Compositions and methods for delivering macromolecules to cells

Abstract

Compounds and methods are provided for facilitating delivery of oligonucleotides, for example, to the nucleus and / or cytosol of a cell.

Description

[Technical Field]

[0001] The present disclosure relates to compositions and methods for delivering macromolecules, e.g., oligonucleotides, polypeptides, or combinations thereof, to cells, e.g., compositions and methods for delivering macromolecules, e.g., oligonucleotides, polypeptides, or combinations thereof, to the cytosol and / or nucleus of a cell. [Background technology]

[0002] Antisense oligonucleotides (ASOs) are short exogenous single-stranded DNA or RNA fragments that have a sequence complementary to the nucleotide sequence of a target nucleic acid. Classical single-stranded antisense oligonucleotides (ASOs) can act in the nucleus to cleave mRNA via an RNase H-dependent mechanism (Juliano, RL (2016) Nuc. Acids. Res. 44(14):6518-6548). Many therapeutic ASOs are "gapmers," which contain a central DNA region that supports RNase H activity, flanked by chemically modified termini to increase affinity and reduce susceptibility to nucleases (Bennett et al. (2010) Annu. Rev. Pharmacol. Toxicol. 50:259-293).

[0003] Splice-switching oligonucleotides (SSOs) are another form of antisense oligonucleotide that disrupt transcript splicing by hybridizing with pre-mRNA and blocking RNA-RNA base pairing or protein-RNA binding interactions between components of the splicing machinery (Havens and Hastings (2016) Nucleic Acids Res. 44(14);6549-63). SSOs can be designed to induce the inclusion or exclusion of introns and exons, ultimately restoring or inhibiting protein function or redirecting splicing to produce alternative protein isoforms. Furthermore, SSOs can be used to mask aberrant splice sites, thereby restoring normal alternative splicing and producing functional proteins. In theory, any pre-mRNA sequence can be targeted with SSOs; however, to date, only four SSOs have been approved by the FDA. (For example, 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 result from mutations affecting splicing. (Bauman et al., (2009) Oligonucleotides. 19(1):1-13) Several diseases, including spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD), currently lack effective treatments that target the underlying genetic defects. (Bestas et al. (2014) Nucleic Acid Ther. 24(1):13-24) SSO is a promising therapeutic approach that targets the underlying causes of these diseases.

[0004] RNA interference (RNAi) is an endogenous regulatory pathway for the control of gene expression, in which short (approximately 15–22 bp) double-stranded RNA fragments known as small interfering RNAs (siRNAs) are loaded into the RNA-induced silencing complex (RISC) and cleave target mRNAs in a sequence-dependent manner (Gavriolv and Saltzman (2012) Yale J. Biol. Med. 85(2):187–200).

[0005] Nucleic acid-based therapeutics have attracted attention as a promising approach for the treatment of various diseases and disorders, but many have failed to meet therapeutic endpoints due to challenges in effective in vivo delivery methods (Juliano, RL (2016) Nuc. Acids. Res. 44(14):6518-6548). One obstacle to the widespread use of oligonucleotide therapeutics is the inability of oligonucleotides to escape the endosomal compartment and reach the cytosol or nucleus at sufficient concentrations (Juliano et al. (2008) Nucleic Acids Res. 36(12):4158-4171).

[0006] Many recent developments have focused on increasing the cellular uptake and endosomal release of therapeutic oligonucleotides, for example, through chemical conjugation to ligands or encapsulation in synthetic nanoparticles (Barton and Medzhitov (2002) Proc. Natl. Acad. Sci. USA 99(23):14943-5, Johannes and Lucchino (2018) Nucleic Acids Ther. 28(3):178-193). Historically, the use of nanocarriers was thought to be required to facilitate the cellular uptake of polyanionic macromolecules such as ASOs. However, it has been discovered that single-stranded oligonucleotides are spontaneously endocytosed by cells in the absence of a carrier, a process called gymnosis (Stein et al. (2009) Nucleic Acids Res. 38(1):10.1093 / nar / gkp841).

[0007] Endosomolytic small molecule compounds (SMCs) are compounds that promote the release of gymnotic-delivered oligonucleotides that would otherwise accumulate in endosomes or lysosomes. Some endosomolytic SMCs induce endosomal membrane destabilization by buffering the endosomal lumen as luminal pH decreases with endosomal maturation. The increase in luminal pH occurs rapidly and can be reversible with appropriate dosing (Maxfield, FR (1982) J. Cell Biol. 95(2):676-681). This buffering leads to an increase in luminal osmolality, engulfing the endosome and inducing membrane rupture, ultimately leaking the endosomal cargo into the cytosol.

[0008] Chloroquine and its derivatives have been widely used to enhance the activity of oligonucleotide-containing nanoparticles by promoting endosomal release. While these compounds demonstrate significant in vitro efficacy, they typically require high micromolar concentrations, resulting in a narrow window between effective and toxic concentrations (Yang et al. (2015) Nucleic Acids Res. 43(4):1987-96; Wang et al. (2017) ACS Chem. Biol. 12(8):1999-2007). For example, chloroquine induces leakage between 40 and 100 μM (Lonn et al. (2016) Sci. Rep. 6:32301; Heath et al. (2019) Nanomedicine. 14(21):2799-2814). Summary of the Invention

[0009] Compositions and methods are provided for delivering macromolecules, such as oligonucleotides, polypeptides, or combinations thereof, to cells. In one aspect, a compound of formula I

[0010] [ka] (In the formula, one of Z1 and Z2 is N and the other is C; 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; R3 is hydro, halo, C1-C4 alkyl, -(CH2) y OH, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl; R6 is hydro or C1-C4 alkyl; R7, R8, R9, R10 , R 11 However, each independently, CHR 12 , C.R. 12 R 17 or NR 13 and R 12 Hydro, C1-C4 alkyl, -OR 14 , or -CO2R 15 and R 13 Hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 14 is hydro or C1-C4 alkyl; R 15 is hydro or C1-C4 alkyl; R 16 is hydro or C1-C4 alkyl; R 17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; wherein one or more of the alkyls are optionally substituted with one or more halo), or a pharmaceutically acceptable salt thereof.

[0011] In another embodiment, a compound of formula Ia

[0012] [ka] (In the formula, one of Z1 and Z2 is N and the other is C; 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; R3 is hydro, halo, C1-C4 alkyl, -(CH2) yOH, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; R4 is C1-C4 alkyl; each R5 is independently hydro or C1-C4 alkyl, for example, methyl or ethyl; R6 is hydro or C1-C4 alkyl, for example, methyl or ethyl; R 13 Hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 14 is hydro or C1-C4 alkyl; R 15 is hydro or C1-C4 alkyl; R 16 is hydro or C1-C4 alkyl; one or more of the alkyls are optionally substituted with one or more halo; or a pharmaceutically acceptable salt thereof.

[0013] In another embodiment, a compound of formula Ib or formula Ic

[0014] [ka] (Wherein R1, X, R3 and R 13 (wherein, as described above) is provided.

[0015] In another embodiment, a compound of Formula I, Ia, Ib, or Ic, 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; R3 is hydro, halo, C1-C4 alkyl, -(CH2) yOH, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; R4 is C1-C4 alkyl; R5 is hydro, methyl or ethyl; R6 is hydro, methyl or ethyl; R 13 Hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 14 is hydro or C1-C4 alkyl; R 15 is hydro or C1-C4 alkyl; R 16 is hydro or C1-C4 alkyl; one or more of the alkyls are optionally substituted with one or more halo; or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, R1 is cyano, halo, or C1-C4 alkyl optionally substituted with one or more chloro or fluoro. In one embodiment, R1 is cyano, methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, fluoro, chloro, or bromo. In one embodiment, R1 is cyano, bromo, chloro, fluoro, or trifluoromethyl. In one embodiment, R1 is fluoro.

[0017] In one aspect, X is -OR2, and R2 is methyl, ethyl, or isopropyl. In one aspect, X is hydro.

[0018] In one aspect, R3 is hydro; cyano; C1-C4 alkyl optionally substituted with one or more chloro, fluoro, or hydroxy; or -OR4, where R4 is C1-C4 alkyl.

[0019] In one embodiment, R7, R8, R 10 , and R 11 are each independently CHR 12 or CR 12 R 17 and R9 is NR 13 In one aspect, each R 12 is hydro. In one aspect, R 13 is hydro, C1-C4 alkyl, -C(=O)R 16 , or -(CH2) y OH and y is 1, 2, or 3. In one aspect, R 13 is —(CH)OH. In one aspect, R 13 is methyl.

[0020] In one aspect, R1 is halo, X is -OR2, R2 is ethyl or methyl, R3 is cyano, and R 13 is hydro, C1-C4 alkyl, -C(=O)CH3, or -(CH2)3OH. In one aspect, R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl, R3 is cyano, and R 13 is hydro or methyl. In one aspect, R is halo, X is —OR, R is ethyl or methyl, R is methyl or methoxy, and R 13 In one aspect, R1 is halo, X is -OR2, R2 is C1-C4 alkyl, R3 is hydro, and R 13 In one aspect, R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl, R3 is hydro, and R 13 is hydro or methyl.

[0021] In one aspect, R1 is halo, X is hydro, R3 is cyano, and R 13 In one aspect, R1 is cyano, X is -OR2, R2 is ethyl or methyl, R3 is haloalkyl, and R 13In one aspect, R1 is ethyl or methyl, X is -OR2, R2 is ethyl or methyl, R3 is hydro, and R 13 In one aspect, R1 is halo, X is -OR2, R2 is ethyl or methyl, and R3 is (CH2) y OH and R 13 is hydro and y is 0, 1, 2, or 3.

[0022] In some embodiments, a compound in Table 1 or a pharmaceutically acceptable salt thereof is disclosed.

[0023] [Table 1-1]

[0024] [Table 1-2]

[0025] [Table 1-3]

[0026] [Table 1-4]

[0027] [Table 1-5]

[0028] [Table 1-6]

[0029] [Table 1-7] or a pharmaceutically acceptable salt thereof.

[0030] In one aspect, a composition is provided comprising an oligonucleotide and a compound of any one of Formula I, Ia, Ib, Ic, or Table 1, a pharmaceutically acceptable salt of a compound of any one of Formula I, Ia, Ib, Ic, or Table 1, or a combination thereof. In one aspect, a pharmaceutical composition is provided comprising an oligonucleotide and a compound of any one of Formula I, Ia, Ib, Ic, or Table 1, a pharmaceutically acceptable salt of a compound of Formula I, Ia, Ib, Ic, or Table 1, or a combination thereof, and a pharmaceutically acceptable diluent or carrier.

[0031] In one embodiment, the oligonucleotide is single-stranded. In one embodiment, the oligonucleotide is double-stranded. In one embodiment, the oligonucleotide comprises DNA. In one embodiment, the oligonucleotide comprises RNA. In one embodiment, the oligonucleotide comprises about 8 to about 30 nucleotides. In one embodiment, the oligonucleotide is an antisense oligonucleotide (ASO), a splice-switching oligonucleotide (SSO), an interfering RNA (RNAi), a small interfering RNA (siRNA), a microRNA (miRNA), an antagomir, a decoy oligonucleotide, or a combination thereof.

[0032] In one embodiment, the oligonucleotide comprises one or more modified nucleotides, including phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), any other 2'-modified oligonucleotide, or a combination thereof.

[0033] In one aspect, a method is provided for introducing an oligonucleotide into the nucleus and / or cytosol of a cell. In one aspect, the method comprises: (a) contacting a cell with an oligonucleotide; (b) contacting the cell with a compound of any one of Formula I, Ia, Ib, Ic, or Table 1, a pharmaceutically acceptable salt of a compound of any one of Formula I, Ia, Ib, Ic, or Table 1, or a combination thereof. In one aspect, the compound promotes entry of the oligonucleotide into the nucleus and / or cytosol of the cell. In one aspect, the oligonucleotide is internalized by the cell via endocytosis and encapsulated in an endosome, and the compound promotes release of the oligonucleotide from the endosome. In one aspect, the oligonucleotide is internalized by transient pore formation induced by the compound.

[0034] In one embodiment, contacting the cell with the oligonucleotide (a) is performed in a composition, wherein the composition (a) comprises about 0.025 μM to about 20 μM of the oligonucleotide. In one embodiment, the composition (a) comprises about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide. In one embodiment, the cell is contacted with a second composition comprising about 1 μM to about 10 μM of the compound.

[0035] In one embodiment, the contacting of the cell with the compound (b) is carried out in a composition, and the composition (b) contains about 1 μM to about 20 μM of the compound. In one embodiment, the composition (b) contains at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 10.5 μM, The compound may be present at about 11 μM, about 11.5 μM, about 12 μM, about 12.5 μM, about 13 μM, about 13.5 μM, about 14 μM, about 14.5 μM, about 15 μM, about 15.5 μM, about 16 μM, about 16.5 μM, about 17 μM, about 17.5 μM, about 18 μM, about 18.5 μM, about 19 μM, about 19.5 μM, or about 20 μM.

[0036] In one embodiment, the cell is contacted with the oligonucleotide of (a) and the compound of (b) at about the same time.

[0037] In one embodiment, the cells are contacted with the oligonucleotide of (a) before contacting with the compound of (b). In one embodiment, the cells are contacted with the oligonucleotide of (a) up to about 48 hours before contacting with the compound of (b). In one embodiment, the cells are contacted with the oligonucleotide of (a) about 12 hours to about 48 hours before contacting with the compound of (b).

[0038] In one embodiment, contacting the cell with the oligonucleotide (a) and contacting the cell with the compound (b) are carried out in the same composition. In one embodiment, the composition comprises about 0.025 μM to about 10 μM of the oligonucleotide. In one embodiment, the composition comprises about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide. In one embodiment, the composition comprises about 1 μM to about 20 μM of the compound. In one embodiment, the composition comprises about 1 μM to about 10 μM of the compound. In one embodiment, the composition comprises a concentration of about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 10.5 μM, about 11 μM M, about 11.5 μM, about 12 μM, about 12.5 μM, about 13 μM, about 13.5 μM, about 14 μM, about 14.5 μM, about 15 μM, about 15.5 μM, about 16 μM, about 16.5 μM, about 17 μM, about 17.5 μM, about 18 μM, about 18.5 μM, about 19 μM, about 19.5 μM, or about 20 μM of the compound.

[0039] In one aspect, the oligonucleotide hybridizes to a target nucleic acid in a cell. In one aspect, 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.

[0040] In one aspect, contacting a cell with a compound of (b) results in endosomal membrane permeabilization as determined by an mCherry-GAL9 recruitment assay, accompanied by reduced oligonucleotide accumulation in endosomes and / or lysosomes.

[0041] In one embodiment, the oligonucleotide alters the activity of a gene expressed by a cell. In one embodiment, the oligonucleotide increases the activity of a gene expressed by a cell. In one embodiment, the activity of a gene expressed by a cell is increased by at least about 10-fold when the cell is contacted with the oligonucleotide and the compound, compared to cells contacted with the oligonucleotide but not the compound. In one embodiment, the activity of a gene expressed by a cell is increased by at least about 100-fold when the cell is contacted with the oligonucleotide and the compound, compared to cells contacted with the oligonucleotide but not the compound.

[0042] In one embodiment, the oligonucleotide reduces the activity of a gene expressed by a cell. In one embodiment, the activity of a gene expressed by a cell is reduced by at least about 10-fold when the cell is contacted with the oligonucleotide and the compound, compared to a cell contacted with the oligonucleotide but not the compound. In one embodiment, the activity of a gene expressed by a cell is reduced by at least about 100-fold when the cell is contacted with the oligonucleotide and the compound, compared to a cell contacted with the oligonucleotide but not the compound.

[0043] In one aspect, the method comprises in vitro delivery of the oligonucleotide to a cell. In one aspect, the method comprises in vivo delivery of the oligonucleotide to a cell.

[0044] In one embodiment, the cells are cultured cells. In one embodiment, the cells are isolated cells. In one embodiment, the cells are cells isolated from a subject in need of treatment. In one embodiment, the cells are part of a tissue or organ. In one embodiment, the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, fat, and / or muscle (e.g., skeletal muscle). In one embodiment, the cells are brain cells, CNS cells, PNS cells, cardiac cells, liver cells, kidney cells, spleen cells, pancreatic cells, lung cells, muscle cells, adipocytes, immune cells, or combinations thereof.

[0045] In one embodiment, the cell is a mammalian cell, hi another embodiment, the cell is a eukaryotic and / or prokaryotic cell.

[0046] In one embodiment, a method for altering expression of a target nucleic acid in a cell is provided. In one embodiment, the method includes contacting the cell with an oligonucleotide capable of hybridizing to the target nucleic acid, wherein the oligonucleotide is internalized by the cell via endocytosis and encapsulated in an endosome; and contacting the cell with a compound of any one of Formula I, Ia, Ib, or Ic, or Table 1, a pharmaceutically acceptable salt of a compound of Formula I, Ia, Ib, or Ic, or Table 1, or a combination thereof, wherein the compound promotes release of the oligonucleotide from the endosome, and hybridization of the oligonucleotide to the target nucleic acid alters expression of the target nucleic acid. In one embodiment, hybridization of the oligonucleotide to the target nucleic acid increases expression of the target nucleic acid. In one embodiment, hybridization of the oligonucleotide to the target nucleic acid decreases expression of the target nucleic acid. In one embodiment, hybridization of the oligonucleotide to the target nucleic acid alters splicing of the target nucleic acid.

[0047] In one aspect, a method of releasing an oligonucleotide from an endosome is provided. In one aspect, the method includes contacting a cell with an oligonucleotide, wherein the oligonucleotide is internalized by the cell by endocytosis and packaged in an endosome, and contacting the cell with a compound of any one of Formula I, Ia, Ib, or Ic, or Table 1, a pharmaceutically acceptable salt of a compound of Formula I, Ia, Ib, or Ic, or Table 1, or a combination thereof, wherein the compound promotes release of the oligonucleotide from the endosome.

[0048] In one aspect, a method for treating and / or preventing a disorder in a subject is provided. In one aspect, the method comprises administering to the subject a therapeutically effective amount of an oligonucleotide and an effective amount of a compound of any one of Formulae I, Ia, Ib, or Ic or Table 1, or a combination thereof. In one aspect, the compound is administered to the subject simultaneously with the oligonucleotide. In one aspect, the compound is administered to the subject after administration of the oligonucleotide. In one aspect, the compound is administered to the subject up to about 48 hours after administration of the oligonucleotide. In one aspect, the compound is administered to the subject about 12 hours to about 48 hours after administration of the oligonucleotide. In one aspect, the administration comprises parenteral administration. In one aspect, the administration comprises intravenous or subcutaneous administration. In one aspect, the subject is a mammal. In one aspect, the subject is human.

[0049] In one aspect, a method for treating and / or preventing a disorder in a subject is provided, comprising isolating cells from the subject; contacting the isolated cells with a therapeutically effective amount of an oligonucleotide and an effective amount of a compound of any one of Formula I, Ia, Ib, or Ic, or Table 1, a pharmaceutically acceptable salt of a compound of Formula I, Ia, Ib, or Ic, or Table 1, or a combination thereof, to produce engineered cells; and transplanting the engineered cells into the subject. In one aspect, the isolated cells are contacted with a composition comprising about 0.025 μM to about 20 μM of the oligonucleotide. In one aspect, the composition comprises about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide. In one aspect, the isolated cells are contacted with a composition comprising about 1 μM to about 20 μM of the compound. In one aspect, the composition comprises about 1 μM to about 10 μM of the compound. In one aspect, the composition comprises a phosphodiesterase inhibitor having a phosphodiesterase activity of at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 10.5 μM, about 1 ... The compound may be present at 1 μM, about 11.5 μM, about 12 μM, about 12.5 μM, about 13 μM, about 13.5 μM, about 14 μM, about 14.5 μM, about 15 μM, about 15.5 μM, about 16 μM, about 16.5 μM, about 17 μM, about 17.5 μM, about 18 μM, about 18.5 μM, about 19 μM, about 19.5 μM, or about 20 μM of the compound. In one embodiment, the isolated cells are contacted with the oligonucleotide and the compound at approximately the same time. In one embodiment, the isolated cells have a composition comprising the oligonucleotide and the compound. In one embodiment, the isolated cells are contacted with the oligonucleotide before contacting the isolated cells with the compound. In one embodiment, the isolated cells are contacted with the oligonucleotide up to about 48 hours before contacting the isolated cells with the compound. In one embodiment, the isolated cells are contacted with the oligonucleotide about 12 to about 48 hours after contacting the isolated cells with the compound. In one aspect, the subject is a mammal, hi one aspect, the subject is a human.

[0050] In one aspect, there is provided the use of a compound of any one of Formula I, Ia, Ib, or Ic, or Table 1, a pharmaceutically acceptable salt of a compound of Formula I, Ia, Ib, or Ic, or Table 1, or a combination thereof, for the manufacture of a medicament for gene therapy.

[0051] In one aspect, a kit is provided that includes a compound of any one of Formula I, Ia, Ib, or Ic, or Table 1, a pharmaceutically acceptable salt of a compound of Formula I, Ia, Ib, or Ic, or Table 1, or a combination thereof, and an oligonucleotide. [Brief explanation of the drawings]

[0052] [Figure 1A] FIG. 1 shows the chemical structure of UNC2383, a small molecule endosomolytic compound described by Wang et al. (2018) ACS Chem. Biol. 12(8):1999-2007. [Figure 1B] 1 shows the chemical structure of compound 2 (also known as AZ4800) described herein. [Figure 1C] 1 shows the chemical structure of compound 6 (also called AZ2467 or A7) described herein. [Figure 2A]

