO4, ASO6, siRNA4, siRNA6, siRNA8
  • the oligo-BCN loaded solid support was placed in a tight closed vial and 2ml of solution of EEE- azide (0.1M in 1,4-dioxane) were added.
  • the solid supports were shaken overnight at room temperature followed by fdtration and solid support wash.
  • the 5 ’-EEE functionalized oligonucleotides were cleaved from solid support by treatment of aq. ammonia (26%) at 55 °C overnight.
  • the crude obtained 5 ’-EEE oligonucleotides were purified by HPLC and used for further 3’ functionalization.
  • oligonucleotides For synthesis of oligonucleotides ASO1, ASO2 and siRNA5-8 corresponding 3’-amino modified oligonucleotides were dissolved in MQ water to obtain 1.5mM solutions. A solution of BCN-PEG-NHS ester in MeCN (10 eq) was added to a solution of oligonucleotide and the mixtures were shaken at 40 °C for 18 h. The solvent was evaporated, and the conjugate was precipitated by addition of NaOAc and EtOH. The precipitate was redissolved in water and the corresponding peptide was added to the same vial as a lOmM DMSO solution. The mixture was shaken at rt overnight. The crude mixture was purified by HPLC.
  • the guide strand and the obtained passenger strand were annealed by mixing the two strands in a 1 : 1 ratio and heating the water solution to 95 °C for 5 min. The reaction was allowed to cool to room temperature where it was shaken for 1 hour. The solvent was evaporated, and the solid product was redissolved in PBS.
  • oligonucleotides For synthesis of oligonucleotides ASO3 and ASO4 corresponding 3 ’-amino modified oligonucleotides were dissolved in MQ water to obtain 1.5mM solutions. Palmitic NHS ester dissolved in DMF was added (10-30 eq.), and the pH adjusted to 9 with triethylamine. The mixtures were shaken at 40°C until full conversion had been confirmed by LCMS. After reaction completion the excess of solvent was removed and the residues were redissolved in MQ water, the final products were precipitated by addition of NaOAc and EtOH.
  • siRNA3 and siRNA4 corresponding 3 ’-amino modified oligonucleotides were dissolved in 0.5 M borate buffer pH 9.6.
  • An equal volume of a solution of GalNAc PFP ester (1.5 equiv.) in DMSO was added and the resulting slightly cloudy mixture was stirred at room temperature for 3 hours.
  • the mixture was subsequently diluted with 3 mL of 25% aqueous ammonia solution and left to stand at room temperature overnight. The next day, the mixture was diluted with water, freeze-dried and the dry residue was purified by reverse phase HPLC.
  • the guide strand and the obtained passenger strand were annealed by mixing the two strands in a 1:1 ratio and heating the water solution to 95 °C for 5 min. The reaction was allowed to cool to room temperature where it was shaken for 1 hour. The solvent was evaporated, and the solid product was redissolved in PBS.
  • HuH7 cells were obtained from Riken, 16HBE cells were ashamedly received from Karl Staples (University of Victoria). Cells were modified to stably express mCherry-GAL9 as earlier described 1 . All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and GlutaMAX (Gibco: 31966-021), all growth media was supplemented with 10% FBS, and 1 pg/ml Puromycin (Gibco: Al 1138-03) to maintain mCherry-GAL9 reporter expression. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. Cells were routinely tested and mycoplasma negative. Knockdown in human primary hepatocytes (GalNAc conjugates)
  • Human hepatocytes were obtained from a commercial supplier (BioIVT, QNT lot) and cultured in rat tail collagen I coated plates.
  • the hepatocytes were maintained in William's E Medium, supplemented with hepatocyte supplemented media and 5C supplements (DAPT, 25 SB431542, Forskolin, IWP2 and LDN193189).
  • the cells were cultured at 37°C in a humidified incubator with 5% CO2.
  • Next after plating Day 1
  • cells were treated with different siRNA conjugates and PBS in maintenance media.
  • media was changed and cells were harvested on Day 4 (72 hr from start of the treatment) for qPCR.
  • Total RNA was extracted according to standard protocol setup in the lab.
  • cDNA synthesis 5 and qPCR were also run according to standard validated protocol.
  • PPIB, and GAPDH TaqMan primers were purchased from Thermo Fischer.
  • HEK293T wt
  • HEK293 Gal9-mCherry
  • Cultured HEK293 cells were seeded directly in assay-ready plates containing ASO compounds as PBS serial dilutions of 1.5 steps. Cells were incubated for 24h at 37°C, 5% CO2 and 95% humidity. One day after plating, cells were lysed and RNA was extracted according to standard protocol. cDNA synthesis and qPCR were also run according to standard validated protocol. MALAT1 and RACK1 TaqMan primers were purchased from Thermo Fisher.
  • Huh7 cells stably expressing mCherry-Gal9 Huh7-mCherry-Gal9 cells were cultured according to standard procedures in DMEM with GlutaMax, 4.5g/L glucose, 10% FBS and I p.g/ml puromycin. Cells were trypsinised, resuspended in standard culture medium and plated at 4000 cells per well in 384 well culture plates. 24h later the media was removed from the cells and replaced with serum free DMEM with Glutamax. 10 point dilution series (concentration range 0.0015-30 ⁇ M) were prepared for both naked and C16 lipid conjugated ASOs. These were dosed into the medium and cells were incubated at 37°C, under 5% CO2 for
  • Real-Time PCR reactions were set up using 2pL cDNA, TaqManTM Fast Advanced Master Mix, and Malatl and Hprtl TaqManTM Gene Expression Assays (Hs00273907_sl and Hs02800695_ml, all Applied Biosystems) in a total volume of lOpL. Amplifications were performed on a QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystems) and were conducted at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
  • Cq Quantification cycle
  • V % knockdown at 10 ⁇ M in Huh7 cells
  • VI Galectin9 response (as fold-change compared to negative control) at 10 uM in 16HBE cells

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Abstract

L'invention concerne des composés et des procédés pour faciliter l'introduction d'un oligonucléotide, par exemple, dans un noyau et/ou un cytosol d'une cellule.
PCT/EP2025/061036 2024-04-24 2025-04-23 Compositions et procédés pour introduire une macromolécule dans une cellule Pending WO2025224155A1 (fr)

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