[0023] Figure 1 shows the functional activity of splice-switching oligonucleotides (SSOs) co-treated with UNC2383 and other selected compounds, including AZ4800 (Compound 2). HeLaLuc-705 cells were pretreated with 1 μM SSO and then co-treated with 10 μM, 5 μM, or 1 μM UNC2383 or other selected compounds, including AZ4800 (Compound 2), for 2 hours. Cells were then rinsed and incubated in growth medium for 4 hours. Values ​​are reported as fold increase over 1 μM naked SSO. [Figure 2B] 1 is a graph showing the increase in functional SSO activity upon co-treatment with select compounds, including AZ4800 (Compound 2) and AZ2467 (Compound 6). HeLaLuc-705 cells were exposed to the same co-treatment regimen as above with 5 μM compound and either 1 μM SSO or 100 nM SSO. [Figure 3] Gal9 body-induced endosomal rupture in HeLa mCherry-Gal9 cells treated with UNC2383 or other selected compounds, including AZ2467 (compound 6) and AZ4800 (compound 2), over a 10-point dose-escalation series for 2 hours and then stained for nuclei (Hoechst 33342). Representative images show the relocalization of Gal9 from the cytosol to puncta in a dose-dependent manner. Scale bar = 20 μm. [Figure 4] Images showing Gal-9 body-induced endosomal rupture in Huh7 mCherry-Gal9 cells treated with UNC2383 or other selected compounds, including AZ2467 (compound 6) and AZ4800 (compound 2), over a 10-point dose-escalation series for 2 hours and then stained for nuclei (Hoechst 33342). Representative images show the relocalization of Gal9 from the cytosol to puncta in a dose-dependent manner. Scale bar = 20 μm. [Figure 5] Figure 1 shows the narrow concentration window between induction of endosomal rupture and cytotoxicity for HeLa and Huh7 cells treated with DMSO, UNC2383, or other selected compounds, including AZ2467 (compound 6) and AZ4800 (compound 2). Gal9 puncta are reported as a percentage of maximum quantification normalized to the nuclear signal. Cell viability is reported as a percentage of total cells. [Figure 6] Images showing Huh7mCherry-Gal9 stable cells treated with 5 μM of selected compounds, including SMC5 / AZ4800 (compound 2), A7 (compound 6), A1 (compound 5), and DMSO, for up to 4 hours in a Gal9 recruitment assay. [Figure 7]Figure 1 shows quantification of Gal9 recruitment in Huh7 cells treated with UNC2383 or other selected compounds, including SMC5 / AZ4800 (compound 2), A7 (compound 6), A1 (compound 5), and DMSO, revealing time-dependent endosomal rupture, even from compounds that showed no detectable functional activity. Values ​​are reported as puncta per relative nuclear signal and plotted normalized to the highest total induced signal (SMC5 / AZ4800 (compound 2), 60 min, Huh7). [Figure 8] Figure 1 shows quantification of Gal9 recruitment in HeLa cells treated with select compounds, including SMC5 / AZ4800 (compound 2), A7 (compound 6), A1 (compound 5), and DMSO, revealing time-dependent endosomal rupture, even from compounds that showed no detectable functional activity. Values ​​are reported as puncta per relative nuclear signal and plotted normalized to the highest total induced signal (SMC5 / AZ4800 (compound 2), 60 min, Huh7). [Figure 9] Figure 1 shows the results of RT-PCR with gel analysis to detect abnormal or functional mRNA copies in cells treated with AZ4800 (compound 2) and AZ2467 (compound 6). Gel images were quantified using Quantity One (BioRad) software. Functional mRNA values ​​were reported as a percentage of total mRNA. [Figure 10] Figure 1 shows a schematic diagram of the treatment regimen in which cells are seeded in growth medium containing DMEM + 10% FBS supplemented with SSO. After 24 hours, endosomolytic compounds diluted in growth medium are added to the desired final concentration. After 2 hours of co-treatment, the medium is removed, the cells are washed with phosphate buffered saline (PBS), and incubated in growth medium for 4 hours. [Figure 11A]1 is a graph showing the increase in functional SSO activity obtained using different treatment regimens with AZ4800 (compound 2). The "positive" group received the treatment described in connection with FIG. 10. The "preload only" group was treated with 1 μM SSO for 24 hours, followed by 5 μM compound for 2 hours. The "no preload" group was treated simultaneously with 1 μM SSO and 5 μM AZ4800 (compound 2) for 2 hours. Values ​​are reported as fold increase over untreated. [Figure 11B] 1 is a graph showing the increase in functional SSO activity in HeLaLuc-705 cells co-treated with 1 μM SSO and various concentrations of AZ2467 (compound 6). The co-treatment period lasted 30 minutes, 1 hour, 2 hours, 4 hours, or 6 hours, followed by the same 4-hour incubation step. Values ​​are reported as fold increase over SSO alone. [Figure 11C] 1 is a graph showing the increase in functional SSO activity in HeLaLuc-705 cells co-treated with 1 μM SSO and various concentrations of AZ4800 (compound 2). The co-treatment period lasted 30 minutes, 1 hour, 2 hours, 4 hours, or 6 hours, followed by the same 4-hour incubation step. Values ​​are reported as fold increase over SSO alone. [Figure 12A] FIG. 11 is a graph showing the increased efficacy of 705-SSO in HeLa cells when treated with different concentrations of AZ4800 (compound 2) according to the treatment scheme outlined in FIG. 10, except that the preloading step was performed for 48 hours instead of 24 hours. Cells were analyzed 2, 4, and 6 hours after treatment with AZ4800 (compound 2). Values ​​are reported as fold increase over treatment with naked oligo alone. [Figure 12B] Figure 11 is a graph showing the increased efficacy of 705-SSO in Huh7 cells when treated with different concentrations of AZ4800 (compound 2) according to the treatment scheme outlined in Figure 10, except that the preloading step was performed for 48 hours instead of 24 hours. Cells were analyzed 2, 4, and 6 hours after treatment with AZ4800 (compound 2). Values ​​are reported as fold increase over treatment with naked oligo alone. [Figure 13A]1 is a graph showing the increased efficacy of 705-SSO in HeLa cells when treated with different concentrations of AZ4800 (compound 2). Cells in the "preloaded" group were treated similarly to the treatment regimen described in FIG. 10, while cells in the "no preloaded" group were co-treated with AZ4800 (compound 2) and 1 μM 705-SSO alone. Values ​​are reported as fold increase over treatment with naked oligo alone. [Figure 13B] 1 is a graph showing the increased efficacy of 705-SSO in Huh7 cells when treated with different concentrations of AZ4800 (compound 2). Cells in the "preloaded" group were treated similarly to the treatment regimen described in FIG. 10, while cells in the "no preloaded" group were co-treated with AZ4800 (compound 2) and 1 μM 705-SSO alone. Values ​​are reported as fold increase over treatment with naked oligo alone. [Figure 14A] 14A and 14B are graphs showing the results of nanoparticle tracking analysis, revealing no significant change in particle size profile when cell growth medium containing 1 μm (FIG. 14A) or 5 μm (FIG. 14B) oligonucleotides was incubated with 5 μm AZ4800 (compound 2) for 2 hours. Composite profiles were generated from 5 × 30-second videos of each condition. [Figure 14B] 14A and 14B are graphs showing the results of nanoparticle tracking analysis, revealing no significant change in particle size profile when cell growth medium containing 1 μm (FIG. 14A) or 5 μm (FIG. 14B) oligonucleotides was incubated with 5 μm AZ4800 (compound 2) for 2 hours. Composite profiles were generated from 5 × 30-second videos of each condition. [Figure 15A] Confocal microscopy images of a live-cell mCherry-Gal9 assay using 488-SSO in HeLa cells, showing Gal9 relocalization and the concomitant reduction of SSO-containing bodies. Huh7Gal9-mCherry cells were preloaded with 488-SSO for 24 hours and then treated with various concentrations of AZ4800 (compound 2) and Hoechst to visualize nuclei. Images were acquired every 10 minutes. Scale bar = 20 μM. [Figure 15B]Confocal microscopy images of live-cell mCherry-Gal9 assays using 488-SSO in Huh7 cells show Gal9 relocalization and the concomitant reduction of SSO-containing bodies. Huh7Gal9-mCherry cells were preloaded with 488-SSO for 24 hours and then treated with various concentrations of AZ4800 (compound 2) and Hoechst to visualize nuclei. Images were acquired every 10 minutes. Scale bar = 20 μM. [Figure 16A] FIG. 15B is a graph showing quantitative analysis of the images in FIG. 15A, showing the time- and dose-dependent decrease in the total number of 488-SSO puncta normalized to the total nuclear signal. [Figure 16B] FIG. 15C is a graph showing quantitative analysis of the images in FIG. 15B, showing the time- and dose-dependent decrease in the total number of 488-SSO puncta normalized to the total nuclear signal. [Figure 17A] FIG. 10 is a graph showing quantitative analysis of Gal9-mCherry puncta in HeLa cells, showing dose- and time-dependent increases in Gal9 bodies normalized to the nuclear signal. [Figure 17B] FIG. 10 is a graph showing quantitative analysis of Gal9-mCherry puncta in Huh7 cells, showing a dose- and time-dependent increase in Gal9 bodies normalized to the nuclear signal. [Figure 18] Schematic diagram of the synthesis of compound 2. [Figure 19] FIG. 1 is a schematic diagram of the synthesis of compound 3. [Figure 20] FIG. 1 is a schematic diagram of the synthesis of compound 4. [Figure 21] FIG. 1 is a schematic diagram of the synthesis of compound 7. [Figure 22] FIG. 1 is a schematic diagram of the synthesis of intermediate A. [Figure 23A] 1 is a graph showing the increased efficacy of Luc 705-SSO in HeLa cells upon treatment with selected compounds, including AZ3325 (compound 7) and AZ3327 (compound 4). [Figure 23B] 1 is a graph showing increased efficacy of 705-SSO in HeLa cells with higher concentrations of AZ3327 (compound 4). [Figure 24]1 is a graph showing the increased efficacy of Luc 705-SSO in HeLa cells upon treatment with select compounds, including AZ2862 (compound 8) and AZ3327 (compound 4). [Figure 25A] 1 is a graph showing the increased efficacy of Luc 705-SSO in U2OS cells upon treatment with select compounds including AZ4374 (compound 21), AZ4376 (compound 22), and AZ2862 (compound 8). [Figure 25B] 1 is a graph showing the increased efficacy of Luc 705-SSO in N2A cells upon treatment with select compounds including AZ4374 (compound 21), AZ4376 (compound 22), and AZ2862 (compound 8). [Figure 26] FIG. 1 is a graph showing the increased efficacy of Luc 705-SSO in HeLa cells by select compounds including AZ4800 (compound 2), AZ3327 (compound 4), AZ4374 (compound 21), AZ2862 (compound 8), and AZ3325 (compound 7). [Figure 27] FIG. 1 is a graph showing the enhanced oligo activity of AZ3327 (compound 4) compared to oligo alone in three cell types (HeLa_Luc705, U2OS_Luc705, and N2A_Luc705 cells). [Figure 28] 1 is a graph comparing the enhanced oligo activity of AZ3327 (compound 4) versus oligo alone in enhancing knockdown of MALAT1 mRNA expression. [Figure 29] 1 is a general scheme for the synthesis or preparation of compounds 2-7 and 19-31. [Figure 30] 29. FIG. 29 is an exploded view of the starting material "X" and the intermediates required for each of compounds 2-7, 19-31 identified in FIG. [Figure 31] FIG. 1 is a general schematic diagram of the synthesis or preparation of compounds 8-17 with an additional alkylation step. [Figure 32] FIG. 1 is a general schematic diagram of the synthesis or preparation of compound 18. [Figure 33] 1 is a graph showing the results of GAL9 responses associated with selected compounds at various doses (1.25-5 μM) in Huh7 cells. [Figure 34] 1 is a graph showing the results of GAL9 responses associated with selected compounds at various doses (1.25-5 μM) in Hela cells. [Figure 35] Images showing Gal9 body-induced endosomal rupture in HeLa, mCherry-Gal9 cells treated with selected compounds are shown. [Figure 36] Images showing Gal9 body-induced endosomal rupture in HeLa, mCherry-Gal9 cells treated with selected compounds are shown. [Figure 37] Images showing Gal9 body-induced endosomal rupture in HeLa, mCherry-Gal9 cells treated with selected compounds are shown. [Figure 38] Images showing Gal9 body-induced endosomal rupture in HeLa, mCherry-Gal9 cells treated with selected compounds are shown. [Figure 39] Images showing Gal9 body-induced endosomal rupture in HeLa mCherry-Gal9 cells treated with various doses (0.3125-10 μM) of selected compounds are shown. [Figure 40] Graph comparing time (0.25-4 hours), dose of selected compounds (0.31-10 μM), and GAL9 response in Huh7 cells. [Figure 41] Graph comparing GAL9 responses 2 hours after administration of selected compounds at various doses (0.31-10 μM) and among different cell lines. [Figure 42] Included are graphs and tables comparing administration of (i) unconjugated siRNA ("duplex-PPIB"), (ii) siRNA conjugated with cholesterol ("Chol-PPIB"), (iii) siRNA conjugated with GalNAc ("GalNAc-PPIB"), and (iv) unconjugated siRNA co-administered with 2 μM Compound 32 ("duplex-PPIB+ERE"). [Figure 43A]Legend for the data shown in Figures 43B and 43C is shown: total cell numbers are shown as open bars (left panel), live cells are shown as light grey shaded bars (center panel), edited cell numbers are shown as green shaded bars, and the percentage of edited cells is shown as a dotted line (right panel). [Figure 43B] Included are graphs showing T-47D cells co-treated with 50 nM Cre protein and various concentrations of selected compounds, with the medium either changed after 2 hours (left panel) or not (right panel). [Figure 43C] Included are graphs showing HeLa cells co-treated with 50 nM Cre protein and various concentrations of selected compounds, with the medium either changed after 2 hours (left panel) or not (right panel). [Figure 44A] Immunofluorescence staining of coronal and sagittal brain segments from mice co-administered with Cre recombinase and various concentrations of selected compounds is shown. [Figure 44B] 1 shows immunofluorescence staining of lumbar spinal cord segments from mice co-administered with Cre recombinase and various concentrations of selected compounds. [Figure 44C] The dissection strategy and immunofluorescence results for each dissection segment are shown. [Figure 44D] IVIS® in vivo imaging and immunofluorescence staining of prefrontal lobe, pre-injection, and incised segments of the injection site are shown. [Figure 45A] Graphs showing percent viability and percent ex vivo editing (as measured by % tdTomato fluorescence) in non-activated T cells are included. [Figure 45B] 1 includes graphs showing percent viability and percent ex vivo editing in pre-activated T cells. [Figure 45C] 1 includes graphs showing percent viability and percent ex vivo editing in T cells after activation. [Figure 46A] A schematic diagram of the editing-based GFP reporter system is shown. [Figure 46B] 1 includes graphs showing HEK cells co-treated with 50 nM of a Cas base editor and various concentrations of selected compounds. [Figure 46C] Included are graphs showing HEK and N2a cells co-treated with 2.5 μM of select compounds and various concentrations of base editors. [Figure 47] Images showing Gal9 body-induced endosomal rupture in Huh7 mCherry-Gal9 cells treated with various doses (0.3125-10 μM) of selected compounds are included. [Figure 48] Graphs comparing GAL9 responses 2 hours after administration of selected compounds at various doses (0.31-10 μM) and among different cell lines are shown. [Figure 49] 1 is a graph showing the increase in functional SSO activity in HeLaLuc-705 cells co-treated with 1 μM SSO and various concentrations of AZ5219 (compound 32). The co-treatment period lasted 30 minutes, 1 hour, 2 hours, 4 hours, or 6 hours, followed by the same 4-hour incubation step. Values ​​are reported as fold increase over SSO alone. DETAILED DESCRIPTION OF THE INVENTION

[0053] A.Definition Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular, for example, "a" or "an" shall include plurals, e.g., "one or more" or "at least one," and the term "or" may mean "and / or" unless otherwise stated. The terms "including," "includes," and "included" are not limiting. Ranges provided herein are of any kind and include all values ​​within the particular ranges described, as well as values ​​related to the endpoints of the particular ranges.

[0054] "Alkyl" refers to a saturated, branched, or straight-chain hydrocarbon group. "Lower alkyl" refers to an alkyl group having 1 up to about 8 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one aspect, the alkyl group contains 1 to 4 carbon atoms (C1-C4 alkyl). Lower alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, and butyl, including n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0055] An alkyl group can be substituted or unsubstituted. In some embodiments, an alkyl group is substituted with one or more halo groups (e.g., F, Cl, Br, I, At, etc.). In some embodiments, an alkyl group is substituted with one or more F or Cl (e.g., mono-, di-, or trifluoro or chloroalkyl). An alkyl group in which one or more hydrogen atoms are replaced with halogen can be referred to as a "haloalkyl," e.g., "halo C1-C4 alkyl" refers to a C1-C4 alkyl substituted with one or more of the same or different halogen atoms. Examples of C1-C4 alkyl substituted with one or more halo groups include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0056] "Alkoxy," also represented as "-OR," where R is an alkyl group, refers to a saturated or unsaturated, branched or straight-chain hydrocarbon group attached to the parent molecule via an oxygen atom. In one aspect, an alkoxy group contains 1 to 4 carbon atoms (C-C alkoxy). Examples of C-C alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, iso-butoxy, and butoxy, including t-butoxy. In some embodiments, an alkoxy group is substituted with one or more halo groups (e.g., F, Cl, Br, I, At, etc.). In some embodiments, an alkoxy group is substituted with one or more F or Cl (e.g., mono-, di-, or trifluoro- or chloroalkoxy).

[0057] "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.

[0058] "Amido" 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), including primary, secondary, and tertiary amides. In one aspect, the alkyl in the amide comprises a C1-C8 alkyl. In one aspect, the alkyl substituent comprises a C1-C4 alkyl. In some embodiments, at least one R5 in the formula "-C(O)NR5R5" is hydro.

[0059] "Ester" refers to the group "-C(O)OR6" (also represented as "-C(=O)OR6"), where R6 is hydro or alkyl. In some embodiments, the ester is a "short chain ester" where R6 is C1-C4 alkyl. In some embodiments, R6 is methyl or ethyl. In some embodiments, R6 is propyl or isopropyl.

[0060] "Cyano" is a carbon atom triple-bonded to a nitrogen atom

[0061] [ka] refers to a group containing

[0062] "Hydro" refers to a hydrogen substituent, also represented by "-H".

[0063] "Endosomolytic" refers to a compound that promotes the release of oligonucleotides from endosomes / lysosomes / autophagosomes / multivesicular bodies or other endosomal vesicles into the cytosol of a cell. In one embodiment, the endosomolytic agent is a small molecule compound (SMC). In one embodiment, the endosomolytic agent has a structure represented by any of Formula I, Ia, or Table 1, or a pharmaceutically acceptable salt thereof.

[0064] "Small molecule compound" or "SMC" refers to an organic molecule having a molecular weight of less than 1000 g / Mol, and includes compounds having a structure represented by any of Formula I, Ia, or Table 1.

[0065] "Nucleic acid" refers to an oligomer or polymer of nucleotides, including naturally occurring or synthetically produced single- or double-stranded deoxyribonucleotides (DNA) or ribonucleotides (RNA). Nucleic acids can include naturally occurring nucleic acid nucleobases such as adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U), as well as non-naturally occurring base analogs or modified nucleobases.

[0066] "Target nucleic acid" refers to the nucleic acid that antisense oligonucleotide hybridizes to.In one aspect, the hybridization of antisense oligonucleotide to target nucleic acid in a cell changes the activity of the gene expressed by the cell.In one aspect, the hybridization of antisense oligonucleotide to target nucleic acid increases the activity of the gene expressed by the cell.In one aspect, the hybridization of antisense oligonucleotide to target nucleic acid decreases the activity of the gene expressed by the cell.

[0067] "Oligonucleotide" refers to an exogenous, natural, or non-natural single- or double-stranded polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA). In one embodiment, the oligonucleotide is about 2 to about 50 nucleotides in length. In one embodiment, the oligonucleotide contains one or more nucleotide analogs or modified backbone residues or linkages, including, but not limited to, phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or combinations thereof. "Locked nucleic acid nucleoside" or "LNA" refers to a nucleoside containing 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.

[0068] A "modified oligonucleotide" refers to an oligonucleotide containing at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0069] An "antisense oligonucleotide" or "ASO" is an oligonucleotide that contains at least a portion that is complementary to a target nucleic acid such that the ASO can hybridize to the target nucleic acid. Antisense oligonucleotides can increase or decrease expression of the target nucleic acid.

[0070] "Splice-switching oligonucleotides" or "SSOs" are short synthetic antisense oligonucleotides that can hybridize to pre-mRNA and disrupt splicing of the transcript, e.g., by blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. A "pre-mRNA" refers to an RNA transcript that contains one or more introns and that has not been fully processed into mRNA.

[0071] "Small interfering RNAs," also known as "short interfering RNAs," "silencing RNAs," or "siRNAs," are a class of double-stranded RNAs that are non-coding, typically about 20 to about 25 base pairs, and have a hydroxylated 3' end and a phosphorylated 5' end. Generally, siRNAs are part of the RNA interference pathway and interfere with the expression of specific genes with complementary nucleotide sequences by post-transcriptionally degrading the mRNA, thereby preventing translation. siRNAs can be conjugated to, for example, sugars such as GalNAc or lipids such as cholesterol to enhance delivery to target cells, e.g., with improved pharmacokinetics and / or efficacy. See, e.g., Osborn et al., Nucleic Acid Ther. 28(3):128-136 (2018). In some aspects, the present disclosure provides methods for enhancing delivery of unconjugated siRNAs.

[0072] "Polypeptide," as used interchangeably herein with "peptide" or "protein," refers to a polymeric form of amino acids of any length and can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. In some aspects, a polypeptide comprises from about 2 to about 5,000 amino acids. In some aspects, a polypeptide is capable of providing site-specific modification in a target nucleic acid. In some embodiments, a polypeptide is a therapeutic polypeptide.

[0073] "Macromolecules" include proteins, nucleic acids, carbohydrates, lipids, nanogels, and macrocycles. In some embodiments, polymers of the present disclosure include oligonucleotides. In some embodiments, polymers of the present disclosure include polypeptides. In some embodiments, polymers of the present disclosure include one or more components of a site-specific modification (SSM) system described herein, e.g., a CRISPR system, a Cre-Lox system, and / or an FLP-FRT system. Throughout this disclosure, reference to an SSM system can refer to any one or more components of the system. In some embodiments, an SSM system includes a CRISPR system, a Cre-Lox system, an FLP-FRT system, any component thereof, or any combination thereof.

[0074] A "CRISPR" system is an SSM system that can perform SSM on a target nucleic acid. In some embodiments, a CRISPR system includes (a) a protein, e.g., a Cas protein, that can provide SSM; 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. In some embodiments, the SSM includes a single-strand break in the target nucleic acid. In some embodiments, the SSM includes a double-strand break in the target nucleic acid. In some embodiments, the SSM includes a deletion. In some embodiments, the SSM includes an insertion. In some embodiments, the SSM includes a mutation. In some embodiments, the SSM includes base editing, e.g., converting a CG base pair to a TA base pair.

[0075] The "Cre-Lox" system is an SSM system that can perform SSM at a target nucleic acid. In some embodiments, the Cre / Lox system comprises a Cre recombinase, which recognizes a pair of Lox (also called LoxP) sequences flanking the target nucleic acid and catalyzes site-specific recombination in the target nucleic acid. A system similar to the Cre-Lox system is the "FLP-FRT" system. The FLP-FRT system is an SSM system that can perform SSM at a target nucleic acid. In some embodiments, the FLP-FRT system comprises an FLP recombinase that recognizes a pair of FRT sequences flanking the target nucleic acid and catalyzes site-specific recombination in the target nucleic acid. In some embodiments, the SSM comprises an inversion. In some embodiments, the SSM comprises an insertion. In some embodiments, the SSM comprises a deletion. In some embodiments, the SSM comprises a translocation. In some embodiments, the position and orientation of the Lox sequence (or FRT sequence) determines the type of SSM (e.g., inversion, deletion, or translocation) performed by the Cre recombinase (or FLP recombinase).

[0076] "Hybridize" refers to the pairing of complementary oligomeric compounds, for example, between an antisense oligonucleotide and its corresponding target nucleic acid. While not limited to any particular 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. Furthermore, some modified nucleobases maintain the ability to pair with corresponding nucleobases. Hybridization can occur between two complementary DNA molecules (DNA-DNA hybridization), between two RNA molecules (RNA-RNA hybridization), or between complementary DNA and RNA molecules (DNA-RNA hybridization). Hybridization can occur between short nucleotide sequences that are 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 do not necessarily have nucleic acid base complementarity at each nucleoside, but sequences with lower sequence complementarity are less stable and less likely to hybridize than sequences with higher sequence complementarity.

[0077] "Specifically hybridize" refers to the ability of an oligonucleotide to hybridize to a target nucleic acid with greater affinity than to a different nucleic acid. In one aspect, an antisense oligonucleotide specifically hybridizes to a target nucleic acid sequence under physiological conditions, e.g., for in vivo or therapeutic use.

[0078] "Targeting" or "targeted to" in the context of an antisense oligonucleotide refers to the association of an antisense oligonucleotide with a specific 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" or "targeted to" in the context of an SSM system (e.g., the CRISPR, Cre-Lox, and / or FLP-FRT systems described herein) refers to the association of a protein of the SSM system (e.g., a Cas protein, Cre recombinase, or FLP recombinase) with a specific target nucleic acid or region of a target nucleic acid. In some aspects, the Cas protein of a CRISPR system targets the target nucleic acid upon hybridization of a guide RNA with the target nucleic acid. In some embodiments, the Cre recombinase of a Cre-Lox system targets the target nucleic acid upon recognition of the Lox sequence flanking the target nucleic acid. In some embodiments, the FLP recombinase of a FLP-FRT system targets the target nucleic acid upon recognition of the FRT sequence flanking the target nucleic acid.

[0079] "Alter" or "modulate" refers to a change in the amount, function, or activity of a molecule, e.g., a macromolecule described herein, compared to the amount, function, or activity before treatment. In one aspect, the compounds described herein increase or decrease the amount, function, or activity of a gene expressed by a target nucleic acid sequence. In one aspect, the compounds increase the activity of antisense oligonucleotides (ASOs) that act on pre-mRNA via RNase H in the nucleus. In one aspect, the compounds increase the activity of siRNAs that act via the RISC complex in the cytosol. In one aspect, the compounds increase the alteration of pre-mRNA splicing by splice-switching oligonucleotides (SSOs), as reflected by an increase in the desired splice variant. In one aspect, the compounds result in a decrease in the level of the corresponding target mRNA and / or protein compared to treatment with an ASO in the absence of the compound. In some aspects, the compounds increase the activity of Cas proteins in a CRISPR system. In some aspects, the compounds increase the activity of Cre recombinase. In some aspects, the compounds increase the activity of FLP recombinase. In some aspects, the compounds increase the frequency of SSMs in a target nucleic acid. In some embodiments, the compound increases the editing efficiency of an SSM system (e.g., a CRISPR, Cre-Lox, and / or FLP-FRT system).

[0080] In one aspect, the compounds described herein "enhance delivery" of macromolecules provided herein, e.g., oligonucleotides and / or polypeptides. In one aspect, the compounds described herein "enhance delivery" of antisense oligonucleotides, increasing the cytosolic and / or nuclear concentration, accumulation, and / or half-life of the oligonucleotide compared to that found without administration of the compound. In one aspect, the compounds described herein "enhance delivery" of one or more components of an SSM system, increasing the cytosolic and / or nuclear concentration, accumulation, and / or half-life of the SSM system compared to that found without administration of the compound. In some aspects, the SSM system comprises a CRISPR system, a Cre-Lox system, an FLP-FRT system, any component thereof, or any combination thereof.

[0081] "Expression" refers to the process by which a protein is produced from a nucleic acid in a host cell, including, but not limited to, transcription, translation, post-translational modification, and secretion. "Increased" expression is in the context of comparing treated cells to an untreated control, e.g., cells treated with a polymer, 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 guide RNA, to untreated cells, or cells treated with a polymer, 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 guide RNA, to cells treated with a polymer, e.g., an antisense oligonucleotide, a recombinase (e.g., Cre or FLP), or a Cas protein and guide RNA only. Similarly, "decreased" expression is in the context of comparing treated cells to an untreated control, e.g., cells 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 guide RNA, to untreated cells, or cells 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 guide RNA, and a compound described herein, to cells treated with a macromolecule, e.g., an antisense oligonucleotide, a recombinase (e.g., Cre or FLP), or a Cas protein and guide RNA only.

[0082] "Disease" refers to any disease, disorder, condition, symptom, or indication.

[0083] "Treating" or "treatment" refers to curative, symptomatic, preventive, and prophylactic treatment, including, but not limited to, preventing or ameliorating a disease or at least one clinical symptom of a disease, reducing the risk of acquiring a disease or at least one clinical symptom of a disease, reducing the onset of a disease or at least one clinical symptom of a disease, reducing the risk of developing a disease or at least one clinical symptom of a disease, or delaying the onset of a disease or at least one clinical symptom of a disease.

[0084] "Subject" and "patient" can be used interchangeably to refer to any animal subject, e.g., a mammalian subject, such as a human, primate, cow, pig, horse, sheep, goat, rodent, cat, and / or dog. In one aspect, the subject is a human.

[0085] The term "cell" can include a single cell, a plurality of cells, or a population of cells, unless otherwise specified, where the context allows. In one aspect, the cell is in vitro, e.g., a cell explanted from a subject. In one aspect, the cell is a cell grown in batch or tissue culture. In one aspect, the cell is located in vivo, e.g., in a subject in need of treatment. In one aspect, the subject is a human subject.

[0086] As used herein, "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0087] A "pharmaceutical composition" comprises 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 guide RNA, and a compound described herein, and a pharmaceutically acceptable carrier or diluent. In one aspect, the carrier or diluent is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration.

[0088] "Pharmaceutically acceptable salts" refers to salts of compounds that are physiologically and pharmaceutically acceptable and possess the desired pharmacological activity of the parent compound, including salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases. The "pharmaceutically acceptable salts" of the compounds described herein can be prepared by methods well known in the art. For a general review of pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002).

[0089] An "effective amount" of a compound refers to an amount sufficient to increase the effectiveness 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. In one aspect, an "effective" amount of a compound refers to an amount sufficient to promote the 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. In one aspect, an "effective" amount of a compound refers to an amount that promotes 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.

[0090] A "therapeutically effective amount" 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 guide RNA, 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 administered 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 guide RNA, the disease, the severity of the disease, the age of the subject being treated, and / or the weight of the subject being treated.

[0091] "Dose" refers to a specified amount of an active agent (e.g., 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 guide RNA; and a compound) provided in a single administration or over a specified period of time. A dose can be administered in one, two, or more boluses or injections. In one aspect, an active agent is administered by infusion over an extended period of time or continuously. A dose can be described as the amount of pharmaceutical agent per hour, day, week, or month. A dose can also be described as the amount per unit body weight of the subject (e.g., mg / kg or g / kg).

[0092] A "dosage unit" refers to the form in which an active agent, e.g., 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 guide RNA, and a compound are provided. In one aspect, the dosage unit is a vial containing a lyophilized active agent. In one aspect, the dosage unit is a vial containing a reconstituted active agent. In one aspect, the active agent is a macromolecule. In one aspect, the active agent is an oligonucleotide. In one aspect, the active agent comprises a polypeptide. In one aspect, the active agent comprises an antisense oligonucleotide. In one aspect, the active agent comprises Cre recombinase. In one aspect, the active agent comprises FLP recombinase. In one aspect, the active agent comprises a Cas protein. In one aspect, the active agent comprises a Cas protein and a guide RNA. In one aspect, the active agent comprises a CRISPR system, a Cre-Lox system, an FLP-FRT system, any components thereof, or any combination thereof.

[0093] The methods and compositions described herein can be used in vitro on a sample or in vivo in a subject.

[0094] The compositions described herein can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. In one embodiment, the compositions are 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-term, or intermittent.

[0095] In one aspect, a compound described herein and a polymer described herein, e.g., an oligonucleotide, e.g., an antisense oligonucleotide, siRNA, or guide RNA, and / or a polypeptide, e.g., a recombinase (e.g., Cre or FLP) or a Cas protein, are administered simultaneously. As used herein, "simultaneously" refers to the simultaneous administration of a compound and a polymer (e.g., an oligonucleotide and / or polypeptide, e.g., an antisense oligonucleotide, siRNA, recombinase (e.g., Cre or FLP), or one or both of a Cas protein and a guide RNA) close enough in time to produce a combined effect. Simultaneous administration does not require that the compound and the polymer (e.g., an oligonucleotide and / or polypeptide, e.g., an antisense oligonucleotide, siRNA, recombinase (e.g., Cre or FLP), or one or both of a Cas protein and a guide RNA) be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. "Concurrently" includes simultaneous administration or sequential administration within a short period of time, for example, where the compound and macromolecule (e.g., an oligonucleotide and / or polypeptide, e.g., an antisense oligonucleotide, siRNA, recombinase (e.g., Cre or FLP), or one or both of a Cas protein and guide RNA) are administered within about 6 hours, about 3 hours, about 1 hour, or about 30 minutes of each other, including administering the compound before, simultaneously with, or after the macromolecule (e.g., an oligonucleotide and / or polypeptide, e.g., an antisense oligonucleotide, siRNA, recombinase (e.g., Cre or FLP), or one or both of a Cas protein and guide RNA).

[0096] In one aspect, the compound and the polymer (e.g., an oligonucleotide and / or polypeptide, e.g., an antisense oligonucleotide, siRNA, recombinase (e.g., Cre or FLP), or one or both of a Cas protein and a guide RNA) are administered sequentially. As used herein, "sequential" administration means that the compound and the polymer (e.g., an oligonucleotide and / or polypeptide, e.g., an antisense oligonucleotide, siRNA, recombinase (e.g., Cre or FLP), or one or both of a Cas protein and a guide RNA) are administered to a patient or a cell at different times. In one aspect, the compound is administered to a patient or a cell at least about 12 hours or 24 hours and up to 36 hours or 48 hours after administration of the polymer (e.g., an oligonucleotide and / or polypeptide, e.g., an antisense oligonucleotide, siRNA, recombinase (e.g., Cre or FLP), or one or both of a Cas protein and a guide RNA).

[0097] B. Overview Delivery of macromolecules, such as oligonucleotides or proteins, to cells requires that the macromolecules, e.g., oligonucleotides or proteins, cross cell membranes, including the plasma membrane and / or endosomal membrane. The use of macromolecules, such as oligonucleotides or proteins, can be hindered by their inability to effectively reach the cytosol and / or nucleus of cells, for example, due to their inability to cross the cell membrane or their inability to escape from the endosomal compartment after endocytosis. Provided herein are compounds that can increase the activity of macromolecules, e.g., antisense oligonucleotides or siRNAs; polypeptides, such as recombinases (e.g., Cre or FLP); or combinations thereof, e.g., oligonucleotides, such as Cas proteins and guide RNAs. In one aspect, the compounds are small molecule compounds (SMCs). In one aspect, the compounds are endosomolytic compounds that promote the release of macromolecules, e.g., oligonucleotides and / or polypeptides, from endosomes to the cytosol of cells. In one aspect, the compounds disclosed herein increase the transfection efficiency of macromolecules, e.g., oligonucleotides and / or polypeptides. Advantageously, the compounds described herein interfere with normal cellular transport mechanisms minimally, i.e., to the point of inducing leakage, and do not induce damage or toxicity that would irreversibly prevent cell growth or result in cell death.

[0098] C. Oligonucleotides In one aspect, compositions and methods are provided for delivering macromolecules (e.g., oligonucleotides) to the cytosol and / or nucleus of a cell. In one aspect, the oligonucleotide is single-stranded. In one aspect, the oligonucleotide is double-stranded. In one aspect, the oligonucleotide comprises deoxyribonucleic acid (DNA). In one aspect, the oligonucleotide comprises ribonucleic acid (RNA). In one aspect, the oligonucleotide is about 5 to about 100 nucleotides, about 5 to about 50 nucleotides, about 8 to about 30 nucleotides, about 10 to about 30 nucleotides, about 15 to about 30 nucleotides, or about 18 to about 30 nucleotides in length. In one aspect, the oligonucleotide has a molecular weight of about 5 kDa to about 15 kDa.

[0099] In one aspect, the oligonucleotide reduces expression of the target nucleic acid, which can be referred to as "gene silencing." In one aspect, the oligonucleotide increases expression of the target nucleic acid, which can be referred to as "gene activation." In one aspect, the oligonucleotide alters the splicing of the target nucleic acid, which can be referred to as "splice switching." In one aspect, the oligonucleotide interacts with the target protein. In one aspect, the oligonucleotide is an agonist or antagonist of the target protein.

[0100] In one embodiment, the oligonucleotide is an antisense oligonucleotide (ASO), a short (approximately 18-30 nucleotide) synthetic single-stranded nucleic acid polymer that regulates gene expression through various mechanisms. In one embodiment, the ASO contains DNA and forms an RNA-DNA heteroduplex that is recognized by endogenous RNase H enzymes, which catalyzes RNA degradation, thereby reducing gene expression. In one embodiment, the ASO binds to the target nucleic acid but does not induce degradation. In one embodiment, the oligonucleotide is a splice-switching oligonucleotide (SSO), which masks sequences within the target nucleic acid, thereby interfering with transcript RNA-RNA and / or RNA-protein interactions. In one embodiment, 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 a terminal 2-nucleotide 3' overhang). In one embodiment, the oligonucleotide is a microRNA (miRNA). In one embodiment, the oligonucleotide targets a non-coding RNA sequence associated with transcriptional repression, reversing the effects of this negative regulation and thereby activating gene expression. Other oligonucleotides include, but are not limited to, interfering RNA (RNAi) and decoy oligonucleotides. In one aspect, the oligonucleotide is a gapmer. In one aspect, the oligonucleotide is an aptamer.

[0101] In some embodiments, the oligonucleotide is part of a site-specific modification (SSM) system, such as a CRISPR system described herein. In some embodiments, the oligonucleotide is a guide RNA. In one embodiment, the guide RNA comprises one or both of: (i) a scaffold region or tracrRNA that can associate with a Cas protein (e.g., a Cas nuclease); and (ii) a spacer region or crRNA that can hybridize to a specific target nucleic acid sequence and thereby direct the Cas protein to perform site-specific modification at the target nucleic acid. Generally, the spacer region is about 15 to about 25 nucleotides in length. In some embodiments, the guide RNA is a single guide RNA (sgRNA) that comprises both a tracrRNA and a crRNA. In some embodiments, the guide RNA comprises a tracrRNA and a crRNA as two separate oligonucleotides that can form a complex with a Cas protein.

[0102] In one embodiment, the oligonucleotide comprises one or more modified nucleotides. In one embodiment, the oligonucleotide comprises one or more modifications to the oligonucleotide phosphate linkage. In one embodiment, the oligonucleotide comprises one or more modifications to the ribose sugar. In one embodiment, the oligonucleotide comprises one or more nucleotides covalently modified to restrict conformation, i.e., locked nucleic acid (LNA). In one embodiment, the oligonucleotide comprises a peptide nucleic acid (PNA). In one embodiment, the oligonucleotide comprises a methylated cytosine at the 5' position. In one embodiment, the oligonucleotide comprises one or more modified nucleotides selected from phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), or a combination thereof. In embodiments, the oligonucleotide is an antisense oligonucleotide targeted to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). In embodiments, the oligonucleotide is an antisense oligonucleotide targeted to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) comprising at least one nucleic acid having an LNA. In such embodiments, the antisense oligonucleotide targets MALAT1 and has the sequence: GM5CAttm5ctaatagm5cAGM5C, where m5c is 5-methylcytidine and the uppercase letters are LNA nucleosides (SEQ ID NO: 4). In embodiments, 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 the expression of MALAT1 in cells by about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 100%.

[0103] D. Polypeptides In one aspect, compositions and methods are provided for delivering macromolecules (e.g., polypeptides) to the cytosol and / or nucleus of a cell. In some aspects, the polypeptide comprises a therapeutic protein, which may be an antibody or a non-antibody protein. In one aspect, the polypeptide is a therapeutic peptide, e.g., as described in Wang et al., Sig Transduct Target Ther. 7:48 (2022).

[0104] In some embodiments, the polypeptide is capable of providing SSM at a target nucleic acid. In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the polypeptide is a nuclease. In some embodiments, the polypeptide is a recombinase. In some embodiments, the polypeptide is part of an SSM system described herein, e.g., a CRISPR system, a Cre-Lox system, or an FLP-FRT system. In some embodiments, the polypeptide is a Cas protein. In some embodiments, the polypeptide is Cas9. In some embodiments, the polypeptide is Cas12a. In some embodiments, the polypeptide is a recombinase. In some embodiments, the polypeptide is Cre. In some embodiments, the polypeptide is FLP.

[0105] In some embodiments, the polypeptide comprises a modified Cas protein. In some embodiments, the modified Cas protein is a Cas nickase that cleaves only one strand of a double-stranded target nucleic acid, such as a Cas9 nickase or a Cas12a nickase. In some embodiments, the modified Cas protein is a catalytically inactivated Cas protein (dead Cas) that does not contain nuclease activity, such as dCas9 or dCas12a. In some embodiments, the dead Cas can bind to the target nucleic acid and prevent other enzymes, such as transcription factors, from binding to the target nucleic acid. Cas nickases and dead Cas proteins are further described, for example, in Xu et al., J Mol Biol. 431(1):34-47 (2019), Qi et al., Cell 152(5):1173-1183 (2013), and Liu et al., Microbial Cell Factories 19:172 (2020).

[0106] In some embodiments, the polypeptide comprises a Cas fusion protein. In some embodiments, the Cas fusion protein comprises a modified Cas protein, e.g., a Cas nickase or a dead Cas, fused to an effector domain. In some embodiments, the polypeptide comprises a Cas nickase or a dead Cas, e.g., dCas9 or dCas12, fused to a nucleotide deaminase, e.g., a cytidine deaminase or an adenosine deaminase, optionally further fused to a DNA glycosylase inhibitor. In some embodiments, the polypeptide comprises a Cas nickase, e.g., a Cas9 nickase or a Cas12 nickase, fused to a reverse transcriptase. Cas fusion proteins are further described, for example, in Rees et al., Nat Rev Genet. 19(12)770-788 (2018), Anzalone et al., Nature 576(7785):149-157 (2019), and Liu et al., Microbial Cell Factories 19:172 (2020).

[0107] In some embodiments, the polypeptide comprises a recombinase. In some embodiments, the polypeptide comprises Cre. In some embodiments, the polypeptide comprises FLP. In some embodiments, the recombinase is a modified recombinase. In some embodiments, the recombinase is an inducible recombinase. Modified (e.g., inducible) recombinases are further described in, e.g., 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(1):82-89 (2011).

[0108] E. Cell invasion In some aspects, the macromolecule is delivered to a target cell. In one aspect, the target cell is a cultured cell. In one aspect, the target cell is an isolated cell. In one aspect, the target cell is a cell isolated from a subject in need of treatment. In one aspect, the target cell is a mammalian cell. In one aspect, the target cell is a eukaryotic cell. In one aspect, the target cell is a prokaryotic cell.

[0109] In one embodiment, the target cell is part of a tissue or organ. In one embodiment, the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, fat, and / or muscle (e.g., skeletal muscle). In one embodiment, the target cell is a brain cell, CNS cell, PNS cell, heart cell, liver cell, kidney cell, spleen cell, pancreatic cell, lung cell, muscle cell, adipocyte, immune cell, or a combination thereof.

[0110] In some embodiments, the target cells are CNS cells. In some embodiments, the CNS cells include glial cells and / or neurons. CNS glial cells include, for example, astrocytes, oligodendrocytes, microglia, and ependymal cells. Neurons include, for example, afferent neurons, efferent neurons, and interneurons.

[0111] In some embodiments, the target cells are hepatocytes, e.g., parenchymal or non-parenchymal hepatocytes. In some embodiments, the target cells include culturable metabolically competent human hepatocytes, culturable induction-competent human hepatocytes, culturable human hepatocytes, suspension-competent human hepatocytes (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-1 and C57B1 / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes).

[0112] In some aspects, the target cells are stem cells, e.g., human stem cells. The stem cells can be pluripotent stem cells, including, for example, 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). In some aspects, the cells are differentiated forms of any of the cells described herein. In some aspects, the eukaryotic cells are cells derived from any primary cell in culture.

[0113] In some embodiments, the target cell is an immune cell, non-limiting examples of which include T cells, B cells, dendritic cells, NK cells, helper T cells, cytotoxic T cells, regulatory T cells, gamma delta T cells, neutrophils, mast cells, monocytes, antigen-presenting cells, lymphocytes, basophils, and phagocytes.

[0114] In some embodiments, the macromolecule delivered to the target cell is an oligonucleotide. In some embodiments, the oligonucleotide is an antisense oligonucleotide. In some embodiments, the oligonucleotide is an siRNA. In some embodiments, the siRNA is unconjugated, i.e., not conjugated to a lipid or sugar. In some embodiments, the siRNA is conjugated to a lipid and / or sugar. In some embodiments, the oligonucleotide is a guide RNA of a CRISPR system. In some embodiments, the macromolecule delivered to the target cell is a polypeptide. In some embodiments, the polypeptide is a Cas protein, e.g., Cas9 or Cas12a. In some embodiments, the Cas protein is a modified Cas protein or a Cas fusion protein described herein. In some embodiments, the polypeptide is a recombinase. In some embodiments, the polypeptide is Cre. In some embodiments, the polypeptide is FLP. Oligonucleotides are hydrophilic polyanions having a molecular weight in the range of about 5 kDa to about 15 kDa and do not readily cross the plasma membrane. Polypeptides also typically do not passively diffuse across the plasma membrane. To be effective, macromolecules such as oligonucleotides and polypeptides must cross the plasma membrane and enter the cytosol and / or nucleus of the cell.

[0115] In one aspect, macromolecules, e.g., oligonucleotides and / or polypeptides, are internalized by endocytosis, where the macromolecules, e.g., oligonucleotides and / or polypeptides, are surrounded by the plasma membrane and then budding within the cell to form vesicles containing the internalized macromolecules, e.g., oligonucleotides and / or polypeptides. In another aspect, macromolecules, e.g., oligonucleotides and / or polypeptides, are internalized by the formation of pores in the plasma membrane.

[0116] The endocytic pathway in mammalian cells involves distinct membrane compartments including early endosomes, late endosomes and lysosomes.

[0117] Early endosomes (EEs) are the first compartment and major 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 heterogeneous with respect to morphology, localization, composition, and function. While most EEs are relatively small and remain close to the plasma membrane, the overall distribution of EEs is cell type-dependent.

[0118] Late endosomes (LEs) typically receive endocytosed material from early endosomes along the endocytic pathway and contain nucleosomes, mitochondria, and proteins characteristic of lysosomal membrane glycoproteins and mRNAs, including acid hydrolases. They are acidic (40, approximately pH 5.5) and are thought to mediate the final sorting of internalized cargo before delivery to lysosomes.

[0119] Lysosomes are compartments of the endocytic pathway that sequester cargo for arrest or degradation. Their primary function is to degrade cellular waste products, lipids, carbohydrates, proteins, and other macromolecules, and return them to the cytoplasm as new cell building materials. To accomplish this, lysosomes contain a variety of hydrolytic enzymes that function in an acidic environment (40, approximately pH 4.8).

[0120] Upon endocytosis, macromolecules, such as oligonucleotides and / or polypeptides, often accumulate in endosomes, particularly late endosomes or lysosomes, where they are pharmacologically inactive. To be active, macromolecules, such as oligonucleotides and / or polypeptides, must escape the endosomal compartment and access their cytosolic or nuclear targets before degradation or export via exocytosis.

[0121] F. Compound Provided herein are compounds that facilitate the entry of macromolecules, e.g., oligonucleotides and / or polypeptides, into the cytosol and / or nucleus of a cell. In one aspect, small molecule compounds (SMCs) that facilitate the entry of macromolecules, e.g., oligonucleotides and / or polypeptides, into the cytosol of a cell are provided. In one aspect, the compounds facilitate the entry of macromolecules, e.g., oligonucleotides and / or polypeptides, into the cytosol by forming pores in the plasma membrane of the cell. In one aspect, the compounds facilitate the release of macromolecules, e.g., oligonucleotides and / or polypeptides, from an endosomal compartment within a cell. In one aspect, the macromolecules are oligonucleotides, and the compounds facilitate the endosomal escape of gymnodal-delivered oligonucleotides. In one aspect, the compounds engorge the endosomal compartment, physically inducing membrane disruption, with concomitant escape of the macromolecules, e.g., oligonucleotides and / or polypeptides, into the cytosol during early-to-late endosomal transit and / or late-to-lysosome transit.

[0122] In one aspect, the compound has the structure represented by Formula I:

[0123] [ka] (In the formula, one of Z1 and Z2 is N and the other is C; 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; R3 is hydro, halo, C1-C4 alkyl, -(CH2) y OH, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; R4 is C1-C4 alkyl; R5 is hydro or C1-C4 alkyl; R6 is hydro or C1-C4 alkyl; R7, R8, R9, R 10 , R 11 However, each independently, CHR 12 , C.R. 12 R 17 or NR 13 and R 12 Hydro, C1-C4 alkyl, -OR 14 , or -CO2R 15 and R 13 Hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 14 is hydro or C1-C4 alkyl; R 15 is hydro or C1-C4 alkyl; R 16 is hydro or C1-C4 alkyl; R 17 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; wherein one or more of the alkyl groups are optionally substituted with one or more halo groups), or a pharmaceutically acceptable salt thereof.

[0124] In one aspect, 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 saturated alkyl. In one aspect, R1 is unsaturated alkyl, such as alkenyl or alkynyl. In some aspects, R1 can be polyunsaturated. In one aspect, R1 is linear alkyl. In one aspect, R1 is branched alkyl. In one aspect, R1 is C1-C4 alkyl. In one aspect, 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 substituted alkyl, for example, substituted with one or more halo. In some embodiments, R1 is C1-C4 alkyl, and at least one carbon of the alkyl is substituted with one or more chloro or fluoro. In some aspects, R1 is substituted with hydroxyl or ketone. In one aspect, R1 is unsubstituted alkyl. In one aspect, R1 is substituted with a halo group, i.e., haloalkyl, such as monohaloalkyl, dihaloalkyl, or trihaloalkyl. In one aspect, R1 is haloC1-C4 alkyl. In one aspect, R1 is haloC1-C4 alkyl, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0125] In one aspect, R1 is alkoxy represented by -OR4. In one aspect, R1 is saturated alkoxy. In one aspect, R1 is 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 hydroxyl or ketone. In one aspect, R1 is alkoxy substituted with a halo group, i.e., haloalkoxy, e.g., monohaloalkoxy, dihaloalkoxy, or trihaloalkoxy. In one aspect, R1 is straight-chain alkoxy. In one aspect, R1 is branched-chain alkoxy. In one aspect, R1 is C1-C4 alkoxy (-OR4), and R4 is C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R1 is alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, or butoxy, including, for example, n-butoxy, sec-butoxy, iso-butoxy, and t-butoxy.

[0126] In one aspect, R1 is an amide, which can be represented by the group "-C(=O)NR5R5," where each R5 is independently hydro or alkyl. In some aspects, at least one R5 of -C(=O)NR5R5 is hydro. In one aspect, R5 is C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, R1 is a primary amide. In one aspect, R1 is a secondary amide. In one aspect, R1 is a tertiary amide. In one aspect, R1 is -C(=O)NHR5.

[0127] In one aspect, R1 is a short chain ester that can be represented by the group "-C(O)OR6" or "-CO2R6", where R6 is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl.

[0128] In one aspect, R1 is cyano.

[0129] In one aspect, X is hydro. In some embodiments, X is alkyl. In one aspect, X is saturated alkyl. In one aspect, X is unsaturated alkyl. In one aspect, X is straight-chain alkyl. In one aspect, X is branched-chain 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 unsubstituted alkyl. In one aspect, X is substituted with a halo group, i.e., haloalkyl, such as monohaloalkyl, dihaloalkyl, or trihaloalkyl. In one aspect, X is haloC1-C4 alkyl. In one aspect, X is haloC1-C4 alkyl, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl. In some embodiments, X is -OR2, where R2 is defined below.

[0130] In one aspect, R2 is hydro. In some embodiments, R2 is alkyl. In one aspect, R2 is saturated alkyl. In one aspect, R2 is unsaturated alkyl. In one aspect, R2 is straight-chain alkyl. In one aspect, R2 is branched-chain 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 methyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R2 is unsubstituted alkyl. In one aspect, R2 is substituted with a halo group, i.e., haloalkyl, for example, monohaloalkyl, dihaloalkyl, or trihaloalkyl. In one aspect, R2 is haloC1-C4 alkyl. In one embodiment, R2 is haloC1-C4 alkyl, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0131] In one aspect, R3 is hydro. In some aspects, R3 is halo, e.g., chloro, fluoro, or bromo. In one aspect, R3 is alkyl. In one aspect, R3 is saturated alkyl. In one aspect, R3 is unsaturated alkyl. In one aspect, R3 is straight-chain alkyl. In one aspect, R3 is branched-chain alkyl. In one aspect, R3 is C1-C4 alkyl. In one aspect, R3 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R3 is haloalkyl, e.g., monohaloalkyl, dihaloalkyl, or trihaloalkyl. In one aspect, R3 is C1-C4 alkyl optionally substituted with one or more halo. In one aspect, R3 is haloC1-C4 alkyl, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, or chloromethyl. In one aspect, R3 is an amide, which can be represented by the group "-C(O)NR5R5," where each R5 is independently hydro or alkyl, such as methyl or ethyl. In one aspect, the alkyl substituent contains from 1 up to about 8 carbon atoms. In one aspect, the alkyl substituent contains from about 1 up to about 4 carbon atoms. In one aspect, R3 is a primary amide. In one aspect, R3 is a secondary amide. In one aspect, R3 is a tertiary amide. In one aspect, 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, such as methyl or ethyl. In one aspect, R3 is cyano. In one embodiment, R3 is "-(CHOH) n In one aspect, R3 is an alkyl substituted with one or more hydroxyl groups, as represented by "-CH2OH", where n is 0, 1, 2, or 3. In one aspect, R3 is a methyl hydroxyl group, as represented by "-CH2OH".

[0132] In one aspect, R3 is an alkoxy represented by "-OR4". In one aspect, R3 is a saturated alkoxy. In one aspect, R3 is an unsaturated alkoxy. In some embodiments, R4 is a 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 an alkoxy substituted with a hydroxyl or a ketone. In one aspect, R3 is an alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a monohaloalkoxy, dihaloalkoxy, or trihaloalkoxy. In one aspect, R3 is a straight-chain alkoxy. In one aspect, R3 is a branched-chain alkoxy. In one aspect, R3 is a C1-C4 alkoxy (-OR4), where R4 is a C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R3 is alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, including n-butoxy, sec-butoxy, iso-butoxy, and t-butoxy. In one aspect, R3 is methoxy.

[0133] In one embodiment, R7, R8, R9, R 10 , or R 11 are each independently CHR 12 , C.R. 12 R 17 or NR 13 In at least one embodiment, R7, R8, R9, R 10 , and R 11 All of this is CHR 12 and R for each atom 12 are independently selected from hydro, C1-C4 alkyl, -OR 14 , or -CO2R 15 In at least one embodiment, R7, R8, R9, R 10 , and R 11 All of this is CHR 12 and each R 12 In at least one embodiment, R, R, R, R 10 , and R 11All of this is CHR 12 and R 12 are each independently selected from hydro or unsubstituted alkyl. 12 can be substituted with a halo group, i.e., haloalkyl, for example, monohaloalkyl, dihaloalkyl, or trihaloalkyl. 12 can be haloC1-C4 alkyl, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl. In at least one embodiment, R7, R8, R9, R 10 , and R 11 All of this is CHR 12 and each R 12 are independently hydro, C1-C4 alkyl, or -OR 14 Selected from R 14 is further selected from hydro or C1-C4 alkyl. In some embodiments, R 12 is alkoxy, e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, including n-butoxy, sec-butoxy, iso-butoxy, and t-butoxy. In at least one embodiment, R, R, R, R 10 , and R 11 All of this is CHR 12 and each R 12 are independently selected from hydro, C1-C4 alkyl, -OR 14 or CO2R 15 Selected from R 14 and R 15 Each of is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl.

[0134] In one embodiment, R7, R8, R9, R 10 , and R 11 At least one of them is CR 12 R 17 and R 12 and R 17 are each independently selected from hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In another embodiment, R7, R8, R9, R10 , and R 11 At least two of them are CR 12 R 17 and R 12 and R 17 are each independently selected from hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In at least one embodiment, R7 is CR 12 R 17 and R 12 is methyl and R 17 is methyl, and R8, R9, R 10 , and R 11 are each independently CHR 12 or NR 13 In at least one embodiment, R8 is selected from CR 12 R 17 and R 12 is methyl and R 17 is methyl, and R7, R9, R 10 , and R 11 are each independently CHR 12 or NR 13 In at least one embodiment, R9 is selected from CR 12 R 17 and R 12 is methyl and R 17 is methyl, and R7, R8, R 10 , and R 11 are each independently CHR 12 or NR 13 In at least one embodiment, R 10 is CR 12 R 17 and R 12 is methyl and R 17 is methyl, and R7, R8, R9, and R 11 are each independently CHR 12 or NR 13 In at least one embodiment, R 11 is CR 12 R 17 and R 12 is methyl and R 17is methyl, and R7, R8, R9 and R 10 are each independently CHR 12 or NR 13 is selected from.

[0135] In one embodiment, R7, R8, R9, R 10 , and R 11 At least one of the 13 and R 13 is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl. In another embodiment, R7, R8, R9, R 10 , and R 11 At least two of them are NR 13 and two R 13 Each of the groups is independently hydro or C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, R7 is NR 13 and R8, R9, R 10 and R 11 is CHR 12 and R 13 is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl, and R 12 In one aspect, R8 is NR 13 and R7, R9, R 10 and R 11 is CHR 12 and R 13 is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl, and R 12 In one aspect, R is NR 13 and R7, R8, R 10 and R 11 is CHR 12 and R 13 is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl, and R 12 is hydro. In one aspect, R 10 is NR 13 and R7, R8, R9 and R 11 is CHR 12 and R13 is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl, and R 12 is hydro. In one aspect, R 11 is NR 13 and R7, R8, R9 and R 10 is CHR 12 and R 13 is hydro or C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl, and R 12 is hydro.

[0136] In one aspect, R7 is NR 13 and R8, R9, R 10 and R 11 is CHR 12 and R 13 is hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 12 In one aspect, R8 is NR 13 and R7, R9, R 10 , and R 11 is CHR 12 and R 13 is hydro, C1-C4 alkyl, -C(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 12 In one aspect, R is NR 13 and R7, R8, R 10 , and R 11 is CHR 12 and R 13 is hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 12 is hydro. In one aspect, R 10 is NR 13and R7, R8, R9, and R 11 is CHR 12 and R 13 is hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 12 is hydro. In one aspect, R 11 is NR 13 and R7, R8, R9, and R 10 is CHR 12 and R 13 is hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 12 In any of these cases, y is selected from 0, 1, 2, or 3, and R 14 , R 15 , and R 16 are each independently selected from hydro and C1-C4 alkyl.

[0137] In one aspect, R9 is NR 13 and R7, R8, R 10 , and R 11 is CHR 12 and each R 12 and R 13 In one aspect, R is NR 13 and R7, R8, R 10 , and R 11 is CHR 12 and each R 12 is hydro and R 13 is C1-C4 alkyl. In one aspect, R9 is NR 13 and R7, R8, R 10 , and R 11 is CHR 12 and each R 12 is hydro and R 13 is methyl.

[0138] In one aspect, R9 is NR 13 and R 13 is hydro, and R7, R8, R 10 , and R 11 At least one of the 12 R 17 and R 12 and R 17 are each C1-C4 alkyl, and R7, R8, R 10 , and R 11 The rest of the CHR 12 and each R 12 is hydro and R 13 is hydro or C1-C4 alkyl. In one aspect, R9 is NR 13 and R 13 is hydro, R, R 10 , and R 11 is CHR 12 and R 12 is hydro and R8 is CR 12 R 17 and R 12 and R 17 In one embodiment, R is NR 13 and R 13 is hydro, and R, R, and R 11 is CHR 12 and R 12 is hydro and R 10 is CR 12 R 17 and R 12 and R 17 In one embodiment, R is NR 13 and R 13 is hydro, and R and R 11 is CHR 12 and R 12 is hydro, and R and R 10 is CR 12 R 17 and R 12 and R 17 Each of is methyl.

[0139] In another embodiment, the compound has the structure represented by formula Ia:

[0140] [ka] (In the formula, one of Z1 and Z2 is N and the other is C; 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; R3 is hydro, halo, C1-C4 alkyl, -(CH2) y OH, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; R4 is C1-C4 alkyl; R5 is hydro or C1-C4 alkyl, for example, methyl or ethyl; R6 is hydro or C1-C4 alkyl, for example, methyl or ethyl; R 13 Hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 16 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; one or more of the alkyls are optionally substituted with one or more halo; or a pharmaceutically acceptable salt thereof.

[0141] In another embodiment, the compound has the structure represented by Formula Ib or Formula Ic:

[0142] [ka] (In the formula, 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; R3 is hydro, halo, C1-C4 alkyl, -(CH2) y OH, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; R4 is C1-C4 alkyl; R5 is hydro or C1-C4 alkyl, for example, methyl or ethyl; R6 is hydro or C1-C4 alkyl, for example, methyl or ethyl; R 13 Hydro, C1-C4 alkyl, -(CH2) y OH, -OR 14 , -CO2R 15 , or -C(=O)R 16 and R 16 is hydro or C1-C4 alkyl; y is 0, 1, 2, or 3; one or more of the alkyls are optionally substituted with one or more halo; or a pharmaceutically acceptable salt thereof.

[0143] In another embodiment, a compound of Formula Ia, Formula Ib, or Formula Ic, wherein: R1 is hydro, halo, C1-C4 alkyl, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; X is -OR2, R2 is hydro or C1-C4 alkyl; R3 is hydro, halo, C1-C4 alkyl, —OR4, —C(═O)NR5R5, —CO2R6, or cyano; R4 is C1-C4 alkyl; R5 is hydro, methyl or ethyl; R6 is hydro, methyl or ethyl; R13 is hydro, one or more of the alkyls are optionally substituted with one or more halo; or a pharmaceutically acceptable salt thereof.

[0144] In one aspect, 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 saturated alkyl. In one aspect, R1 is unsaturated alkyl, such as alkenyl or alkynyl. In some aspects, R1 can be polyunsaturated. In one aspect, R1 is linear alkyl. In one aspect, R1 is branched alkyl. In one aspect, R1 is C1-C4 alkyl. In one aspect, 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 substituted alkyl, for example, substituted with one or more halo. In some embodiments, R1 is C1-C4 alkyl, and at least one carbon of the alkyl is substituted with one or more chloro or fluoro. In some aspects, R1 is substituted with hydroxyl or ketone. In one aspect, R1 is unsubstituted alkyl. In one aspect, R1 is substituted with a halo group, i.e., haloalkyl, such as monohaloalkyl, dihaloalkyl, or trihaloalkyl. In one aspect, R1 is haloC1-C4 alkyl. In one aspect, R1 is haloC1-C4 alkyl, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, and chloromethyl.

[0145] In one aspect, R1 is an alkoxy represented by "-OR4". In one aspect, R1 is a saturated alkoxy. In one aspect, R1 is an unsaturated alkoxy. In some embodiments, R4 is a 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 an alkoxy substituted with a hydroxyl or a ketone. In one aspect, R1 is an alkoxy substituted with a halo group, i.e., a haloalkoxy, e.g., a monohaloalkoxy, dihaloalkoxy, or trihaloalkoxy. In one aspect, R1 is a straight-chain alkoxy. In one aspect, R1 is a branched-chain alkoxy. In one aspect, R1 is a C1-C4 alkoxy (-OR4), and R4 is a C1-C4 alkyl, e.g., methyl or ethyl. In one aspect, R1 is alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, or butoxy, including, for example, n-butoxy, sec-butoxy, iso-butoxy, and t-butoxy.

[0146] In one aspect, R1 is an amide, which can be represented by the group "-C(=O)NR5R5," where each R5 is independently hydro or alkyl. In some aspects, at least one R5 of "-C(=O)NR5R5" is hydro. In one aspect, R5 is C1-C4 alkyl, e.g., methyl, ethyl, propyl, or butyl. In one aspect, R1 is a primary amide. In one aspect, R1 is a secondary amide. In one aspect, R1 is a tertiary amide. In one aspect, R1 is -C(=O)NHR5.

[0147] In one aspect, R1 is a short chain ester that can be represented by the group "-C(O)OR6" or "-CO2R6", where R6 is hydro or C1-C4 alkyl, for example, methyl or ethyl.

[0148] In one aspect, R1 is cyano.

[0149] In one aspect, X is hydro. In one aspect, X is C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl. In one aspect, X is -OR2.

[0150] In one aspect, R2 is hydro. In some embodiments, R2 is alkyl. In one aspect, R2 is saturated alkyl. In one aspect, R2 is unsaturated alkyl. In one aspect, R2 is straight chain alkyl. In one aspect, R2 is branched chain 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 methyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0151] In one aspect, R3 is hydro. In some aspects, R3 is halo, such as chloro, fluoro, or bromo. In one aspect, R3 is alkyl. In one aspect, R3 is saturated alkyl. In one aspect, R3 is unsaturated alkyl. In one aspect, R3 is straight-chain alkyl. In one aspect, R3 is branched-chain alkyl. In one aspect, R3 is C1-C4 alkyl. In one aspect, R3 is methyl, ethyl, propyl, isopropyl, or butyl. In one aspect, butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl. In one aspect, R3 is haloalkyl, such as monohaloalkyl, dihaloalkyl, or trihaloalkyl. In one aspect, R3 is C1-C4 alkyl optionally substituted with one or more halo. In one aspect, R3 is haloC1-C4 alkyl, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, or chloromethyl. In one aspect, R3 is an amide, which can be represented by the group "-C(O)NR5R5," where each R5 is independently hydro or alkyl, such as methyl or ethyl. In one aspect, the alkyl substituent contains from 1 up to about 8 carbon atoms. In one aspect, the alkyl substituent contains from about 1 up to about 4 carbon atoms. In one aspect, R3 is a primary amide. In one aspect, R3 is a secondary amide. In one aspect, R3 is a tertiary amide. In one aspect, 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, such as methyl or ethyl. In one aspect, R3 is cyano. In one embodiment, R3 is "-(CHOH) n In one aspect, R3 is an alkyl substituted with one or more hydroxyl groups, as represented by "-CH2OH", where n is 0, 1, 2, or 3. In one aspect, R3 is a methyl hydroxyl group, as represented by "-CH2OH".

[0152] In one aspect, compounds of Formula Ia, Formula Ib, and Formula Ic described herein can include, by way of non-limiting example, any of the following found in Table 2A below:

[0153] [Table 2-1]

[0154] [Table 2-2]

[0155] In one aspect, the compound has a structure represented by formula Ia, Ib, or Ic, wherein R, X, R, and R 13 as can be seen in Table 2B.

[0156] [Table 3]

[0157] In one aspect, the compound has a structure represented by Formula I, wherein R1 is halo, X is —OR2, R2 is methyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 is C1-C4 alkyl or -(CH2) y OH, and y is 1 to 3.

[0158] In one aspect, the compound has a structure represented by Formula I, wherein R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 is C1-C4 alkyl.

[0159] In one aspect, the compound has a structure represented by Formula I, wherein R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 is methyl.

[0160] In one aspect, the compound has a structure represented by Formula I, wherein Z1 is N, Z2 is C, R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 But it is hydro.

[0161] In one aspect, the compound has a structure represented by Formula I, wherein Z1 is N, Z2 is C, R1 is fluoro, X is —OR2, R2 is ethyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 is methyl.

[0162] In one aspect, the compound has a structure represented by Formula I, wherein Z1 is N, Z2 is C, R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, R7, R 10 , and R 11 But CHR 12 and R 12 is hydro and R8 is CR 12 R 17 and R 12 and R 17are each methyl, and R9 is NR 13 and R 13 is methyl.

[0163] In one aspect, the compound has a structure represented by Formula I, wherein R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 But (CH2) y OH and y is 1.

[0164] In one aspect, the compound has a structure represented by Formula I, wherein R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 But (CH2) y OH and y is 2.

[0165] In one aspect, the compound has a structure represented by Formula I, wherein R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R7, R8, R 10 , and R 11 But CHR 12 and R 12 is hydro and R9 is NR 13 and R 13 But (CH2) y OH and y is 3.

[0166] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl, R3 is cyano, and R 13is hydro, C1-C4 alkyl, -C(=O)R 16 , or (CH2) y In some embodiments, R is fluoro, R is ethyl, and R 13 In some embodiments, R is fluoro, R is ethyl, and R 13 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R is fluoro, R is ethyl, and R 13 is -C(=O)R 16 In some embodiments, R is fluoro, R is ethyl, and R 13 is -(CH2) y OH, for example, —(CH)OH. In some embodiments, R is fluoro, R is methyl, and R 13 In some embodiments, R is fluoro, R is methyl, and R 13 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R is fluoro, R is methyl, and R 13 is -C(=O)R 16 In some embodiments, R is fluoro, R is methyl, and R 13 is -(CH2) y OH, for example, —(CH)OH. In some embodiments, R is chloro, R is ethyl, and R 13 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R is chloro, R is ethyl, and R 13 is -C(=O)R 16 In some embodiments, R is chloro, R is ethyl, and R 13 is -(CH2) y OH, for example, —(CH)OH. In some embodiments, R is chloro, R is methyl, and R 13In some embodiments, R is chloro, R is methyl, and R 13 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R is chloro, R is methyl, and R 13 is -C(=O)R 16 In some embodiments, R is chloro, R is methyl, and R 13 is -(CH2) y OH, for example, —(CH2)3OH.

[0167] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl, R3 is cyano, and R 13 In some embodiments, R is haloalkyl, e.g., trifluoromethyl, R is ethyl, R is cyano, and R 13 In some embodiments, R is haloalkyl, e.g., trifluoromethyl, R is ethyl, R is cyano, and R 13 In some embodiments, R is haloalkyl, e.g., trifluoromethyl, R is methyl, R is cyano, and R 13 In some embodiments, R is haloalkyl, e.g., trifluoromethyl, R is methyl, R is cyano, and R 13 is methyl.

[0168] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is halo, X is -OR2, R2 is ethyl or methyl, R3 is methyl or methoxy, and R 13 In some embodiments, R is fluoro, R is ethyl, R is methyl, and R 13In some embodiments, R is fluoro, R is methyl, R is methoxy, and R 13 In some embodiments, R is fluoro, R is methyl, R is methyl, and R 13 In some embodiments, R is fluoro, R is ethyl, R is methoxy, and R 13 In some embodiments, R is chloro, R is ethyl, R is methyl, and R 13 In some embodiments, R is chloro, R is methyl, R is methoxy, and R 13 In some embodiments, R is chloro, R is methyl, R is methyl, and R 13 In some embodiments, R is chloro, R is ethyl, R is methoxy, and R 13 is hydro.

[0169] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is halo, X is -OR2, R2 is C1-C-4 alkyl, R3 is hydro, and R 13 In some embodiments, R is fluoro, R is methyl, R is hydro, and R 13 In some embodiments, R is chloro, R is methyl, R is hydro, and R 13 In some embodiments, R is bromo, R is methyl, R is hydro, and R 13 In some embodiments, R is fluoro, R is ethyl, R is hydro, and R 13 In some embodiments, R is chloro, R is ethyl, R is hydro, and R 13In some embodiments, R is bromo, R is ethyl, R is hydro, and R 13 In some embodiments, R is fluoro, R is propyl, e.g., isopropyl, R is hydro, and R 13 In some embodiments, R1 is chloro, R2 is propyl, e.g., isopropyl, R3 is hydro, and R 13 In some embodiments, R is bromo, R is propyl, e.g., isopropyl, R is hydro, and R 13 is hydro.

[0170] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is haloalkyl, X is -OR2, R2 is ethyl or methyl, R3 is hydro, and R 13 In some embodiments, R is trifluoromethyl, R is methyl, R is hydro, and R 13 In some embodiments, R is trifluoromethyl, R is methyl, R is hydro, and R 13 In some embodiments, R is trifluoromethyl, R is ethyl, R is hydro, and R 13 In some embodiments, R is trifluoromethyl, R is ethyl, R is hydro, and R 13 In some embodiments, R is trifluoromethyl, R is methyl, R is hydro, and R 13 In some embodiments, R is trifluoromethyl, R is methyl, R is hydro, and R 13 In some embodiments, R is trifluoromethyl, R is ethyl, R is hydro, and R 13In some embodiments, R is trifluoromethyl, R is ethyl, R is hydro, and R 13 is methyl.

[0171] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is halo, X is hydro, R3 is cyano, and R 13 is hydro. In some embodiments, R1 is fluoro. In some embodiments, R1 is chloro. In some embodiments, R1 is bromo.

[0172] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is cyano, X is —OR2, R2 is ethyl or methyl, R3 is haloalkyl, and R 13 In some embodiments, R is cyano, R is methyl, R is trifluoromethyl, and R 13 In some embodiments, R is cyano, R is ethyl, R is trifluoromethyl, and R 13 is hydro.

[0173] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, wherein R1 is ethyl or methyl, X is -OR2, R2 is ethyl or methyl, R3 is hydro, and R 13 In some embodiments, R1 is methyl, R2 is methyl, R3 is hydro, and R 13 In some embodiments, R is methyl, R is ethyl, R is hydro, and R 13 In some embodiments, R is ethyl, R is methyl, R is hydro, and R 13 In some embodiments, R is ethyl, R is ethyl, R is hydro, and R13 is hydro.

[0174] In one aspect, the compound has a structure represented by Formula Ia, Formula Ib, or Formula Ic, where R1 is halo, X is -OR2, R2 is ethyl or methyl, and R3 is -(CH2) y OH and R 13 is hydro and y is 0, 1, 2, or 3. In some embodiments, R is fluoro, R is methyl, R is —OH, and R 13 In some embodiments, R is fluoro, R is methyl, R is —CHOH, and R 13 In some embodiments, R is fluoro, R is methyl, R is —(CH)OH, and R 13 In some embodiments, R is fluoro, R is methyl, R is —(CH)OH, and R 13 In some embodiments, R1 is chloro, R2 is methyl, R3 is —OH, and R 13 In some embodiments, R is chloro, R is methyl, R is —CHOH, and R 13 In some embodiments, R is chloro, R is methyl, R is —(CH)OH, and R 13 In some embodiments, R is chloro, R is methyl, R is —(CH)OH, and R 13 In some embodiments, R is fluoro, R is ethyl, R is —OH, and R 13 In some embodiments, R is fluoro, R is ethyl, R is —CHOH, and R 13 In some embodiments, R is fluoro, R is ethyl, R is —(CH)OH, and R 13In some embodiments, R is fluoro, R is ethyl, R is —(CH)OH, and R 13 In some embodiments, R is chloro, R is ethyl, R is —OH, and R 13 In some embodiments, R is chloro, R is ethyl, R is —CHOH, and R 13 In some embodiments, R is chloro, R is ethyl, R is —(CH)OH, and R 13 In some embodiments, R is chloro, R is ethyl, R is —(CH)OH, and R 13 is hydro.

[0175] In one aspect, the compound has a structure represented by formula Ia, Ib, or Ic, where R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R 13 But it is hydro.

[0176] In one aspect, the compound has a structure represented by formula Ia, wherein R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R 13 But it is hydro.

[0177] In one aspect, the compound has a structure represented by formula Ia, wherein R1 is fluoro, X is —OR2, R2 is ethyl, R3 is cyano, and R 13 is methyl.

[0178] In one aspect, the compound has a structure represented by formula Ib, wherein R1 is fluoro, X is —OR2, R2 is methyl, R3 is cyano, and R 13 But it is hydro.

[0179] In one aspect, the compound has a structure represented by formula Ib, wherein R1 is fluoro, X is —OR2, R2 is ethyl, R3 is cyano, and R 13 is methyl.

[0180] In some embodiments, the compound comprises a structure shown in Table 1. In some embodiments, the compound is any one of compounds 2-7 shown in Table 1. In some embodiments, the compound is compound 4 shown in Table 1. In some embodiments, the compound is compound 8 shown in Table 1. In some embodiments, the compound is compound 32 shown in Table 1. In some embodiments, the compound is any one of compounds 32a or 32b shown below.

[0181] In one aspect, compounds represented by Formula I, Ia, Ib, or Ic may contain one or more stereocenters and may exist as racemates or racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. Stereoisomers can be separated using conventional techniques, such as chromatography or fractional crystallization, or stereoisomers can be prepared by stereoselective synthesis. For example, compounds 32-35 contain at least one stereocenter, and all of their individual isomers (shown further below) are disclosed herein.

[0182] [ka]

[0183] [ka]

[0184] [ka]

[0185] [ka]

[0186] G. Composition In one aspect, a composition is provided comprising a polymer and a compound described herein. In some aspects, the polymer comprises an oligonucleotide. In some aspects, the polymer is an antisense oligonucleotide. In some aspects, the polymer is an siRNA. In some aspects, the siRNA is conjugated to a lipid and / or a sugar. In some aspects, the siRNA is unconjugated. In some aspects, the polymer comprises a polypeptide. In some aspects, the polymer comprises one or more components of an SSM system described herein. In some aspects, the SSM system is a CRISPR system. In some aspects, the SSM system is a Cre-Lox system. In some aspects, the SSM system is an FLP-FRT system. In some aspects, the polymer comprises a Cas protein and / or a guide RNA. In some aspects, the polymer comprises Cre. In some aspects, the polymer comprises FLP.

[0187] In one aspect, a composition is provided comprising (i) an oligonucleotide and / or polypeptide, and (ii) a compound described herein. In one aspect, a composition is provided comprising (i) an oligonucleotide and / or polypeptide, and (ii) a compound represented by Formula I or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided comprising (i) an oligonucleotide and / or polypeptide, and (ii) a compound represented by Formula Ia or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided comprising (i) an oligonucleotide and / or polypeptide, and (ii) a compound represented by Formula Ib or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided comprising (i) an oligonucleotide and / or polypeptide, and (ii) a compound represented by Formula Ic or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided comprising (i) an oligonucleotide and / or polypeptide, and (ii) any one of Compounds 2-35, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided comprising (i) an oligonucleotide and / or polypeptide, and (ii) any one of Compounds 4, 8, and 32, or a pharmaceutically acceptable salt thereof. In one aspect, a pharmaceutical composition is provided comprising (i) an oligonucleotide and / or polypeptide, (ii) a compound described herein, and (iii) a pharmaceutically acceptable carrier or diluent. In one aspect, a pharmaceutical composition is provided comprising (i) an oligonucleotide and / or polypeptide, a compound represented by any one of Formulas I, Ia, Ib, and Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, and (iii) a pharmaceutically acceptable carrier or diluent. In one aspect, a pharmaceutical composition is provided comprising (i) an oligonucleotide and / or polypeptide; (ii) any one of Compounds 2-35, a pharmaceutically acceptable salt thereof, or a combination thereof; and (iii) a pharmaceutically acceptable carrier or diluent. In one aspect, a pharmaceutical composition is provided that includes: (i) an oligonucleotide and / or a polypeptide; (ii) any one of compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof; and (iii) a pharmaceutically acceptable carrier or diluent.Pharmaceutical compositions can be formulated to be compatible with the intended route of administration, including, but not limited to, intravenous (IV) or subcutaneous (SC or SQ) administration; parenteral administration, such as intraperitoneal, intramuscular, oral, transdermal, or transmucosal administration.

[0188] In one aspect, a composition comprising an oligonucleotide is provided. In one aspect, a composition comprising an antisense oligonucleotide (ASO) is provided. In one aspect, a composition comprising a splice-switching oligonucleotide (SSO) is provided. In one aspect, a composition comprising an siRNA is provided. In some aspects, the siRNA is conjugated to a lipid and / or a sugar. In some aspects, the siRNA is unconjugated. In one aspect, a composition comprising a guide RNA is provided. In one aspect, a composition comprising about 0.025 μM to about 20 μM of an oligonucleotide is provided. In one aspect, the composition comprises at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of an oligonucleotide. In one embodiment, the composition comprises about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide.

[0189] In one embodiment, a composition comprising a polypeptide is provided. In one embodiment, a composition comprising a Cas protein, such as a Cas9 or Cas12a protein, is provided. In some embodiments, the Cas protein is a modified Cas protein or a Cas fusion protein described herein. In one embodiment, a composition comprising Cre is provided. In one embodiment, a composition comprising FLP is provided. In one embodiment, a composition comprising about 0.001 μM to about 20 μM of a polypeptide is provided. In one embodiment, a composition comprises at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of a polypeptide. In one embodiment, a composition comprises about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of a polypeptide.

[0190] In one aspect, a composition is provided comprising an oligonucleotide and a polypeptide. In one aspect, a composition is provided comprising a Cas protein and a guide RNA. In some aspects, the Cas protein is Cas9. In some aspects, the Cas protein is Cas12a. In some aspects, the Cas protein is a modified Cas protein or a Cas fusion protein described herein. In one aspect, a composition is provided comprising about 0.001 μM to about 20 μM of each of an oligonucleotide and a polypeptide. In one aspect, a composition comprises at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of each of an oligonucleotide and a polypeptide. In one embodiment, the composition comprises about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of each of the oligonucleotide and polypeptide.

[0191] In one aspect, a composition is provided comprising a compound described herein. In one aspect, a composition is provided comprising a small molecule compound described herein. In one aspect, a composition is provided comprising a compound represented by Formula I, Ia, Ib, or Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided comprising a compound represented by any one of Formula I, Ia, Ib, or Ic, or a compound listed in Table 1, a pharmaceutically acceptable salt thereof, or a combination thereof. In one aspect, a composition is provided comprising any one of Compounds 4, 8, and 32, or a pharmaceutically acceptable salt thereof. In one aspect, a composition is provided comprising any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof. In one aspect, a composition is provided comprising about 1 μM to about 20 μM of a compound described herein. In one aspect, a composition comprises about 1 μM to about 10 μM of a compound described herein. In one aspect, a composition comprises about 1 μM to about 5 μM of a compound described herein. In one aspect, the composition comprises at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein.

[0192] In one aspect, a composition is provided comprising about 0.025 μM to about 20 μM of an oligonucleotide and about 1 μM to about 20 μM of a compound described herein. In one embodiment, a composition comprises at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of an oligonucleotide and at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein. In one embodiment, the composition comprises about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide and about 1 μM to about 1 μM to about 20 μM, about 1 μM to about 10 μM, or about 1 μM to about 5 μM of a compound described herein. In some embodiments, 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 siRNA is conjugated to a lipid and / or a sugar. In some embodiments, the siRNA is unconjugated. In some embodiments, the oligonucleotide is a guide RNA.

[0193] In one aspect, a composition is provided comprising about 0.001 μM to about 20 μM of a polypeptide and about 1 μM to about 20 μM of a compound described herein. In one aspect, a composition comprises at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of a polypeptide and at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein. In one embodiment, the composition comprises about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of the polypeptide and about 1 μM to about 1 μM to about 20 μM, about 1 μM to about 10 μM, or about 1 μM to about 5 μM of a compound described herein. 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.

[0194] In one aspect, a composition is provided comprising about 0.001 μM to about 20 μM each of an oligonucleotide and a polypeptide, and about 1 μM to about 20 μM of a compound described herein. In one embodiment, a composition comprises at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of each of an oligonucleotide and a polypeptide, and at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein. In one embodiment, the composition comprises about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of each of the oligonucleotide and polypeptide, and about 1 μM to about 1 μM to about 20 μM, about 1 μM to about 10 μM, or about 1 μM to about 5 μM of a compound described herein. In some embodiments, the polypeptide is a Cas protein and the oligonucleotide is a guide RNA.

[0195] A "composition comprising an oligonucleotide and / or a polypeptide" and / or a "composition comprising a compound of Formula I, Ia, Ib, or Ic, or a compound listed in Table 1" can include biological cells, e.g., in vitro cells. For example, a composition comprising an oligonucleotide, a polypeptide, and / or a compound of Formula I, Ia, Ib, or Ic, or a compound listed in Table 1, can include an in vitro cell culture, e.g., a mammalian cell culture, and the above concentrations can refer to the concentration of the oligonucleotide, polypeptide, and / or a compound of Formula I, Ia, Ib, or Ic, or a compound listed in Table 1, in the cell culture. In another aspect, a composition comprising an oligonucleotide, polypeptide, and / or compound of Formula I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, can include in vitro cells isolated from a subject, e.g., a mammalian subject, e.g., a human subject, and the concentrations above can refer to the concentration of the oligonucleotide, polypeptide, and / or compound of Formula I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, in combination with cells isolated from a subject.

[0196] H. Treatment Method In one aspect, a method is provided for introducing a macromolecule into a cell, e.g., a target cell described herein. In one aspect, the cell is a cultured cell. In one aspect, the cell is an isolated cell. In one aspect, the cell is a cell isolated from a subject in need of treatment. In one aspect, the cell is a mammalian cell. In one aspect, the cell is a eukaryotic and / or prokaryotic cell.

[0197] In one embodiment, the cell is part of a mammalian, e.g., human, tissue or organ. In one embodiment, the organ or tissue is the brain, central nervous system (CNS) or peripheral nervous system (PNS), heart, liver, kidney, spleen, pancreas, lung, fat, and / or muscle (e.g., skeletal muscle). In one embodiment, the cell is a brain cell, CNS cell, PNS cell, cardiac cell, liver cell, kidney cell, spleen cell, pancreatic cell, lung cell, muscle cell, adipocyte, immune cell, or a combination thereof.

[0198] In some embodiments, the cells are CNS cells. In some embodiments, the CNS cells include glial cells and / or neurons. CNS glial cells include, for example, astrocytes, oligodendrocytes, microglia, and ependymal cells. Neurons include, for example, afferent neurons, efferent neurons, and interneurons.

[0199] In some embodiments, the cells are hepatocytes, e.g., parenchymal or non-parenchymal cells. In some embodiments, the cells include culturable metabolically competent human hepatocytes, culturable induction-competent human hepatocytes, culturable human hepatocytes, suspension-competent human hepatocytes (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-1 and C57B1 / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes).

[0200] In some aspects, the cells are human stem cells. The stem cells can be pluripotent stem cells, including, for example, 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). In some aspects, the cells are differentiated forms of any of the cells described herein. In some aspects, the eukaryotic cells are cells derived from any primary cell in culture.

[0201] In some embodiments, the cell is an immune cell, non-limiting examples of which include T cells, B cells, dendritic cells, NK cells, helper T cells, cytotoxic T cells, regulatory T cells, gamma delta T cells, neutrophils, mast cells, monocytes, antigen-presenting cells, lymphocytes, basophils, and phagocytes.

[0202] In one aspect, a method is provided 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 the nucleus and / or cytosol of a cell. In one aspect, a method is provided for introducing a polypeptide into the nucleus and / or cytosol of a cell. In one aspect, the oligonucleotide is an ASO, SSO, and / or siRNA. In one aspect, the oligonucleotide is an siRNA, and the siRNA is unconjugated. In one aspect, the oligonucleotide is an siRNA, and the siRNA is conjugated to a lipid and / or sugar. In one aspect, the oligonucleotide is a guide RNA. In one aspect, the oligonucleotide hybridizes to a target nucleic acid in a cell. In one aspect, the polypeptide is capable of providing an SSM to a target nucleic acid in a cell. In one aspect, 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.

[0203] In one aspect, the method comprises (a) contacting a cell with an oligonucleotide and / or a polypeptide; and (b) contacting the cell with a compound described herein. In one aspect, the oligonucleotide is an antisense oligonucleotide (ASO). In one aspect, the oligonucleotide is a splice-switching oligonucleotide (SSO). In one aspect, the oligonucleotide is an siRNA. In some aspects, the siRNA is conjugated to a lipid and / or sugar. In some aspects, the siRNA is unconjugated. In one aspect, the cell is contacted with an oligonucleotide. In one aspect, the cell is contacted with a polypeptide. In one aspect, the cell is contacted with both an oligonucleotide and a polypeptide. In one aspect, the oligonucleotide is a guide RNA. In one aspect, the polypeptide is a recombinase. In one aspect, the polypeptide is Cre. In one aspect, the polypeptide is FLP. In one aspect, the polypeptide is a Cas protein. In one aspect, the polypeptide is a Cas protein and the oligonucleotide is a guide RNA. In one aspect, the compound is a small molecule compound (SMC). In one aspect, the compound is an endosomolytic compound. In one aspect, the compound has a structure represented by Formula I, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula Ia, or a pharmaceutically acceptable salt thereof. In one aspect, the compound has a structure represented by Formula Ib, 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 I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof. In one aspect, the compound is any one of Compounds 4, 8, and 32, or a pharmaceutically acceptable salt thereof.

[0204] In one aspect, the method comprises in vitro delivery of a macromolecule, e.g., an oligonucleotide and / or polypeptide, and / or a compound, to a cell. In one aspect, the method comprises in vivo delivery of a macromolecule, e.g., an oligonucleotide and / or polypeptide, and / or a compound, to a cell. In one aspect, the cell is a cultured cell. In one aspect, the cell is a cell isolated 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 prokaryotic cell.

[0205] In one aspect, the macromolecule, e.g., oligonucleotide and / or polypeptide, is internalized by the cell via endocytosis and becomes encapsulated in an endosome, hi one aspect, the compound promotes the release of the macromolecule, e.g., oligonucleotide and / or polypeptide, from the endosome.

[0206] In one aspect, macromolecules, such as oligonucleotides and / or polypeptides, are internalized by cells via transient pore formation. In one aspect, the compound promotes transient pore formation in the plasma membrane.

[0207] In one embodiment, cells are contacted with a composition comprising an oligonucleotide. In one embodiment, cells are contacted with a composition comprising an antisense oligonucleotide (ASO). In one embodiment, cells are contacted with a composition comprising a splice-switching oligonucleotide (SSO). In one embodiment, cells are contacted with a composition comprising an siRNA. In some embodiments, the siRNA is conjugated to a lipid and / or sugar. In some embodiments, the siRNA is unconjugated. In one embodiment, cells are contacted with a composition comprising a guide RNA. In one embodiment, cells are contacted with a composition comprising an oligonucleotide in an amount sufficient to provide a therapeutic effect. In one embodiment, cells are contacted with a composition comprising an oligonucleotide in an amount sufficient to provide an SSM at the target nucleic acid. In one embodiment, cells are contacted with a composition comprising about 0.025 μM to about 20 μM of the oligonucleotide. In one embodiment, the cells are contacted with a composition comprising at least about 0.1 μM and up to about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, or 20 μM of the oligonucleotide. In one embodiment, the cells are contacted with a composition comprising about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide.

[0208] In one embodiment, a cell is contacted with a composition comprising a polypeptide. In one embodiment, a cell is contacted with a composition comprising a Cas protein. In one embodiment, a cell is contacted with a composition comprising a recombinase (e.g., Cre or FLP). In one embodiment, a cell is contacted with a composition comprising a polypeptide in an amount sufficient to provide a therapeutic effect. In one embodiment, a cell is contacted with a composition comprising a polypeptide in an amount sufficient to provide an SSM at a target nucleic acid. In one embodiment, a cell is contacted with a composition comprising about 0.3 μM to about 20 μM of the polypeptide. In one embodiment, a cell is contacted with a composition comprising at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of the polypeptide. In one embodiment, the cells are contacted with a composition comprising about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of the polypeptide.

[0209] In one embodiment, cells are contacted with a composition comprising an oligonucleotide and a polypeptide. In one embodiment, cells are contacted with a composition comprising a Cas protein and a guide RNA. In one embodiment, cells are contacted with a composition comprising an oligonucleotide and a polypeptide in an amount sufficient to provide a therapeutic effect. In one embodiment, cells are contacted with a composition comprising an oligonucleotide and a polypeptide in an amount sufficient to provide SSM at a target nucleic acid. In one embodiment, cells are contacted with a composition comprising about 0.001 μM to about 20 μM each of an oligonucleotide (e.g., a guide RNA) and a polypeptide (e.g., a Cas protein). In one embodiment, cells are contacted with a composition comprising at least about 0.01 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM each of an oligonucleotide and a polypeptide. In one embodiment, the cells are contacted with a composition comprising about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM, or about 0.1 μM to about 1 μM of each of the oligonucleotide and the polypeptide.

[0210] In one embodiment, cells are contacted with a composition comprising a compound described herein. In one embodiment, cells are contacted with a composition comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, cells are contacted with a composition comprising a compound represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, a pharmaceutically acceptable salt thereof, or a combination thereof. In one embodiment, cells are contacted with a composition comprising any one of compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof. In one embodiment, cells are contacted with a composition at a concentration sufficient to (i) reduce accumulation of oligonucleotides in endosomes and / or lysosomes, (ii) induce physical disruption of endosomes, and / or (iii) promote transient pore formation in the plasma membrane, thereby promoting release of macromolecules (e.g., oligonucleotides and / or polypeptides) into the cytosol or nucleus.

[0211] In one embodiment, cells are contacted with a composition comprising about 1 μM to about 20 μM of a compound described herein. In one embodiment, cells are contacted with a composition comprising about 1 μM to about 10 μM of a compound described herein. In one embodiment, cells are contacted with a composition comprising about 1 μM to about 5 μM of a compound described herein. In one embodiment, cells are contacted with a composition comprising at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein. In some embodiments, the compound has a structure represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0212] In one embodiment, the cells are contacted with a composition comprising about 0.025 μM to about 20 μM of an oligonucleotide and about 1 μM to about 20 μM of a compound described herein. In one aspect, a cell is contacted with a composition comprising at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of an oligonucleotide and at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein. In one aspect, a cell is contacted with a composition comprising about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of an oligonucleotide and about 1 μM to about 1 μM to about 20 μM, about 1 μM to about 10 μM, or about 1 μM to about 5 μM of a compound described herein. In one aspect, the composition comprises an oligonucleotide. In one aspect, the composition comprises an antisense oligonucleotide (ASO). In one aspect, the composition comprises a splice-switching oligonucleotide (SSO). In one aspect, the composition comprises an 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 composition comprises a guide RNA. In one aspect, the composition comprises a compound described herein. In one aspect, the composition comprises a compound represented by Formula I, or a pharmaceutically acceptable salt thereof. In one aspect, the composition comprises a compound represented by Formula I, Formula Ia, Formula Ib, or Formula Ib, or a compound listed in Table 1, a pharmaceutically acceptable salt thereof, or a combination thereof. In one aspect, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0213] In one aspect, the cells are contacted with a composition comprising about 0.025 μM to about 20 μM of a polypeptide and about 1 μM to about 20 μM of a compound described herein. In one aspect, a cell is contacted with a composition comprising at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of a polypeptide and at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein. In one embodiment, a cell is contacted with a composition comprising about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of a polypeptide and about 1 μM to about 1 μM to about 20 μM, about 1 μM to about 10 μM, or about 1 μM to about 5 μM of a compound described herein. In one embodiment, the composition comprises a polypeptide. In one embodiment, the composition comprises a Cas protein, e.g., Cas9 or Cas12a. In one embodiment, the composition comprises a recombinase, e.g., Cre or FLP. In one embodiment, the composition comprises a compound described herein. In one embodiment, the composition comprises a compound represented by Formula I, or a pharmaceutically acceptable salt thereof. In one embodiment, the composition comprises a compound represented by Formula I, Formula Ia, Formula Ib, or Formula Ib, or a compound listed in Table 1, a pharmaceutically acceptable salt thereof, or a combination thereof. In one embodiment, the compound is any one of compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0214] In one embodiment, the cells are contacted with a composition comprising about 0.025 μM to about 20 μM each of an oligonucleotide and a polypeptide, and about 1 μM to about 20 μM of a compound described herein. In one aspect, cells are contacted with a composition comprising at least about 0.1 μM and up to about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM of each of oligonucleotides and polypeptides, and at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 15 μM, or about 20 μM of a compound described herein. In one embodiment, a cell is contacted with a composition comprising about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of each of an oligonucleotide and a polypeptide, and about 1 μM to about 1 μM to about 20 μM, about 1 μM to about 10 μM, or about 1 μM to about 5 μM of a compound described herein. In one embodiment, the composition comprises an oligonucleotide and a polypeptide. In one embodiment, the composition comprises a Cas protein, e.g., Cas9 or Cas12a, and a guide RNA. In one embodiment, the composition comprises a compound described herein. In one embodiment, the composition comprises a compound represented by Formula I, or a pharmaceutically acceptable salt thereof. In one embodiment, the composition comprises a compound represented by Formula I, Formula Ia, Formula Ib, or Formula Ib, or a compound listed in Table 1, a pharmaceutically acceptable salt thereof, or a combination thereof. In one embodiment, the compound is any one of compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0215] In one embodiment, the cells are contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide, and the compound simultaneously, i.e., approximately simultaneously. In one embodiment, the cells are contacted with a composition comprising the macromolecule, e.g., oligonucleotide and / or polypeptide, and the compound. In one embodiment, the macromolecule, e.g., oligonucleotide and / or polypeptide, and the compound are in the same composition. In one embodiment, the cells are contacted with a first composition comprising the macromolecule, e.g., oligonucleotide and / or polypeptide, and a second composition comprising the compound. In one embodiment, the cells are contacted with the first composition comprising the macromolecule, e.g., oligonucleotide and / or polypeptide, and the second composition comprising the compound approximately simultaneously. In one embodiment, the cells are contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide, and the compound within about 6 hours, about 3 hours, about 2 hours, about 1 hour, about 30 minutes, or less than about 15 minutes of each other.

[0216] In one embodiment, cells are contacted sequentially with a polymer, e.g., an oligonucleotide and / or polypeptide, and a compound. In one embodiment, cells are contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, before contacting with a compound. In one embodiment, cells are contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, after contacting with a compound. In one embodiment, cells are contacted with a first composition comprising a polymer, e.g., an oligonucleotide and / or polypeptide, and then with a second composition comprising a compound. In one embodiment, cells are contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, up to about 48 hours before contacting the cells with a compound. In one embodiment, cells are contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, about 12 hours to about 48 hours before contacting the cells with a compound. In one embodiment, cells are contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, at least about 12 hours or about 24 hours and up to about 36 hours or about 48 hours before contacting the cells with a compound.

[0217] In one aspect, contacting cells with a compound described herein results in endosomal membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting cells with a compound having the structure shown in Formula I or a pharmaceutically acceptable salt thereof results in endosomal membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting cells with a compound having the structure shown in Formula I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting cells with any one of Compounds 2-35, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting cells with a compound having the structure of Compound 2, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting a cell with a compound having the structure of Compound 4, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting a cell with a compound having the structure of Compound 8, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting a cell with a compound having the structure of Compound 32, or a pharmaceutically acceptable salt thereof, results in membrane permeabilization as determined by an mCherry-GAL9 recruitment assay. In one aspect, contacting a cell with a compound having the structure shown in Formula I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, results in endosomal membrane permeabilization as determined by an mCherry-GAL9 recruitment assay.

[0218] In one embodiment, the polymer, e.g., an oligonucleotide and / or polypeptide, alters the activity of a gene expressed by a cell. In one embodiment, the gene is an endogenous gene. In one embodiment, the gene is an exogenous gene. In one embodiment, the polymer, e.g., an oligonucleotide and / or polypeptide, increases the activity of a gene expressed by a cell. In one embodiment, the activity of a gene expressed by a cell is increased by at least about 10-fold when the cell is contacted with the polymer, e.g., the oligonucleotide and / or polypeptide, and the compound, compared to a cell contacted with the polymer, e.g., the oligonucleotide and / or polypeptide, but not the compound. In one embodiment, the activity of a gene expressed by a cell is increased by at least about 10-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold, and up to about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 1000-fold. In one embodiment, the activity of a gene expressed by a cell is increased by about 10-fold to about 1000-fold, about 10-fold to about 500-fold, about 10-fold to about 400-fold, about 10-fold to about 300-fold, about 10-fold to about 200-fold, about 10-fold to about 100-fold, about 100-fold to about 300-fold, or about 100-fold to about 200-fold. In one embodiment, the activity of a gene expressed by a cell is increased by about 10-fold to about 1000-fold when the cell is contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, and a compound, compared to a cell contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, but not with a compound. In one embodiment, the activity of a gene expressed by a cell is increased by about 100-fold to about 500-fold. In one embodiment, the activity of a gene expressed by a cell is increased by about 100-fold to about 300-fold. In one embodiment, the activity of a gene expressed by the cell is increased by about 100-fold to about 200-fold. In one embodiment, the activity of a gene expressed by the cell is increased by about 10-fold to about 500-fold. In one embodiment, the activity of a gene expressed by the cell is increased by about 10-fold to about 300-fold. In one embodiment, the activity of a gene expressed by the cell is increased by about 10-fold to about 200-fold. In one embodiment, the activity of a gene expressed by the cell is increased by about 10-fold to about 100-fold. In one embodiment, the activity of a gene expressed by the cell is increased by about 10-fold to about 50-fold.

[0219] In one embodiment, the polymer, e.g., oligonucleotide and / or polypeptide, reduces the activity of a gene expressed by a cell. In one embodiment, the activity of a gene expressed by a cell is reduced by at least about 10-fold when the cell is contacted with the polymer, e.g., oligonucleotide and / or polypeptide, and the compound, compared to a cell contacted with the polymer, e.g., oligonucleotide and / or polypeptide, but not the compound. In one embodiment, the activity of a gene expressed by the cell is reduced by at least about 10-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold, and up to about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 1000-fold. In one embodiment, the activity of a gene expressed by a cell is reduced by about 10-fold to about 1000-fold, about 10-fold to about 500-fold, about 10-fold to about 400-fold, about 10-fold to about 300-fold, about 10-fold to about 200-fold, about 10-fold to about 100-fold, about 100-fold to about 300-fold, or about 100-fold to about 200-fold. In one embodiment, the activity of a gene expressed by a cell is reduced by about 10-fold to about 1000-fold when the cell is contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, and a compound, compared to a cell contacted with a polymer, e.g., an oligonucleotide and / or polypeptide, but not with a compound. In one embodiment, the activity of a gene expressed by a cell is reduced by about 100-fold to about 500-fold. In one embodiment, the activity of a gene expressed by a cell is reduced by about 100-fold to about 300-fold. In one embodiment, the activity of a gene expressed by the cell is decreased by about 100-fold to about 200-fold. In one embodiment, the activity of a gene expressed by the cell is decreased by about 10-fold to about 500-fold. In one embodiment, the activity of a gene expressed by the cell is decreased by about 10-fold to about 300-fold. In one embodiment, the activity of a gene expressed by the cell is decreased by about 10-fold to about 200-fold. In one embodiment, the activity of a gene expressed by the cell is decreased by about 10-fold to about 100-fold. In one embodiment, the activity of a gene expressed by the cell is decreased by about 10-fold to about 50-fold.

[0220] In one aspect, a method for altering expression of a target nucleic acid in a cell is provided. In one aspect, the method includes contacting the cell with an oligonucleotide capable of hybridizing to the target nucleic acid, wherein the oligonucleotide is internalized by the cell via endocytosis and encapsulated in an endosome; and contacting the cell with a compound described herein, wherein the compound promotes release of the oligonucleotide from the endosome, wherein hybridization of the oligonucleotide to the target nucleic acid alters expression of the target nucleic acid. In one aspect, 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. In some embodiments, 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 sugar and / or peptide. In some aspects, the oligonucleotide is unconjugated. In some aspects, the combination of the unconjugated oligonucleotide and the compound provides a similar level of modified expression of the target nucleic acid compared to an oligonucleotide conjugated to a lipid and / or sugar and / or peptide. In some embodiments, the compound has a structure represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof. In one aspect, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0221] In one aspect, a method for altering expression of a target nucleic acid in a cell is provided. In one aspect, the method includes contacting the cell with a polypeptide capable of performing SSM at the target nucleic acid, wherein the polypeptide is internalized by the cell via endocytosis and encapsulated in an endosome; and contacting the cell with a compound described herein, wherein the compound promotes release of the polypeptide from the endosome, and wherein SSM at the target nucleic acid alters expression of the target nucleic acid. In one aspect, the SSM increases expression of the target nucleic acid. In one aspect, the SSM decreases expression of the target nucleic acid. In some embodiments, the polypeptide is a Cas protein. In some embodiments, 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 Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof. In one aspect, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0222] In one aspect, a method for altering expression of a target nucleic acid in a cell is provided. In one aspect, the method includes contacting a cell with a polypeptide and an oligonucleotide, wherein the polypeptide is capable of performing SSM, the oligonucleotide is capable of hybridizing to the target nucleic acid, and the polypeptide and oligonucleotide are internalized by the cell via endocytosis and encapsulated in an endosome; and contacting the cell with a compound described herein, wherein the compound promotes release of the polypeptide and oligonucleotide from the endosome, wherein hybridization of the oligonucleotide to the target nucleic acid causes the polypeptide to perform SSM, thereby altering expression of the target nucleic acid. In one aspect, the SSM increases expression of the target nucleic acid. In one aspect, the SSM decreases expression of the target nucleic acid. In some embodiments, the oligonucleotide is a guide RNA and the polypeptide is a Cas protein. In some embodiments, the compound has a structure represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof. In one embodiment, the compound is any one of compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0223] In one aspect, a method is provided for releasing a macromolecule, e.g., an oligonucleotide and / or a polypeptide, from an endosome. In one aspect, the method includes contacting a cell with a macromolecule, e.g., an oligonucleotide and / or a polypeptide, where the macromolecule, e.g., the oligonucleotide and / or the polypeptide, is internalized by the cell via endocytosis and encapsulated in an endosome; and contacting the cell with a compound described herein, where the compound promotes release of the macromolecule, e.g., the oligonucleotide and / or the polypeptide, from the endosome. In some embodiments, 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 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. In some embodiments, the compound has a structure represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0224] In one aspect, a method for treating and / or preventing a disorder (e.g., a genetic disorder) in a subject is provided. In one aspect, the method comprises administering to the subject a therapeutically effective amount of a macromolecule, e.g., an oligonucleotide and / or polypeptide, and an effective amount of a compound described herein. In one aspect, the compound is administered to the subject simultaneously with the macromolecule, e.g., the oligonucleotide and / or polypeptide. In one aspect, the compound is administered to the subject after administration of the macromolecule, e.g., the oligonucleotide and / or polypeptide. In one aspect, the compound is administered to the subject up to about 48 hours after administration of the macromolecule, e.g., the oligonucleotide and / or polypeptide. In one aspect, the compound is administered to the subject about 12 hours to about 48 hours after administration of the macromolecule, e.g., the oligonucleotide and / or polypeptide. In one aspect, the compound is administered to the subject at least about 12 hours or about 24 hours and up to about 36 hours or about 48 hours after administration of the macromolecule, e.g., the oligonucleotide and / or polypeptide. In one aspect, the administration comprises parenteral administration. In one aspect, parenteral administration comprises intravenous (IV) or subcutaneous (SC) administration. In one aspect, the subject is a mammal. In one aspect, the subject is a human. In some embodiments, 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 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. In some embodiments, the compound has a structure represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof.In some embodiments, the compound is any one of compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0225] In one aspect, a method for treating and / or preventing a disorder in a subject is provided. In one aspect, the method includes isolating cells from the subject, contacting the isolated cells with a therapeutically effective amount of a macromolecule, e.g., an oligonucleotide and / or polypeptide, and an effective amount of a compound described herein to produce an engineered cell, and transplanting the engineered cell into the subject. In one aspect, the isolated cells are contacted with a composition comprising about 0.025 μM to about 20 μM of the macromolecule, e.g., an oligonucleotide and / or polypeptide. In one aspect, the isolated cells are contacted with about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the macromolecule, e.g., an oligonucleotide and / or polypeptide. In one aspect, the isolated cells are contacted with a composition comprising about 1 μM to about 20 μM of a compound described herein. In one aspect, the isolated cells are contacted with a composition comprising about 1 μM to about 10 μM of a compound described herein. In one aspect, the isolated cells are irradiated with a phospholipid-containing agonist at a concentration of at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 10.5 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about The subject is contacted with a composition comprising 1.5 μM, about 12 μM, about 12.5 μM, about 13 μM, about 13.5 μM, about 14 μM, about 14.5 μM, about 15 μM, about 15.5 μM, about 16 μM, about 16.5 μM, about 17 μM, about 17.5 μM, about 18 μM, about 18.5 μM, about 19 μM, about 19.5 μM, or about 20 μM of a compound described herein. In some embodiments, 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 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. In some embodiments, the compound has a structure represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0226] In one aspect, the isolated cells are contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide, and the compound at approximately the same time. In one aspect, the isolated cells are contacted with a composition comprising the macromolecule, e.g., oligonucleotide and / or polypeptide, and the compound. In one aspect, the isolated cells are contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide, before contacting the isolated cells with the compound. In one aspect, the isolated cells are contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide, up to about 48 hours before contacting the isolated cells with the compound. In one aspect, the isolated cells are contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide, about 12 hours to about 48 hours before contacting the isolated cells with the compound. In one aspect, the isolated cells are contacted with the macromolecule, e.g., oligonucleotide and / or polypeptide, at least about 12 hours or about 24 hours and up to about 36 hours or about 48 hours before contacting the isolated cells with the compound. In one aspect, the subject is a mammal. In one aspect, the subject is human. In some embodiments, 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 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. In some embodiments, the compound has a structure represented by Formula I, Formula Ia, Formula Ib, or Formula Ic, or is a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the compound is any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0227] Provided herein is the use of a compound described herein in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound described herein in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula Ia or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula Ib or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula Ic or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof, in the manufacture of a medicament for gene therapy. Provided herein is the use of any one of Compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof, in the manufacture of a medicament for gene therapy.

[0228] Provided herein is the use of a compound described herein and a polymer, e.g., an oligonucleotide and / or a polypeptide, in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula I or a pharmaceutically acceptable salt thereof and a polymer, e.g., an oligonucleotide and / or a polypeptide, in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula Ia or a pharmaceutically acceptable salt thereof and a polymer, e.g., an oligonucleotide and / or a polypeptide, in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula Ib or a pharmaceutically acceptable salt thereof and a polymer, e.g., an oligonucleotide and / or a polypeptide, in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula Ic or a pharmaceutically acceptable salt thereof and a polymer, e.g., an oligonucleotide and / or a polypeptide, in the manufacture of a medicament for gene therapy. Provided herein is the use of a compound of Formula I, Formula Ia, Formula Ib, Formula Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof, and a polymer, e.g., an oligonucleotide and / or a polypeptide, in the manufacture of a medicament for gene therapy. Provided herein is the use of any one of compounds 4, 8, and 32, a pharmaceutically acceptable salt thereof, or a combination thereof, and a polymer, e.g., an oligonucleotide and / or a polypeptide, in the manufacture of a medicament for gene therapy.

[0229] Provided herein are kits comprising a compound described herein and a polymer, e.g., an oligonucleotide and / or a polypeptide. In one embodiment, the kit comprises a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a polymer, e.g., an oligonucleotide and / or a polypeptide. In one embodiment, the kit comprises a compound of Formula Ia, or a pharmaceutically acceptable salt thereof, and a polymer, e.g., an oligonucleotide and / or a polypeptide. In one embodiment, the kit comprises a compound of Formula Ib, or a pharmaceutically acceptable salt thereof, and a polymer, e.g., an oligonucleotide and / or a polypeptide. In one embodiment, the kit comprises a compound of Formula Ic, or a pharmaceutically acceptable salt thereof, and a polymer, e.g., an oligonucleotide and / or a polypeptide. In one embodiment, the kit comprises a compound of Formula I, Formula Ia, Formula Ib, or Formula Ic, or a compound listed in Table 1, or a pharmaceutically acceptable salt thereof, or a combination thereof, and a polymer, e.g., an oligonucleotide and / or a polypeptide. In one embodiment, the kit comprises any one of compounds 4, 8, and 32, or a pharmaceutically acceptable salt thereof, or a combination thereof, and a macromolecule, eg, an oligonucleotide and / or a polypeptide.

[0230] The entire contents of all publications, patents, and patent applications referenced herein are hereby incorporated by reference.

[0231] The specific examples contained herein are for illustrative purposes only and should not be construed as limiting the present disclosure. Furthermore, the compositions and methods provided herein have been described in connection with specific embodiments thereof, and numerous details have been set forth for illustrative purposes. It will be apparent to those skilled in the art that the present disclosure is susceptible to additional embodiments and that some of the details described herein may be modified without departing from the basic principles of the present disclosure. [Example]

[0232] Example 1. Preparation of splice-switching oligonucleotides (SSOs) An 18-nt splice-switching oligonucleotide binding to Luc705 (5'-CCUCUUACCUCAGUUACA-3' SEQ ID NO: 1) (705-SSO) was synthesized using 2'-0-methylated (2'-OMe) modified bases and phosphorothioate (PS) saturated backbone linkages. Luc705-SSO labeled with Alexa Fluor™ 488 (green) fluorescent label was synthesized as shown in Table 3. For non-fluorescent oligos, HPLC purification and Na+ salt exchange steps were included in the synthesis. SSO was delivered at 100 μM in IDTE buffer (nuclease-free HO supplemented with 10 mM Tris and 0.1 mM EDTA) at pH 8.0.

[0233] [Table 4] * , PS bond; m, 2-OMe nucleotide

[0234] Example 2. Preparation of a small molecule compound (SMC) library Small molecule compounds (SMCs) were screened for their ability to increase the activity of gapmer antisense oligonucleotides (ASOs) by co-treating cells with SMCs and ASOs, as shown in the subsequent Examples. A representative procedure for synthesizing SMCs is provided below.

[0235] General Procedure for the Preparation of Compounds 2-7 and 19-31 The general procedures for the preparation of compounds 2-7 and 19-31 are shown generally in Figures 29-30, where Figure 29 provides an overall general scheme and includes starting material X shown in Step 3. Figure 30 sets forth the definitions of starting material X and necessary intermediates for each of the compounds shown (e.g., compound II shown in Figure 30 is starting material "X" in the preparation described in Figure 29 for compound 2). Further description of exemplary intermediates and compounds is provided below. The aminopyrazole and aminoisopropoxypyrazole derivative intermediates identified in Figure 30 used to make compounds 19 and 30, respectively, were prepared according to the same procedures described below for intermediates 3 (aminomethoxypyrazole) and 7 (aminoethoxypyrazole), respectively.

[0236] General procedure for the preparation of intermediate 2

[0237] [ka] 1. Compound 1 (9.00 g, 62.8 mmol, 1.00 equiv.) and compound 1A (28.8 g, 81.1 mmol, 1.29 equiv.) are added to a flask charged with DMF (63 mL). 2. Add Cs2CO3 (30.5 g, 93.7 mmol, 1.49 equiv.) to the mixture. 3. Degas with N2 three times. 4. Stir at 120°C for 2 hours. 5. TLC shows that Compound 1 has been consumed and a new spot has formed. 6. Adjust the pH of the mixture to 7-8 with HCl (1M). 7. Extract with a mixture of DCM (200 mL x 3). 8. The organic layer was washed with brine (100 mL x 2) and dried over magnesium sulfate. 9. Concentrate the organic layer in vacuo to remove DCM. 10. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 30 / 1 to 3 / 1). 11. Compound 2 (18.1 g, 70.5% yield, 80.0% purity) is obtained as a yellow solid.

[0238] 1 H NMR: 400MHz DMSO-d6 δ8.746(s,1H),7.94(s,1H),4.69-4.68(m,1H),4.31-4.26(m,2H),4.05(s,3H),3.33 (s,4H),2.88(s,3H),2.72(s,1H),2.01-1.98(m,2H),1.81-1.74(m,2H),1.40(s,9H).

[0239] General procedure for the preparation of intermediate 3

[0240] [ka] 1. To a flask charged with EtOAc (110 mL) is added compound 2 (17.0 g, 52.0 mmol, 1.00 equiv.). 2. Add Pd / C (1.70 g, 52.0 mmol, 10% purity, 1.00 equiv.) to the mixture. 3. Add H2 (15 Psi) to the mixture. 4. Stir at 25°C for 10 hours. 5. TLC (petroleum ether / ethyl acetate=3 / 1, product Rf=0.42) shows that compound 3 was formed. 6. Filtered through Celite. 7. Wash the filter cake with EtOAc (150 mL x 3). 8. Concentrate the filtrate in vacuo to remove EtOAc. 9. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 30 / 1 to 3 / 1). 10. Compound 3 (10.9 g, 70.6% yield) is obtained as a dark blue oil.

[0241] 1 H NMR: 400MHz DMSO-d6 δ7.00(s,1H),4.01-3.94(m,3H),3.52(s,3H),2.88-2.73(m,2H),1.88-1.84(m,2H),1.66-1.60(m,2H),1.40(s,9H).

[0242] Alternative Method for Preparing Intermediate Compound 3 A solution of compound 2 and iron(III) chloride (3.41 g, 1.05 mmol) in MeOH (21.14 mL) was treated with hydrazine (2.197 mL, 70.00 mmol) and refluxed overnight. The reaction was filtered. The filtrate was shrunk to give a residue. The residue was redissolved in DCM / MeOH (10:1) and washed with water. The organic layer was concentrated to give tert-butyl 4-(4-amino-3-methoxy-1H-pyrazol-1-yl)piperidine-1-carboxylate (2.000 g, 96%).

[0243] General Procedure for the Preparation of Intermediate 4-1

[0244] [ka] 1. Compound 3 (0.50 g, 1.69 mmol, 1.00 equiv.) and compound 3b-10 (444 mg, 1.54 mmol, 0.91 equiv.) charged with i-PrOH (10.0 mL) are added to a microwave vial. 2. Add TsOH (529 mg, 3.08 mmol, 1.82 equiv) to the mixture. 3. Stir at 120°C for 2 hours. 4. LCMS (ET37912-40-P1a) shows that compound 4-1 was formed. 5. Adjust the pH of the mixture to 7-8 with saturated NaHCO3. 6. The mixture is extracted with EtOAc (50 mL x 3). 7. Wash the organic layer with brine (30 mL x 2). 8. Concentrate in vacuo to remove EtOAc. 9. Compound 4-1 is obtained as a yellow solid (0.40 g, crude).

[0245] Exemplary Procedure for the Preparation of Compound 2 The procedure for the preparation of compound 2 is outlined in FIG. 1. Compound 4-1 (0.90 g, 1.64 mmol, 1.00 equiv) is dissolved in HCl / dioxane (4 M, 9.00 mL, 21.9 equiv). 2. Stir at 25°C for 3 hours. 3. LCMS (product Rt=0.582 min) shows that compound 2 was formed. 4. Add saturated NaHCO3 (50.0 mL) to the solution pH=8. 5. Extract with EtOAc (100.0 mL x 2), separate the organic layer, dry over Na2SO4 and concentrate in vacuo. 6. Preparative HPLC (Column: Phenomenex Gemini-NX 80 * 40mm * 3um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 20%-40%, 8 min). 7. Compound 2 (0.062 g, 8.34% yield) is obtained as a white solid.

[0246] 1 H NMR: 400MHz DMSO-d6 δ 8.44(s,1H),8.08(s,1H),7.85(d,J=8.0Hz,1H),7.73(s,1H),7.61(d,J=8.0Hz,1H),7.34 (t,J=8.0Hz,1H),3.90(s,1H),3.77(m,3H),2.96(s,2H),1.84(s,2H),1.67-1.60(m,2H).

[0247] Exemplary Procedure for the Preparation of Compound 6 The procedure for preparing compound 6 is the same as that described above for compound 2, except that starting from intermediate 1, the methoxy substituent is replaced with modified compound 3b-10 (i.e., the indole intermediate) lacking the ethoxy and cyano substituents.

[0248] General Procedure for the Preparation of Intermediate 3b-10

[0249] [ka] 1. Compound 3C (4.84 g, 26.3 mmol, 1.50 equiv.) charged with DCE (100 mL) is added to a flask. 2. Add AlCl3 (3.52 g, 26.3 mmol, 1.44 mL, 1.50 equiv) to the mixture, which will turn yellow in color. 3. Stir at 80°C for 0.5 hours. 4. Compound 3b_Int (2.5 g, 17.59 mmol, 1.00 equiv) is added to the mixture, which turns orange-red in color. 5. Stir at 80°C for 12 hours. 6. LCMS shows that compound 3b_10 was produced. 7. Add the mixture to an ice-water mixture (200 mL). 8. The mixture is extracted with EtOAc (250 mL x 3). 9. Wash the organic layer with brine (100 mL x 2). 10. Concentrate in vacuo to remove EtOAc. 11. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 30 / 1 to 1 / 1). 12. Compound 3b_10 (0.50 g, 9.83% yield) is obtained as an orange solid.

[0250] 1 H NMR: 400MHz DMSO-d6 δ 8.95(s,1H),8.37(s,1H),7.91(d,J=8.0Hz,1H),7.69(d,J=8.0Hz,1H),7.41(t,J=8Hz,1H).

[0251] General procedure for the preparation of intermediate 4-2

[0252] [ka] 1. Compound 3 (0.50 g, 1.69 mmol, 1.00 equiv.) and compound 7a-10 (545 mg, 1.69 mmol, 1.00 equiv.) charged with i-PrOH (10.0 mL) are added to a microwave vial. 2. Add TsOH (529.65 mg, 3.08 mmol, 1.82 equiv) to the mixture. 3. Stir at 120°C for 2 hours. 4. LCMS shows that compound 4-2 was formed. 5. Adjust the pH of the mixture to 7-8 with saturated NaHCO3. 6. The mixture is extracted with EtOAc (30 mL x 3). 7. Wash the organic layer with brine (15 mL x 2). 8. Concentrate in vacuo to remove EtOAc. 9. Compound 4-2 is obtained as a yellow solid (0.40 g, crude).

[0253] General procedure for the preparation of compound 3 The procedure for the preparation of compound 3 is outlined in FIG.

[0254] Compound 4-2 (0.90 g, 1.54 mmol, 1.00 equiv) was dissolved in HCl / dioxane (4 M, 9.00 mL, 23.3 equiv). 1. Stir at 25°C for 3 hours. 2. LCMS (product Rt=0.602 min) shows that compound 3 was formed. 3. Add saturated NaHCO3 (10.0 mL) to the solution pH=8. 4. Extract with EtOAc (50.0 mL x 2), separate the organic layer, dry over Na2SO4 and concentrate in vacuo. 5. Preparative HPLC (Column: Phenomenex Gemini-NX 80 * 40mm * 3um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 15%-35%, 8 min). 6. Compound 3 (0.08 g, 10.7% yield, 100% purity) is obtained as a yellow solid.

[0255] 1 H NMR: 400MHz DMSO-d6 δ 8.84(brs,1H),7.84(d,J=8.0Hz,1H),7.72(s,1H),7.56(d,J=7.2Hz),7.33(t,J=8Hz,1H) ,3.91(brs,1H),3.90(s,3H),2.99(s,2H),2.53-2.50(m,1H),1.85(s,1H),1.62(brs,1H).

[0256] General Procedure for the Preparation of Intermediates 7a-10

[0257] [ka] 1. Compound 7a_1 (5.72 g, 26.3 mmol, 1.50 equiv.) is added to a flask charged with DCE (100 mL). 2. Add AlCl3 (2.34 g, 17.5 mmol, 961 μL, 1.00 equiv) to the mixture, which will turn yellow in color. 3. Stir at 80°C for 0.5 hours. 4. Compound 3C (2.50 g, 17.5 mmol, 1.00 equiv) is added to the mixture, which turns orange-red in color. 5. Stir at 80°C for 12 hours. 6. LCMS shows that compound 7a_10 is formed. 7. Add the mixture to an ice-water mixture (200 mL). 8. The mixture is extracted with EtOAc (200 mL x 3). 9. Wash the organic layer with brine (100 mL x 2). 10. Concentrate in vacuo to remove EtOAc. 11. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 1 / 1). 12. Compound 7a_10 (1.60 g, 28.2% yield) is obtained as a yellow solid.

[0258] 1H NMR: 400MHz DMSO-d6 δ 9.26(s,1H),8.03(s,1H),7.91(d,J=8.0Hz,1H),7.73(d,J=8.0Hz,1H),7.41(t,J=8Hz,1H).

[0259] General procedure for the preparation of intermediate 4-3

[0260] [ka] 1. Add tert-butyl compound 3 (399 mg, 1.47 mmol, 0.91 equiv.) charged with i-PrOH (7.00 mL) to a microwave vial. 2. Add TsOH (504 mg, 2.93 mmol, 1.82 equiv) to the mixture. 3. Stir at 120°C for 2 hours. 4. LCMS (product Rt=0.857 min) shows that compound 4-3 was formed. 5. Adjust the pH of the mixture to 7-8 with saturated NaHCO3. 6. The mixture is extracted with EtOAc (100 mL x 3). 7. Wash the organic layer with brine (100 mL x 2). 8. Concentrate the organics in vacuo to remove EtOAc. 9. Compound 4-3 is obtained as a white solid (0.6 g, crude).

[0261] General Procedure for the Preparation of Compound 4 and Compounds 32-35 The procedure for the preparation of compound 4 and compounds 32-35 is shown schematically in Figure 20 using compound 4 as an example. 1. Dissolve compound 4-3 (0.60 g, 1.13 mmol, 1.00 equiv) in HCl / dioxane (4 M, 4.20 mL, 14.9 equiv). 2. Stir at 25°C for 2 hours. 3. LCMS (product Rt=0.348 min) shows that compound 4 was formed. 4. Add saturated NaHCO3 (50.0 mL) to the solution pH=8. 5. Extract with EtOAc (100.0 mL x 2), separate the organic layer, dry over Na2SO4 and concentrate in vacuo. 6. Preparative HPLC (Column: Phenomenex Gemini-NX 80 * 40mm * 3um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 20%-50%, 8 min).

[0262] Compound 4 (0.05 g, 10.0% yield, 98.3% pure) is obtained as a yellow solid. Note that compounds 32-35 follow the same procedure as described above for compound 4, except that intermediate 1A (in the preparation of intermediate 2->intermediate 3->intermediate 4-3) is replaced with the following compound:

[0263] [ka] (Compound 32, 0.03 g, 4% yield, 96.5% purity obtained as a yellow solid);

[0264] [ka] (Compound 33, 0.05 g, 5% yield, 99.6% purity obtained as a yellow solid);

[0265] [ka] (Compound 34, 0.03 g, 2% yield, 97% purity obtained as a yellow solid); and

[0266] [ka] (Compound 35, 0.03 g, 3% yield, 98.5% purity obtained as a yellow solid)

[0267] Compounds 32–35 can be further separated into their isomeric portions by supercritical fluid chromatography (SFC). For example, compound 32 was separated into its enantiomers by SFC using a mobile phase consisting of ethanol / DEA / CO2 and a YMC SA(IA) column (5 μm particle size). The preparation conditions consisted of a flow rate of 3.5 mL / min at 40 °C, and the compounds were detected at 272 nm. LC / MS analysis revealed that the enantiomeric excess of both enantiomers was greater than 99% ee. The fractions containing the separated enantiomers were evaporated to obtain the final compounds as solids.

[0268] 1H NMR: 400MHz DMSO-d6 Compound 4:δ 8.81(brs,1H),8.60(d,J=4.0Hz,1H),8.08(s,1H),7.73(s,1H),7.32(brs,1H),6.95(d,J=4.0Hz,1H),3.99 -3.94(m,1H),3.79(s,1H),3.01(d,J=8.0Hz,1H),2.57-2.54(m,2H),1.90-1.87(m,2H),1.71-1.68(m,2H).

[0269] Compound 32: δ 8.82(br s,1H),8.77(s,1H),8.09(s,1H),7.74(m,2H),7.34(br s,1H),6.96(s,1H),4.17(m,1H),3.79(s,3H),2.80(m,2H),1.75-1.87(dd,2H),1.51-1.61(m,3H),1.10(s,3H),1.06(s,3H).

[0270] Compound 33: δ 8.81(br s,1H),8.60(s,1H),8.09(s,1H),7.75(m,2H),7.33(br s,1H),6.96(s,1H),3.90(m,1H),3.79(s,3H),3.12(d,1H),2.84(d,1H) ,2.65(t,1H),2.44(t,1H),2.01(m,1H),1.70-1.81(m,2H),1.45(m,1H).

[0271] Compound 34:δ 8.56(m,3H),8.07(m,1H),7.70(m,2H),7.35(t,1H),6.94(m,1H),3.99(m,1H),3.82( s, 3H), 3.05 (m, 2H), 2.66 (m, 2H), 1.93 (t, 2H), 1.68 (m, 1H), 1.36 (m, 1H), 1.05 (d, 3H).

[0272] Compound 35: δ 8.55(br s,3H),8.05(s,1H),7.64-7.71(m,2H),7.33(m,1H),6.93(br s,1H),4.16(br s,1H),3.83(br s,3H),3.15(br s,1H),2.80(m,2H),2.65(m,1H),2.21(br s,1H),1.97(m,1H),1.74(m,1H),0.71(d,3H).

[0273] General procedure for the preparation of compounds 8-17 The procedure for the preparation of compounds 8-17 follows that described above for compound 4 with the addition of an additional alkylation step as shown in FIG. 1. A small molecule (i.e., any of compounds 2-4, 21, 22, or 24) with a free NH (1.00 equiv.) is added to a flask charged with DMF (3 mL). 2. Triethylamine (2.0 equiv.) is added to the mixture. 3. Add alkyl bromide or alkyl iodide (1.00-2.00 equivalents) to the mixture. 4. Stir at 20°C for 18 hours. 5. LCMS shows that the starting material has been consumed and the reaction is complete. 6. Add 30% aqueous ammonia (3 drops) to quench the excess alkyl halide. 7. The reaction mixture is concentrated in vacuo to remove the DMF. 8. The residue was purified by column chromatography (SiO2, heptane / ethyl acetate = 100 / 0 to 0 / 100, followed by DCM / MeOH = 100 / 0 to 60 / 40). 9. Compounds 8~17 were obtained as yellow solids.

[0274] 1H NMR: 500MHz DMF-d7 Compound 8: δ 1.94–2.1 (m, 6H), 2.24 (s, 3H), 2.89 (d, J = 10.7 Hz, 2H), 3.89 (s, 3H), 3.96 (dd, J = 11.1, 5.4 Hz, 1H), 7.02 (d, J = 3.6 Hz, 1H), 7.44 (s, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.89 (s, 1H), 8.18 (dd, J = 3.6, 2.2 Hz, 1H), 8.63 (d, J = 4.3 Hz, 1H).

[0275] Compound 9: δ 1.10 (s, 6H), 1.95–2.5 (m, 6H), 3.06 (bs, 2H), 3.89 (s, 3H), 4.03 (m, 1H), 7.03 (d, J = 3.6 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H), 8.16–8.21 (m, 1H), 8.64 (d, J = 4.3 Hz, 1H).

[0276] Compound 10: δ 0.91 (t, J = 7.4 Hz, 3H), 1.52 (s, 2H), 2.04 (s, 6H), 2.32 (s, 2H), 2.99 (s, 2H), 3.89 (s, 3H), 4.01 (s, 1H), 7.03 (d, J = 3.6 Hz, 1H), 7.45 (s, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H), 8.17–8.21 (m, 1H), 8.64 (d, J = 4.2 Hz, 1H).

[0277] Compound 11: δ 1.05 (t, J = 7.2 Hz, 3H), 1.93–2.09 (m, 6H), 2.38 (q, J = 7.2 Hz, 2H), 3.01 (d, J = 10.4 Hz, 2H), 3.89 (s, 3H), 3.98 (dt, J = 10.8, 5.8 Hz, 1H), 7.02 (d, J = 3.7 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.89 (s, 1H), 8.18 (dd, J = 3.6, 2.2 Hz, 1H), 8.63 (d, J = 4.3 Hz, 1H).

[0278] Compound 12: δ 1.99 (s, 2H), 2.29–2.49 (m, 4H), 3.00–3.27 (m, 4H), 3.64 (m, 4H), 3.90 (s, 3H), 4.39 (m, 1H), 7.03 (s, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.78 (d, J = 7.3 Hz, 1H), 7.95 (s, 1H), 8.19 (s, 1H), 8.64 (d, J = 4.2 Hz, 1H).

[0279] Compound 13: δ 1.29 (t, J = 7.0 Hz, 3H), 1.91–2.14 (m, 6H), 2.23 (s, 3H), 2.88 (d, J = 11.3 Hz, 2H), 3.95 (m, 1H), 4.24 (q, J = 7.0 Hz, 2H), 7.03 (d, J = 3.6 Hz, 1H), 7.44 (s, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H), 8.18–8.2 (m, 1H), 8.64 (d, J = 4.2 Hz, 1H).

[0280] Compound 14: δ 1.95-2.07 (m, 6H), 2.22 (s, 3H), 2.87 (d, J = 10.7 Hz, 2H), 3.90 (s, 3H), 3.97 (s, 1H), 7.03 (d, J = 3.6 Hz, 1H), 7.63 (dd, J = 8.1, 1.4 Hz, 1H), 7.91 (d, J = 8.1 Hz, 2H), 8.18 (s, 1H), 8.62 (d, J = 4.4 Hz, 1H).

[0281] Compound 15: δ 1.29 (t, J = 7.0 Hz, 3H), 1.90–2.05 (m, 6H), 2.22 (s, 3H), 2.87 (d, J = 2.8 Hz, 2H), 4.01 (m, 1H), 4.24 (q, J = 7.0 Hz, 2H), 7.02 (s, 1H), 7.12 (s, 1H), 7.94 (s, 1H), 8.26 (s, 1H), 8.47 (s, 1H), 8.57 (s, 1H).

[0282] Compound 16:δ 2.19(s,2H),2.40(s,4H),2.95-3.17(m,6H),3.77(bs,1H),3.83-3.94(m,6H),4.23(m,1H),4.48(m,1H),7.41(t, J=7.8Hz,2H),7.66(d,J=7.3Hz,2H),7.98(d,J=8.2Hz,2H),8.04(m,2H),8.77(s,2H),9.20(bs,1H),9.27(bs,1H).

[0283] Compound 17:δ 2.11(s,3H),2.57(m,6H),3.17(bs,2H),3.89(s,3H),4.08(m,1H),7.42(t,J=7.8Hz,1H), 7.69(d,J=7.3Hz,1H),7.96(s,1H),7.97(dd,J=8.3,1.0Hz,1H),8.33(s,1H),8.49(s,1H).

[0284] Procedure for the preparation of compound 18 The procedure for the preparation of compound 18 is described below and shown schematically in FIG. 1. Small molecule compound 4 (1.00 equiv.) with a free NH is added to a flask charged with DMF (3 mL). 2. Acetyl chloride (1.05 eq) is added to the mixture. 3. Stir at 20°C for 18 hours. 4. LCMS shows that the starting material has been consumed and the reaction is complete. 5. The reaction mixture is concentrated in vacuo to remove the DMF. 6. The residue was purified by column chromatography (SiO2, heptane / ethyl acetate = 100 / 0 to 0 / 100, followed by DCM / MeOH = 100 / 0 to 60 / 40). 7. Acetylated molecular compound 18 is obtained as a yellow solid.

[0285] 1H NMR: 500MHz DMF-d7 Compound 18:δ 1.80(qd,J=12.3,4.5Hz,1H),1.94(qd,J=12.2,11.5,3.8Hz,1H),2.05(m,2H),2.10(s,3H ),2.72-2.8(m,1H),3.27(td,J=13.8,13.1,2.8Hz,1H),3.89(s,3H),4.03(d,J=13.9Hz,1 H),4.30(tt,J=11.3,4.1Hz,1H),4.58(d,J=13.2Hz,1H),7.02(d,J=3.6Hz,1H),7.45(d,J =7.6Hz,1H),7.78(d,J=7.4Hz,1H),7.93(s,1H),8.17-8.2(m,1H),8.64(d,J=4.2Hz,1H).

[0286] General Procedure for the Preparation of Intermediate 3b-11

[0287] [ka] 1. Compound 3b_6 (6.81 g, 47.9 mmol, 1.00 equiv.) is added to a flask charged with THF (300 mL). 2. Add iodine(methyl)magnesium (3M, 15.97 mL, 1.00 equiv.) to the mixture. 3. Stir at 0°C for 30 minutes. 4. Compound 3C (8.00 g, 47.9 mmol, 1.00 equiv.) is added to the mixture. 5. Stir at 70°C for 15 hours. 6. TLC (petroleum ether:ethyl acetate=3:1, Rf=0.60) showed that compound 3b_6 was consumed and the reaction was complete. 7. Wash the residue with saturated NH4Cl until pH=7-8. 8. The mixture is extracted with EtOAc (300 mL x 3). 9. Wash the organic layer with brine (200 mL x 2). 10. Concentrate the organic layer in vacuo to remove EtOAc. 11. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 30 / 1 to 3 / 1). 12. Compound 3b_11 (5.00 g, 19.1% yield, 50% purity) is obtained as a yellow solid.

[0288] General procedure for the preparation of intermediate 6

[0289] [ka] 1. Compound 5 (1.00 g, 6.36 mmol, 1.00 equiv.) and compound 1A (2.92 g, 8.21 mmol, 1.29 equiv.) are added to a flask charged with DMF (7.00 mL). 2. Add Cs2CO3 (3.09 g, 9.48 mmol, 1.49 equiv.) to the mixture. 3. Degas with N2 three times. 4. Stir at 120°C for 2 hours. 5. TLC (petroleum ether / ethyl acetate=3 / 1, product Rf=0.42) shows that compound 6 was formed. 6. Adjust the pH of the mixture to 7-8 with HCl (1M). 7. Extract with a mixture of DCM (50 mL x 3). 8. Concentrate in vacuo to remove DCM. 9. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 15 / 1 to 2 / 1). 10. Compound 6 (1.50 g, 69.2% yield) is obtained as a yellow solid.

[0290] 1 H NMR: 400MHz DMSO-d6 δ8.73(s,1H),4.32-4.26(m,2H),4.06-4.00(m,2H),2.04-1.98(m,2H),1.77-1.76(m,2H),1.39(s,9H),1.35-1.33(m,3H).

[0291] General procedure for the preparation of intermediate 7

[0292] [ka] 1. Compound 6 (1.00 g, 2.94 mmol, 1.00 equiv) is added to a flask charged with EtOAc (5 mL). 2. Add Pd / C (0.10 g, 10% purity, 1.00 equivalents) to the mixture. 3. Add H2 (15 Psi) to the mixture. 4. Stir at 25°C for 2 hours. 5. TLC (petroleum ether:ethyl acetate=2:1, Rf=0.47) showed that compound 6 was consumed and compound 7 was formed. 6. Filter through Celite. 7. Wash the filter cake with EtOAc. 8. Concentrate the filtrate in vacuo to remove EtOAc. 9. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 15 / 1 to 2 / 1). 10. Compound 7 (0.77 g, 84.4% yield) is obtained as a pale yellow oil.

[0293] General procedure for the preparation of intermediate 8

[0294] [ka] 1. Compound 7 (0.30 g, 966 umol, 1.00 equiv.) and compound 7a (261 mg, 879 umol, 0.91 equiv.) charged with i-PrOH (7 mL) are added to a microwave vial. 2. Add TsOH (302 mg, 1.76 mmol, 1.82 equiv) to the mixture. 3. Stir at 120°C for 2 hours. 4. LCMS (ET37912-42-p1a) shows that compound 8 was formed. 5. Adjust the pH of the mixture to 7-8 with saturated NaHCO3. 6. The mixture is extracted with EtOAc (60 mL x 3). 7. Wash the organic layer with brine (30 mL x 2). 8. Concentrate the organics in vacuo to remove EtOAc. 9. Compound 8 is obtained as a yellow solid (0.3 g, crude).

[0295] General procedure for the preparation of compound 7 The procedure for the preparation of compound 7 is outlined in FIG. 1. Dissolve compound 8 (0.50 g, 874 umol, 1.00 equiv) in HCl / dioxane (4 M, 5.00 mL, 22.8 equiv). 2. Stir at 20°C for 3 hours. 3. TLC (petroleum ether / ethyl acetate=5 / 1, product Rf=0.1) shows that compound 7 was formed. 4. Add saturated NaHCO3 (50.0 mL) to the solution pH=8. 5. Extract with EtOAc (50.0 mL x 2), separate the organic layer, dry over Na2SO4 and concentrate in vacuo. 6. The crude product was triturated with EtOAc (100 mL) at 25° C. for 30 minutes. 7. Filter and collect the cake. 8. Compound 7 (0.068 g, 16.0% yield) is obtained as a white solid.

[0296] 1H NMR: 400MHz DMSO-d6 δ9.03-8.88(s,1H),8.60(s,1H),8.52-7.96(s,1H),7.85(s,1H),7.74(s,1H),7.45-7.43(s,1H),7.10-6.96(m,2H) ,4.30(s,2H),3.85(s,1H),3.02-2.99(m,2H),2.66-2.55(m,2H),1.88(m,2H),1.70-1.67(m,2H),1.26-1.17(m,3H).

[0297] General Procedure for the Preparation of Intermediate A 3M MgICH3 (1.781 mL, 5.34 mmol) was added to a yellow solution of 1J-indole-4-carbonitrile (0.760 g, 5.34 mmol) in TFH (30 mL). The yellow suspension turned to a white solid, which formed the indole Mg complex. After 15 min, 2,4,5-trichloropyrimidine (1 g, 5.34 mmol) was added at 0 °C, and the mixture was allowed to warm to room temperature. LCMS showed little product. The mixture was heated to 70 °C, and the reaction proceeded for 2 h. After stirring overnight, the reaction was complete. The mass of the product was [M+1]: 288.9 with a retention time of RT = 3.05 min. The reaction was maintained at 70 °C overnight. The reaction did not go to completion. The solid was filtered off to give the crude product, 3-(2,5-dichloropyrimidin-4-yl)-1H-indole-4-carbonitrile (0.546 g, 35.0%). 25 mg of the crude compound was purified by Gilson chromatography using a UV lamp at 222 nm and a mobile phase of 25-80% CAN in 0.1% TFA in water. 17 mg of the solid product was the TFA salt. LCMS and NMR confirmed the target compound. 1H NMR (300 MHz, DMSO-d6): δ 6.94 (d, J = -3.58 Hz, 1H), 7.32-7.53 (m, 1H), 7.72 (d, J = 7.54 Hz, 1H), 8.09 (dd, J = 5.93, 2.35 Hz, 2H), 9.09 (s, 1H).

[0298] Example 3. Cells and culture methods All stable cell lines derived from HeLa and Huh7 cells were maintained and cultured in T75 TC flasks (Sarstedt, 83.3911.002) in Dulbecco's modified Eagle's medium (DMEM) containing high glucose, GlutaMAX (Gibco, catalog no. 31966-021), 10% fetal bovine serum (Gibco, catalog no. 10270-106), and 1% penicillin-streptomycin (Gibco, catalog no. 15140-122) at 37°C, 5% CO2, and 95% humidity. Cells were detached (trypsin 0.25% EDTA, 10 min) and passaged twice weekly. HEK293, HeLa, HepG2, Huh7, and U2OS mCherry-GAL9 cell lines were maintained in DMEM, NCI-H358 mCherry-GAL9 was maintained in RPMI (Gibco: 32430-027), and SH-SY5Y mCherry-GAL9 was maintained in DMEM / F12 (Gibco: 32430-027). All growth media were supplemented with 10% FBS and 1 μg / ml puromycin (Gibco: A11138-03) to maintain reporter expression. All cells were routinely tested negative for mycoplasma.

[0299] Example 4. Splice switching activity assay Example 4A Splice switching activity assay in HeLa Luc705 and Huh7 Luc705 cells Using HeLa Luc705 and Huh7 Luc705 cells, the SMCs generated in Example 2 were screened and characterized for their ability to increase the levels of SSO-induced splice variants by simultaneous treatment of cells with SSO and SMCs. The Luc705 reporter construct was used to quantify luciferase protein produced as a result of functional SSO delivery. (Kang et al. (1998) Biochemistry, 37(18):6235-6239; Rocha et al. (2016) Four Novel Splice-Switch Reporter Cell Lines: Distinct Impact of Oligonucleotide Chemistry and Delivery Vector on Biological Activity. Nucleic Acid Ther. 26(6):381) Briefly, cells were stably transfected with a plasmid containing a luciferase coding sequence interrupted by the insertion of intron 2 from β-globin pre-mRNA, which contains a cryptic splice site. Unless the aberrant slice sites were masked by antisense oligonucleotides, luciferase pre-mRNA was improperly processed.

[0300] UNC2383 is a small molecule compound described by Wang et al. (2017) ACS Chem. Biol. 12(8):1999-2007) that enhances the pharmacological efficacy of antisense and splice-switching oligonucleotides.

[0301] HeLa Luc705 and Huh7 Luc705 cells were seeded at 10,000 cells / well in 96-well plates in growth medium containing 1 μM Luc705-SSO and incubated to allow adhesion and oligo-internalization. After 24 hours, 10 μl of SMC diluted in growth medium (DMEM + 10% FBS) was added to each well. After 2 hours, the medium was removed from the wells and replaced with fresh growth medium for 4 hours to allow translation of the mutant proteins. The medium was then removed, and the cells were lysed with 0.1% Triton-PBS.

[0302] To detect luciferase activity, 30 μL of cell lysate was transferred to a white-walled 96-well plate. After automatic injection of 25 μL of luciferin substrate per well, the luciferase intensity of each well was measured using a GloMax® 96 Microplate Luminometer machine (Promega) (Firefly Luciferase Assay System: Promega). Photon measurements were acquired for 10 seconds, starting 2 seconds after injection.

[0303] As shown in Figure 2A, AZ4800 (compound 2, Figure 1B) induced a substantially higher increase in SSO activity than UNC2383 (Figure 1A).

[0304] Select compounds were tested essentially as described above with cotreatment of 1 μM SSO and 100 nM SSO to identify compounds that were effective at submicromolar SSO concentrations, as shown in Figure 2B. Most compounds, with the exception of AZ4800 (compound 2, Figure 1B) and AZ2467 (compound 6, Figure 1C), did not induce an increase in activity.

[0305] To determine whether AZ4800 (Compound 2, Figure 1B) and SSO must be simultaneously present in the cell culture medium for Compound 2 to exert its synergistic effect, a treatment plan was modified from the UNC2383 treatment plan detailed by Wang et al. (2017) ACS Chem. Biol. 12(8):1999-2007 to include a 24-hour pretreatment with SSO, followed by a 2-hour addition of Compound 2, followed by removal of SSO and Compound 2, and finally a 4-hour protein translation period (Figure 10). When the pretreatment step was extended to 48 hours, the increase in SSO activity was more pronounced (Figures 12A and 12B). Importantly, no activity was observed when SSO was only pretreated and then removed when the compound was added (Figure 11A). Importantly, significant activity was observed even without the pretreatment step (Figures 13A and 13B).

[0306] Using the Luc705 model, functional concentration and time curves were generated for both compounds 2 (AZ4800) and 6 (AZ2467). See Figures 11B and 11C. Co-treatment with compound 2 (AZ4800) increased SSO activity up to 5 μM, resulting in a 100-fold increase in activity, which then plateaued after 2 hours. Compound 6 (AZ2467) produced a 200-fold increase in SSO activity at 10 μM for up to 6 hours.

[0307] Example 4B Splice switching activity assay in HeLa Luc705 cells HeLa_Luc705 cells were seeded at 10,000 cells / well in 96-well plates in growth medium containing 2 μM Luc705-SSO or 1 μM Luc705-SSO. Various SMCs were diluted directly into the prepared growth medium. After 24 h to allow for adhesion and SSO internalization, 10 μl of diluted SMCs were added to each well at the indicated concentrations. For positive controls, SSOs were complexed with Lipofectamine 2000 for 30 min at room temperature, and cells were treated at a final concentration of 200 nM. ON concentrations above 200 nM require LF2000 to be used above the toxic threshold of the cells. After 24 h of treatment, cells were lysed with 0.1% Triton X-100 (Sigma-Aldrich, Cat. No. X100) in 1x PBS (Gibco, Cat. No. 10010023).

[0308] For luciferase activity detection, 30 μL of cell lysate was transferred to a white-walled plate. Following automated injection of 25 μL of luciferin substrate, luciferase activity in each well was immediately measured using a luminometer according to the Promega Firefly Luciferase Assay System. The photon measurement timing parameters were optimized for this procedure.

[0309] As shown in Figure 23A, select compounds were tested essentially as described above with 2 μM SSO in HeLa705 cells to identify compounds effective in SSO. Most compounds, with the exception of AZ3325 (compound 7) and AZ3327 (compound 4), did not induce an increase in SSO activity. As shown in Figure 23B, higher concentrations of AZ3327 (compound 4) resulted in an increase in SSO activity relative to 1 μM naked SSO in HeLa705 cells. As shown in Figure 24, select compounds were tested essentially as described above with 2 μM Luc705 SSO in HeLa705 cells to identify compounds effective in SSO. With the exception of AZ4374 (compound 21), AZ2862 (compound 8), and AZ3327 (compound 4), no increase in SSO activity was induced. As shown in Figure 26, select compounds were tested essentially as described above with 2 μM Luc705 SSO in HeLa705 cells to identify compounds effective in SSO. Several compounds, including AZ4800 (compound 2), AZ3327 (compound 4), AZ4374 (compound 21), AZ2862 (compound 8), and AZ3325 (compound 7), induced increased SSO activity. Figure 49 shows the results of combining AZ5219 (compound 32) with 1 μM Luc705 SSO in HeLa705 cells at various time intervals.

[0310] Example 4C Splice switching activity assay in U2OS_Luc705 and N2A_Luc705 cells U2OS_Luc705 and N2A_Luc705 cells were seeded at 10,000 cells / well in 96-well plates in growth medium containing 1 μM Luc705-SSO. Various SMCs were diluted directly into the prepared growth medium. After 24 h to allow for adhesion and SSO internalization, 10 μl of diluted SMCs were added to each well at the indicated concentrations. For positive controls, SSO was complexed with Lipofectamine 2000 for 30 min at room temperature, and cells were treated at a final concentration of 200 nM. ON concentrations above 200 nM require LF2000 to be used above the cytotoxic threshold of the cells. After 24 h of treatment, cells were lysed with 0.1% Triton X-100 (Sigma-Aldrich, Cat. No. X100) in 1x PBS (Gibco, Cat. No. 10010023).

[0311] For luciferase activity detection, 30 μL of cell lysate was transferred to a white-walled plate. Following automated injection of 25 μL of luciferin substrate, luciferase activity in each well was immediately measured using a luminometer according to the Promega Firefly Luciferase Assay System. The photon measurement timing parameters were optimized for this procedure.

[0312] As shown in Figure 25A, 5 μM of select compounds were tested essentially as described above with 1 μM Luc705 SSO in U2OS_Luc705 cells to identify compounds effective in SSO. AZ4374 (compound 21), AZ4376 (compound 22), compounds AZ5738 (compound 25), AZ5739 (compound 20), and AZ2862 (compound 8) induced increased SSO activity compared to oligo alone. As shown in Figure 25B, 5 μM of select compounds were tested essentially as described above with 1 μM Luc705 SSO in N2A705 cells to identify compounds effective in SSO. AZ4374 (compound 21), AZ4376 (compound 22), and AZ2862 (compound 8) induced increased SSO activity compared to oligo alone.

[0313] Example 4D Splice switching activity assay in HeLa Luc705 cells, U2OS_Luc705 cells, and N2A_Luc705 cells HeLa_Luc705, U2OS_Luc705, and N2A_Luc705 cells were seeded at 10,000 cells / well in 96-well plates in growth medium containing 1 μM Luc705-SSO. Various SMCs were diluted directly into the prepared growth medium. After 24 h to allow for adhesion and SSO internalization, 10 μl of diluted SMCs were added to each well at the indicated concentrations. For positive controls, SSO was complexed with Lipofectamine 2000 for 30 min at room temperature, and cells were treated at a final concentration of 200 nM. ON concentrations above 200 nM require LF2000 to be used above the toxic threshold of the cells. After 24 h of treatment, cells were lysed with 0.1% Triton X-100 (Sigma-Aldrich, Cat. No. X100) in 1x PBS (Gibco, Cat. No. 10010023).

[0314] For luciferase activity detection, 30 μL of cell lysate was transferred to a white-walled plate. Following automated injection of 25 μL of luciferin substrate, luciferase activity in each well was immediately measured using a luminometer according to the Promega Firefly Luciferase Assay System. The photon measurement timing parameters were optimized for this procedure.

[0315] As shown in Figure 27, 2.5 μM AZ3327 (compound 4) was tested essentially as described above in conjunction with 1 μM SSO in three cell types (HeLa_Luc705, U2OS_Luc705, and N2A_Luc705 cells) to demonstrate enhanced activity of the oligo compared to the oligo alone. We can conclude that the oligo-enhanced activity exhibited by these compounds is not necessarily cell type dependent.

[0316] Example 5. Reverse transcription polymerase chain reaction (RT-PCR) The percentage of corrected luciferase mRNA was quantified using a previously validated RT-PCR protocol (Saher et al. (2019) Pharmaceuticals. 11(12):666). Total RNA was extracted from HeLa Luc705 and Huh7 Luc705 cells using TRI Reagent (Sigma-Aldrich) according to the manufacturer's instructions.

[0317] Three nanograms of isolated RNA was used for RT-PCR reactions using the ONE STEP RT-PCR kit (QIAGEN). The total reaction volume was 20 μL, and the primer sequences used were as follows: Fwd-5'-TTGATATGTGGATTTCGAGTCGTC-3' (SEQ ID NO: 2) Rev-5'-TGTCAATCAGAGTGCTTTTGGCG-3' (SEQ ID NO: 3)

[0318] The RT-PCR program was as follows: a reverse transcription step at 55°C for 35 min, followed by 95°C for 15 min, immediately followed by 30 cycles of PCR (94°C for 30 s, then 55°C for 30 s, then 72°C for 30 s) and a final extension at 72°C for 10 min. PCR products were analyzed using 1% agarose gels in 0.5x TAE buffer and visualized by SYBR Gold (Invitrogen, Molecular products) staining.

[0319] A Versadoc imaging system (BioRad, Hercules, CA, USA) equipped with a cooled CCD camera was used to analyze the gel. Band intensities were analyzed with Quantity One software (BioRad, Hercules, CA, USA). The percentage of correction was calculated using this formula: (corrected RNA band intensity) * Calculations were made using 100 / (band intensity of corrected RNA + band intensity of uncorrected RNA).

[0320] RT-PCR confirmed that the increased functional luciferase production was the result of antisense SSO activity against the target Luc705 pre-mRNA. Quantification of mRNA splice variants revealed an increase in the proportion of alternatively spliced ​​mRNA (Figure 9). Notably, treatment with AZ4800 (compound 2) and SSO induced higher levels of splice switching in both HeLa and Huh7 cells than transfection with SSO using Lipofectamine 2000, a widely used transfection reagent.

[0321] Example 6. mCherry-GAL9 Recruitment Assay All compounds were subjected to the mCherry-GAL9 recruitment assay. Under normal conditions, mCherry-GAL9 is uniformly dispersed throughout the cytosol. Upon detection of an endosomal leakage event, mCherry-GAL9 translocates to damaged endosomes (Du Rietz et al. (2020) Nat. Commun. 11:1809). The resulting distinct punctate localization pattern can be visualized and quantified.

[0322] HeLa and Huh7 cell lines stably expressing mCherry-GAL9 were generated as described (bioRxiv) (Munson, M. et al. (2021) Communications Biology, 4(211):1-14) and seeded at 3000 or 3500 cells / well, respectively, into 384-well CellCarrier Ultra plates (PerkinElmer: 6007558) 16 hours before experimental use.

[0323] Dose-response curves for selected compounds were generated by dispensing the indicated compounds into source plates (Greiner: 781280) containing growth medium using an Echo 655T acoustic dispenser (Labcyte). At the start of the experiment, medium containing the appropriate compound and dose was transferred to the plate using a liquid handling robot (Agilent Bravo).

[0324] At the assay endpoint, cells were washed 2x with PBS at room temperature and fixed in 4% PFA (VWR:9713.1000) for 15 min at room temperature. Cells were washed a further 3x with PBS, then PBS + 1 μg / ml Hoechst 33342 (ThermoFisher Scientific:H21492) was added for a minimum of 1 h before imaging.

[0325] Plates were imaged using a spinning disk confocal microscope (Yokogawa CV7000) equipped with a 20x objective (NA 0.75). Images were processed using Columbus image analysis software (PerkinElmer v2.9.0) to identify and quantitate cells and mCherry-GAL9 constructs. The resulting data were processed and normalized in Spotfire (Tibco v10.3) and plotted in Prism (Graphpad v8.0.1). Image panels were assembled using the FigureJ plugin for FIJI (Mutterer and Zinck (2013) J. Microsc. 252(1):89-91).

[0326] Cells were subjected to 10-point concentration dilutions (0-15 μM) for 2 h, then fixed and imaged (Figures 3 and 4). Images were analyzed, and curves were generated from both mCherry-GAL9 point quantification and cell viability, as determined by nuclear morphology (Figure 5). The GAL9 recruitment response supports the functional splice switching by SSO data, validates the mCherry-GAL9 assay as a powerful tool for screening endosomolytic compounds, and supports the hypothesis that AZ2467 (compound 6) and AZ4800 (compound 2) promote SSO activity by inducing endosome disruption. The GAL9 assay was performed again, this time in live cells, for 4 h with compounds selected for screening. Compounds were screened at 5 μM (Figures 6-8) or 0.3125-10 μM (Figures 35-38). Figures 39 and 47 provide results for selected compounds at various doses, from 0.3125 to 10 μM.

[0327] This assay revealed that GAL9 translocation was activated for several compounds within 4 hours. Compounds SMC5 (compound 2), A7 (compound 6), and A1 (compound 5) were selected for further characterization due to their efficacy at shorter time points.

[0328] HeLa and Huh7 cells were subjected to mCherry-GAL9 recruitment analysis after co-treatment with AZ4800 (compound 2) and 1 μM A488-SSO. After preloading with A488-SSO, A488-positive structures could be detected, likely reflecting endosomal localization. These A488-positive structures gradually disappeared over 2 h after treatment with AZ4800 (compound 2), indicating endosomal rupture and subsequent leakage of A488-SSO into the cytosol, where it was too diffuse to be detected (Figures 15A and 15B). This disruption occurred in a time- and concentration-dependent manner, consistent with the initial findings of functional AZ4800 (compound 2) (Figures 16A and 16B). Concomitantly, GAL9 relocalization resulted in the formation of mCherry-GAL9 puncta (Figures 17A and 17B). Quantification of GAL9 also revealed a time- and concentration-dependent response, supporting the hypothesis that A488-SSO endosomal escape occurs in a manner sufficient to induce GAL9 recruitment.

[0329] HeLa and Huh7 cells were further subjected to mCherry-GAL9 recruitment analysis after treatment with selected compounds and observed for endosomal rupture at various time points (1 h, 2 h, and 16 h). The GAL9 response results at different doses (1.25–5 μM) of selected compounds are shown in Figure 33 (Huh7 cells) and Figure 34 (HeLa cells). As shown in both Figures 33 and 34, AZ3327 (compound 4) provided the most potent response indicative of endosomal remodeling from 1 h after administration of AZ2862 (compound 8), demonstrating the second most potent response among the exemplified compounds. Figure 40 provides additional results comparing time (0.25–4 h), doses (0.31–10 μM) of selected compounds, and GAL9 responses in Huh7 cells. As shown in Figure 40, AZ3327 (compound 4) was the most potent, with a GAL9 response from 1.25 μM up to 15 min after administration. AZ2862 also potently induces GAL9+ remodeling from 2.5 μM+. Figures 41 and 48 provide further data showing the interplay between dose (0.31-10 μM) and GAL9 responses across different cell lines at 2 hours post-dose. Potent compounds such as AZ3327 (compound 4) demonstrate GAL9 responses across multiple cell lines from diverse backgrounds.

[0330] Example 7. Nanoparticle tracking analysis Many SSO transfection reagents act through complexation or association with SSOs. Nanoparticle tracking analysis (NTA) was performed to determine whether compound 2 induces aggregation of oligonucleotides in cell culture media.

[0331] SSOs were incubated in cell growth medium with and without Compound 2 for 2 hours at 37°C, mimicking the treatment regimen shown in Figure 10. Analysis was performed using a Nanosight NS500 instrument running NTA2.3 analysis software. A script was run to record 5 x 30-second videos in light scattering mode with a camera level of 13. The videos were then analyzed with a screen gain set to 10 and a detection threshold set to 7.

[0332] No significant differences in particle size were detected (Figures 14A and 14B).

[0333] Example 8. Improvement of MALAT1 activity by SMC cell culture N2A cells were cultured in Dulbecco's modified Eagle's medium (Gibco, 31966) containing 10% fetal bovine serum (Gibco, 10270) and maintained at 37°C in a humidified atmosphere containing 5% CO , 21% O .

[0334] Cell lysis, reverse transcription, and real-time polymerase chain reaction For gapmer knockdown analysis, 50,000 N2A cells were plated in a 24-well plate in a total volume of 500 μL of medium per well. After 24 hours, cells were treated with 2 μM MALAT1-targeting gapmer oligonucleotide (oligo only) having the sequence GM5CAttm5ctaatagm5cAGM5C (where m5c is 5-methylcytidine and uppercase letters are LNA nucleosides (SEQ ID NO: 4)), 2 μM MALAT1-targeting gapmer oligonucleotide simultaneously with 2 μM AZ3327 (2 μM AZ3327), or 200 nM lipofectamine-conjugated MALAT1 oligonucleotide (LF2000). All treatments were performed in biological triplicates. After 24 hours, the gapmer-containing medium was removed from the wells, and the cells were washed once with Dulbecco's phosphate-buffered saline (Gibco, 14040). RNA isolation was performed using a Promega Maxwell RSC RNA extraction system according to the manufacturer's instructions. RNA concentration was measured via Nanodrop, and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit according to the manufacturer's instructions (ThermoFisher Scientific, 4368814).

[0335] Real-time PCR reactions were performed on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) using the MALAT1 TaqMan assay (mouse Mm01227912, Applied Biosystems) according to the manufacturer's instructions. PCR reactions were performed in technical triplicates using the following primer and probe sequences: GAGGAATCAGATGAGGATATGGGA (forward sequence) (SEQ ID NO: 5), AAGCAGGCTGACTTGGTTGC (reverse sequence) (SEQ ID NO: 6), and TCGGTCTCTTCGACTAATCCCGCCAA (probe sequence) (SEQ ID NO: 7). Absolute mRNA expression levels for MALAT1 were calculated by linear regression fitting the PCR products to a standard curve. Expression values ​​were then normalized to the corresponding values ​​from the negative control (untreated cells) and reported as percentages.

[0336] As shown in Figure 28, the addition of 2 μM AZ3327 (compound 4) enhanced the knockdown of MALAT1 mRNA expression compared to the oligo alone.

[0337] Example 9. RNA silencing assay Peptidylpropyl isomerase B (PPIB) mRNA was used in the following assay. PPIB siRNA: Guide strand: 5'-UCACGAUGGAAUUUGCUGUU-3' (SEQ ID NO: 8) Passenger strand: 5'-CAGCAAAUUCCAUCGUGA-3' (SEQ ID NO: 9) In vitro silencing of peptidylpropyl isomerase B (PPIB) mRNA in hepatocytes by lipid-conjugated siRNA or siRNA co-administered with 2 μM of the endosomal release enhancer (ERE) compound, Compound 32. Primary human hepatocytes (PHH) were plated at 50,000 cells / well in 2D culture and cultured in serum-free medium. On day 1, cells were treated with (i) unconjugated siRNA ("duplex-PPIB"), (ii) cholesterol-conjugated siRNA ("Chol-PPIB"), (iii) siRNA conjugated with GalNAc ("GalNAc-PPIB"), and (iv) unconjugated siRNA co-administered with 2 μM of Compound 32 ("duplex-PPIB+ERE"). siRNA concentrations are shown in Table 4. Conditions were performed in triplicate. Medium was changed on day 2, and samples were collected for analysis on day 4. The results are shown in Figure 42.

[0338] [Table 5]

[0339] As shown in Figure 42, co-administration of siRNA with an ERE resulted in similar changes in target PPIB RNA expression as siRNA conjugated to cholesterol or GalNAc moieties. Co-administration of siRNA bearing an ERE and siRNA conjugated to cholesterol or GalNAc significantly reduced target PPIB RNA expression compared to unconjugated siRNA alone.

[0340] Example 10. Co-delivery of Cre recombinase and Compound 4 for in vitro editing Cre recombinase was co-administered with Compound 4 to evaluate the improvement of editing efficiency in an in vitro reporter system in which edited cells express GFP. Figure 43A shows the legend for the data shown in Figures 43B and 43C. Total cell numbers are shown as open bars (Figure 43A, left panel). Viable cells are shown as light gray shaded bars (Figure 43A, center panel). Edited cell numbers are shown as green shaded bars, and the percentage of edited cells is shown as a dotted line (Figure 43A, right panel).

[0341] Figure 43B shows the results of T-47D cells co-administered with 50 nM Cre protein and 0, 1, 2, or 3 μM Compound 4, with or without a medium change after 2 hours (left panel) or not (right panel). Medium change increased cell viability, as indicated by the difference between the left and right panels of Figures 43B and 43C. Furthermore, as shown in Figure 43B, co-administration of Compound 4 improved editing efficiency in T-47D cells in a dose-dependent manner. When Cre protein was co-administered with 2 μM or 3 μM Compound 4 under medium change conditions, and with 3 μM Compound 4 under medium non-change conditions, over 80% editing was observed. A similar trend was observed when the experiment was repeated in HeLa cells, as shown in Figure 43C.

[0342] Example 11. Co-delivery of Cre recombinase and Compound 4 for in vivo and ex vivo editing Cre recombinase (MGSSHHHHHHSSGLVPRGSHGGGSAAAMGTRLPKKKRKVSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPA EPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRV KDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD (SEQ ID NO: 10)) was co-administered with Compound 4 to evaluate improved editing efficacy in an in vivo reporter system in which edited mice express tdTomato. Floxed mice were administered 60 pmol of Cre recombinase and various concentrations of Compound 4 (2.5, 5.0, 7.5, 10.0, 12.5, and 15 μM) calculated in a volume of 40 μL of CSF via intracerebroventricular injection (ICV). Brain segments were analyzed by immunofluorescence staining 7 days after treatment.

[0343] Figure 44A shows coronal and sagittal brain segments, and Figure 44B shows lumbar spinal cord segments. Both Figure 44A and Figure 44B show increased tdTomato staining at higher concentrations of Compound 4, demonstrating that in vivo editing in the brain is dose-dependently enhanced by Compound 4. Furthermore, only mice treated with Compound 4 showed clear staining in lumbar sections of the spinal cord.

[0344] Figure 44C shows the dissection strategy and results, demonstrating that editing was primarily observed at the injection site. Figure 44D further confirms editing by IVIS® in vivo imaging and immunofluorescence staining.

[0345] The combination of Cre recombinase and Compound 4 was also tested in the ex vivo editing of anti-CD3 / 28 stimulated T cells. Cells were obtained from mice, then subjected to anti-CD3 / 28 stimulation, and then treated with 2.5 μM Compound 4 along with various concentrations of Cre recombinase (50, 100, 200, 500, and 1000 nM) on a U-plate without mixing (T1) or with mixing in a tube shaker during treatment (T2).

[0346] The percent viability and editing efficiency of rested, pre-activated, and post-activated cells are shown in Figures 45A-C, respectively. Figure 45C shows an editing efficiency of over 90% in activated T cells without affecting their proliferative capacity. Figure 45A shows that rested T cells were also highly edited, but proliferation rates were impaired due to the nature of the experiment.

[0347] Example 12. Co-delivery of a base editor and compound 4 The ABE8e base editor (Richter et al., Nature Biotechnol. 38:883-891 (2020)), which contains SpCas9 D10A nickase and a modified Tada adenosine deaminase, was co-administered with Compound 4 to evaluate the improved editing effect in an in vitro reporter system in which edited cells express GFP. Figure 46A shows a schematic diagram of the editing-based reporter system, which converts the TAA stop codon to a CAA codon, thereby enabling transcription of the GFP gene. The bar shading and lines indicating total cell number, live cells, and edited cells are the same as in Figure 43A.

[0348] HEK cells were co-administered with a fixed concentration (50 nM) of ABE8e and various concentrations of AZ3327 (compound 4). The results in Figure 46B show editing efficiencies of over 90% at compound 4 concentrations above 2.5 μM. The left, center, and right panels of Figure 46B show media replaced 1 hour, 2 hours, and 4 hours after co-administration, respectively.

[0349] HEK and N2a cells were co-treated with a fixed concentration (2.5 μM) of Compound 4 and various concentrations of ABE8e. The results are shown in Figure 46C (left panel: HEK; right panel: N2a) and demonstrate ABE8e dose-dependent editing efficiency, with ABE8e concentrations below 5 nM showing reduced efficiency.

Claims

1. A compound comprising a structure represented by Formula I: 【Chemistry 1】 (In the formula, Z １ and Z ２ one of which is N and the other is C, R １ But, Hydro, Halo, C １ -C ４ Alkyl, -OR ４ , —C(═O)NR ５ R ５ , -CO ２ R ６ or cyano, X is hydro, C １ -C ４ Alkyl, or -OR ２ and R ２ but hydro or C １ -C ４ is alkyl, R ３ But, Hydro, Halo, C １ -C ４ Alkyl, -(CH ２ ) ｙ OH, -OR ４ , —C(═O)NR ５ R ５ , -CO ２ R ６ or cyano, R ４ But C １ -C ４ is alkyl, Each R ５ are independently hydro or C １ -C ４ is alkyl, R ６ but hydro or C １ -C ４ is alkyl, R ７ , R ８ , R ９ , R １０ , R １１ are each independently CHR １２ , C.R. １２ R １７ or NR １３ and R １２ But Hydro, C １ -C ４ Alkyl, -OR １４ , or CO ２ R １５ and R １３ But Hydro, C １ -C ４ Alkyl, -(CH ２ ) ｙ OH, -OR １４ , -CO ２ R １５ , or -C(=O)R １６ and R １４ but hydro or C １ -C ４ is alkyl, R １５ but hydro or C １ -C ４ is alkyl, R １６ but hydro or C １ -C ４ is alkyl, R １７ but hydro or C １ -C ４ is alkyl, y is 0, 1, 2, or 3; wherein one or more of said alkyl is optionally substituted with one or more halo), or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 , wherein the compound comprises a structure represented by formula Ia: 【Chemistry 2】

3. The compound of claim 1 , wherein the compound comprises a structure represented by Formula Ib or Formula Ic: 【Transformation 3】

4. R １ is optionally substituted with cyano, halo, or one or more chloro or fluoro; １ -C ４ The compound of any one of claims 1 to 3, which is alkyl.

5. R １ The compound of any one of claims 1 to 3, wherein is selected from cyano, methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, fluoro, chloro, and bromo.

6. R １ The compound of any one of claims 1 to 3, wherein is cyano, bromo, chloro, fluoro, or trifluoromethyl.

7. R １ The compound of any one of claims 1 to 3, wherein is fluoro.

8. X is -OR ２ and R ２ The compound of any one of claims 1 to 7, wherein is methyl, ethyl, or isopropyl.

9. The compound of any one of claims 1 to 7, wherein X is hydro.

10. R ３ is optionally substituted with hydro; cyano; one or more chloro, fluoro, or hydroxy; １ -C ４ alkyl; or -OR ４ and R ４ But C １ -C ４ The compound of any one of claims 1 to 9, which is alkyl.

11. R ７ , R ８ , R １０ , and R １１ are each independently CHR １２ and R ９ But NR １３ 2. The compound of claim 1, wherein:

12. Each R １２ The compound of claim 11 , wherein is hydro.

13. R １３ But Hydro, C １ -C ４ Alkyl, —C(═O)R １６ , or -(CH ２ ) ｙ 13. The compound of any one of claims 2 to 12, wherein y is OH and y is 1, 2, or 3.

14. R １３ But -(CH ２ ) ３ 14. The compound of claim 13, wherein:

15. R １３ The compound of claim 13, wherein is methyl.

16. R １ is halo and X is -OR ２ and R ２ is ethyl or methyl, and R ３ is cyano and R １３ But Hydro, C １ -C ４ Alkyl, —C(═O)CH ３ , or -(CH ２ ) ３ 4. The compound of claim 2 or 3, wherein:

17. R １ is haloalkyl and X is -OR ２ and R ２ is ethyl or methyl, and R ３ is cyano and R １３ 4. The compound of claim 2 or 3, wherein is hydro or methyl.

18. R １ is halo and X is -OR ２ and R ２ is ethyl or methyl, and R ３ is methyl or methoxy, and R １３ The compound of claim 2 or 3, wherein is hydro.

19. R １ is halo and X is -OR ２ and R ２ But C １ -C ４ alkyl, and R ３ is hydro and R １３ The compound of claim 2 or 3, wherein is hydro.

20. R １ is haloalkyl and X is -OR ２ and R ２ is ethyl or methyl, and R ３ is hydro and R １３ 4. The compound of claim 2 or 3, wherein is hydro or methyl.

21. R １ is halo, X is hydro, and R ３ is cyano and R １３ The compound of claim 2 or 3, wherein is hydro.

22. R １ is cyano and X is -OR ２ and R ２ is ethyl or methyl, and R ３ is haloalkyl, and R １３ The compound of claim 2 or 3, wherein is hydro.

23. R １ is ethyl or methyl, and X is -OR ２ and R ２ is ethyl or methyl, and R ３ is hydro and R １３ The compound of claim 2 or 3, wherein is hydro.

24. R １ is halo and X is -OR ２ and R ２ is ethyl or methyl, and R ３ However, (CH ２ ) ｙ OH and R １３ 4. The compound of claim 2 or 3, wherein is hydro and y is 0, 1, 2, or 3.

25. R １ is fluoro and X is -OR ２ and R ２ is methyl, and R ３ is cyano and R １３ The compound of claim 2 or 3, wherein is hydro.

26. R １ is fluoro and X is -OR ２ and R ２ is ethyl, and R ３ is cyano and R １３ The compound according to claim 2 or 3, wherein is methyl.

27. Z １ is N and R １ is fluoro and X is -OR ２ and R ２ is methyl, and R ３ is cyano and R １３ is hydro and R ７ , R ７ , R ８ , and R １１ Each of ２ and R ８ But, CR １２ R １７ and R １２ and R １７ 10. The compound of claim 1, wherein each of is methyl.

28. The compound is any one of the following compounds 2-8 or 32: 【Chemistry 4】 【Transformation 5】 or a pharmaceutically acceptable salt thereof.

29. The compound is Compound 4: 【Transformation 6】 or a pharmaceutically acceptable salt thereof.

30. The compound is Compound 8: 【Transformation 7】 or a pharmaceutically acceptable salt thereof.

31. The compound is Compound 32: 【Transformation 8】 or a pharmaceutically acceptable salt thereof.

32. oligonucleotides and / or polypeptides, A compound according to any one of claims 1 to 31, and a pharmaceutically acceptable diluent or carrier.

33. 33. The pharmaceutical composition of claim 32, wherein the oligonucleotide is single-stranded.

34. 33. The pharmaceutical composition of claim 32, wherein the oligonucleotide is double-stranded.

35. The pharmaceutical composition of any one of claims 32 to 34, wherein the oligonucleotide comprises DNA.

36. The pharmaceutical composition of any one of claims 32 to 34, wherein the oligonucleotide comprises RNA.

37. 37. The pharmaceutical composition of any one of claims 32 to 36, wherein the oligonucleotide comprises from about 8 to about 30 nucleotides.

38. 38. The pharmaceutical composition of any one of claims 32 to 37, wherein the oligonucleotide is an antisense oligonucleotide (ASO), a splice-switching oligonucleotide (SSO), an interfering RNA (RNAi), a small interfering RNA (siRNA), a microRNA (miRNA), an antagomir, a decoy oligonucleotide, or a combination thereof.

39. The pharmaceutical composition of any one of claims 32 to 38, wherein the oligonucleotide comprises one or more modified nucleotides.

40. 40. The pharmaceutical composition of claim 39, wherein the one or more modified nucleotides comprise phosphodiester (PO), phosphorothioate (PS), 2'O-methyl (2'OMe), 2'O-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoramidate morpholino (PMO), locked nucleic acid (LNA), 2'-deoxy-2'-fluoro (2'-F), any other 2'-modified oligonucleotide, or a combination thereof.

41. 1. A method for introducing oligonucleotides and / or polypeptides into the nucleus and / or cytosol of a cell, comprising: (a) contacting the cell with the oligonucleotide and / or the polypeptide; (b) contacting the cell with a compound according to any one of claims 1 to 31, wherein the compound promotes entry of the oligonucleotide and / or the polypeptide into the nucleus and / or cytosol of the cell.

42. 42. The method of claim 41, wherein the oligonucleotide and / or the polypeptide are internalized by the cell via endocytosis and encapsulated in an endosome, and the compound promotes release of the oligonucleotide and / or the polypeptide from the endosome.

43. 42. The method of claim 41, wherein the oligonucleotide and / or the polypeptide is internalized by the cell via transient pore formation, and the compound promotes transient pore formation in the plasma membrane.

44. 44. The method of any one of claims 41 to 43, wherein the contacting of the cell with the oligonucleotide and / or the polypeptide of (a) is carried out in a composition, and the composition of (a) comprises from about 0.025 μM to about 20 μM of the oligonucleotide and / or the polypeptide.

45. 45. The method of claim 44, wherein the composition of (a) comprises about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide and / or the polypeptide.

46. 46. ​​The method of any one of claims 41 to 45, wherein the cell is contacted with a second composition comprising about 1 μM to about 20 μM of the compound.

47. 47. The method of claim 46, wherein the contacting of the cell with the compound of (b) is carried out in a composition, and the composition of (b) comprises about 1 μM to about 10 μM of the compound.

48. 48. The method of claim 47, wherein the composition of (b) comprises at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM of the compound, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM of the compound.

49. 49. The method of any one of claims 41 to 48, wherein the cell is contacted with the oligonucleotide and / or polypeptide of (a) and the compound of (b) at about the same time.

50. 49. The method of any one of claims 41 to 48, wherein the cell is contacted with the oligonucleotide and / or the polypeptide of (a) before being contacted with the compound of (b).

51. 51. The method of claim 50, wherein the cell is contacted with the oligonucleotide and / or the polypeptide of (a) up to about 48 hours before contacting the cell with the compound of (b).

52. 51. The method of claim 50, wherein the cell is contacted with the oligonucleotide and / or the polypeptide of (a) about 12 hours to about 48 hours before contacting the cell with the compound of (b).

53. 49. The method of any one of claims 41 to 48, wherein said contacting of (a) and said contacting of (b) occur in the same composition.

54. 54. The method of claim 53, wherein the composition comprises from about 0.025 μM to about 10 μM of the oligonucleotide and / or the polypeptide.

55. 55. The method of claim 54, wherein the composition comprises about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM, of the oligonucleotide and / or the polypeptide.

56. 56. The method of any one of claims 53-55, wherein the composition comprises from about 1 μM to about 20 μM of the compound.

57. 57. The method of claim 56, wherein the composition comprises about 1 μM to about 10 μM of the compound.

58. 58. The method of claim 57, wherein the composition comprises at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM of the compound.

59. 59. The method of any one of claims 41 to 58, wherein the oligonucleotide hybridizes to a target nucleic acid in the cell.

60. 60. The method of claim 59, wherein the target nucleic acid is in the nucleus of the cell.

61. 60. The method of claim 59, wherein the target nucleic acid is in the cytosol of the cell.

62. 62. The method of any one of claims 41-61, wherein contacting the cell with the compound of (b) results in endosomal membrane permeabilization as determined by an mCherry-GAL9 recruitment assay.

63. 63. The method of any one of claims 41 to 62, wherein said oligonucleotide and / or said polypeptide alters the activity of a gene expressed by said cell.

64. 64. The method of claim 63, wherein the oligonucleotide and / or the polypeptide increases the activity of a gene expressed by the cell.

65. 65. The method of claim 64, wherein the activity of the gene expressed by the cell is increased by at least about 10-fold when the cell is contacted with the oligonucleotide and / or the polypeptide and the compound, compared to a cell contacted with the oligonucleotide and / or the polypeptide but not the compound.

66. 66. The method of claim 65, wherein the activity of the gene expressed by the cell is increased by at least about 100-fold when the cell is contacted with the oligonucleotide and / or the polypeptide and the compound, compared to a cell contacted with the oligonucleotide and / or the polypeptide but not the compound.

67. 64. The method of claim 63, wherein the oligonucleotide and / or the polypeptide reduces the activity of a gene expressed by the cell.

68. 68. The method of claim 67, wherein the activity of the gene expressed by the cell is reduced by at least about 10-fold when the cell is contacted with the oligonucleotide and / or the polypeptide and the compound, compared to cells contacted with the oligonucleotide and / or the polypeptide but not the compound.

69. 69. The method of claim 68, wherein the activity of the gene expressed by the cell is reduced by at least about 100-fold when the cell is contacted with the oligonucleotide and / or the polypeptide and the compound, compared to a cell contacted with the oligonucleotide and / or the polypeptide but not the compound.

70. 70. The method of any one of claims 41 to 69, comprising in vitro delivery of said oligonucleotide and / or said polypeptide to said cell.

71. 70. The method of any one of claims 41 to 69, comprising in vivo delivery of said oligonucleotide and / or said polypeptide to said cell.

72. The method of any one of claims 41 to 69, wherein the cells are cultured cells.

73. 70. The method of any one of claims 41 to 69, wherein the cells are isolated cells obtained from a subject in need of treatment.

74. 70. The method of any one of claims 41 to 69, wherein the cell is part of a tissue or organ.

75. 70. The method of any one of claims 41 to 69, wherein the cell is a mammalian cell.

76. 1. A method for altering expression of a target nucleic acid in a cell, comprising: contacting the cell with an oligonucleotide capable of hybridizing to the target nucleic acid, wherein the oligonucleotide is internalized by the cell via endocytosis and packaged within an endosome; and contacting the cell with a compound of any one of claims 1-31, wherein the compound promotes release of the oligonucleotide from the endosome, wherein hybridization of the oligonucleotide to the target nucleic acid alters expression of the target nucleic acid.

77. 77. The method of claim 76, wherein hybridization of the oligonucleotide to the target nucleic acid increases expression of the target nucleic acid.

78. 77. The method of claim 76, wherein hybridization of the oligonucleotide to the target nucleic acid reduces expression of the target nucleic acid.

79. 1. A method for releasing oligonucleotides and / or polypeptides from endosomes, comprising: contacting the cell with the oligonucleotide and / or the polypeptide, wherein the oligonucleotide and / or the polypeptide is internalized by the cell via endocytosis and encapsulated within the endosome; and contacting the cell with a compound according to any one of claims 1 to 31, wherein the compound promotes release of the oligonucleotide and / or the polypeptide from the endosome.

80. 1. A method for the treatment and / or prevention of a disorder in a subject, comprising:

32. A method comprising administering to the subject (i) a therapeutically effective amount of an oligonucleotide and / or polypeptide, and (ii) an effective amount of a compound according to any one of claims 1 to 31.

81. 81. The method of claim 80, wherein the compound is administered to the subject simultaneously with the oligonucleotide and / or the polypeptide.

82. 81. The method of claim 80, wherein the compound is administered to the subject after administration of the oligonucleotide and / or the polypeptide.

83. 83. The method of claim 82, wherein the compound is administered to the subject up to about 48 hours after administration of the oligonucleotide and / or the polypeptide.

84. 83. The method of claim 82, wherein the compound is administered to the subject about 12 hours to about 48 hours after administration of the oligonucleotide and / or the polypeptide.

85. 80. The method of any one of claims 80 to 79, wherein administering comprises parenteral administration.

86. 86. The method of claim 85, wherein administering comprises intravenous or subcutaneous administration.

87. 87. The method of any one of claims 80 to 86, wherein the subject is a mammal.

88. 87. The method of any one of claims 80 to 86, wherein the subject is a human.

89. 1. A method for the treatment and / or prevention of a disorder in a subject, comprising: isolating cells from the subject; and contacting the isolated cells with a therapeutically effective amount of (i) an oligonucleotide and / or polypeptide and (ii) an effective amount of a compound of any one of claims 1-31 to generate engineered cells; and transplanting the engineered cells into the subject.

90. 90. The method of claim 89, wherein the isolated cells are contacted with a composition comprising about 0.025 μM to about 20 μM of the oligonucleotide and / or the polypeptide.

91. 91. The method of claim 90, wherein the composition comprises about 0.1 μM to about 10 μM, about 0.1 μM to about 5 μM, or about 0.1 μM to about 1 μM of the oligonucleotide and / or the polypeptide.

92. 92. The method of any one of claims 89-91, wherein the isolated cells are contacted with a composition comprising about 1 μM to about 20 μM of the compound.

93. 93. The method of claim 92, wherein the composition comprises about 1 μM to about 10 μM of the compound.

94. 94. The method of claim 93, wherein the composition comprises at least about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM, and up to about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM of the compound.

95. 93. The method of any one of claims 89 to 92, wherein the isolated cell is contacted with (i) the oligonucleotide and / or the polypeptide and (ii) the compound at about the same time.

96. 96. The method of claim 95, comprising contacting the isolated cell with a composition comprising (i) the oligonucleotide and / or the polypeptide and (ii) the compound.

97. 93. The method of any one of claims 89 to 92, wherein the isolated cells are contacted with the oligonucleotide and / or the polypeptide before contacting the isolated cells with the compound.

98. 98. The method of claim 97, wherein the isolated cells are contacted with the oligonucleotide and / or the polypeptide up to about 48 hours before contacting the isolated cells with the compound.

99. 99. The method of claim 98, wherein the isolated cells are contacted with the oligonucleotide and / or the polypeptide for about 12 hours to about 48 hours during which the isolated cells are contacted with the compound.

100. 100. The method of any one of claims 89 to 99, wherein the subject is a mammal.

101. The method of any one of claims 89 to 100, wherein the subject is a human.

102. 32. Use of (i) a compound according to any one of claims 1 to 31 and (ii) an oligonucleotide and / or a polypeptide in the manufacture of a medicament for gene therapy.

103. A kit comprising (i) a compound according to any one of claims 1 to 31 and (ii) an oligonucleotide and / or a polypeptide.

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