Formulations for regulating MYC expression

Abstract

The present disclosure relates to compositions and methods for reducing the expression of the MYC gene in cells. In some embodiments, the expression repressor comprises a targeting moiety that binds to the MYC promoter, an anchor sequence, or a super enhancer. In some embodiments, the expression repressor comprises an effector moiety that suppresses transcription or methylates DNA. A system comprising two expression repressors is also disclosed. The compositions can be used, for example, for the treatment of cancers such as HCC.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 366,833, filed Jun. 22, 2022, and U.S. Provisional Patent Application No. 63 / 377,148, filed Sep. 26, 2022, the entire contents of which are hereby incorporated by reference herein.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety herein. The XML copy created on Jun. 19, 2023, is named O2057 - 7033WO_SL.xml and is 344,197 bytes in size.

Background Art

[0003] Dysregulation of gene expression is the underlying cause of many diseases (e.g., mammalian, e.g., human diseases), such as neoplasia, neurological disorders, metabolic disorders, and obesity. Dysregulation of the transcription factor MYC plays a central role in various human tumors and chronic liver diseases. The MYC protein is considered "undruggable" due to various factors, such as the absence of a distinct ligand - binding site and the essential role of its physiological function in maintaining normal tissues. Techniques aimed at regulating MYC gene expression provide viable alternative approaches in the treatment of these diseases. There is a need for new tools, systems, and methods for stably changing, e.g., reducing, the expression of disease - related genes such as MYC.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure provides, inter alia, therapeutic compositions comprising expression repressors and expression repressor systems that can be used to modulate, e.g., reduce, the expression of a target gene, such as MYC, encapsulated in lipid nanoparticles (LNPs). In some embodiments, the LNP comprises one or more (e.g., all) of an ionizable lipid, a PEGylated lipid, phosphatidylcholine, and a sterol.

[0005] In some aspects, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a target gene promoter, such as the MYC promoter, and optionally an effector moiety, wherein the expression repressor can reduce the expression of a target gene, such as MYC.

[0006] In some aspects, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a target locus, such as MYC, and an effector moiety comprising MQ1 or a fragment or variant thereof, wherein the expression repressor can reduce the expression of a target gene, such as MYC.

[0007] In some aspects, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a regulatory element located in the super-enhancer region of MYC and optionally an effector moiety, wherein the expression repressor can reduce the expression of MYC.

[0008] In some aspects, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a regulatory element located in the super-enhancer region of a target gene, such as MYC, and an effector moiety (e.g., KRAB, or MQ1, or a fragment or variant thereof), wherein the expression repressor can reduce the expression of the target gene, such as MYC.

[0009] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a regulatory element located in the super-enhancer region of a target gene, such as MYC, the targeting moiety comprising a zinc finger domain, wherein the expression repressor is capable of reducing the expression of the target gene, such as MYC.

[0010] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a regulatory element located in the super-enhancer region of MYC, the targeting moiety comprising a zinc finger domain or a TAL effector domain, and an effector moiety comprising a transcriptional repressor (e.g., KRAB or a fragment or variant thereof) or a DNA methyltransferase (e.g., MQ1 or a fragment or variant thereof); wherein the expression repressor is capable of reducing the expression of MYC.

[0011] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a target locus, such as MYC, the targeting moiety comprising a zinc finger domain, wherein the expression repressor is capable of reducing the expression of the target gene, such as MYC.

[0012] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequences of SEQ ID NO: 1, 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, or 75, 76, 78, 79, 80, 81, 84, 85, 86, wherein the expression repressor is capable of reducing the expression of MYC.

[0013] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2 or 77, 82, 83, wherein the expression repressor is capable of reducing the expression of a target gene, such as MYC. In some embodiments, the expression repressor comprises an effector moiety.

[0014] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a genomic locus within 1400 nt upstream or downstream of SEQ ID NO: 4.

[0015] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, or 83.

[0016] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, 96, or 108.

[0017] In some embodiments, the present disclosure provides a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcriptional regulatory element (e.g., a promoter or a transcription start site (TSS)) operably linked to a target gene, such as MYC, or to a sequence proximal to the transcriptional regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising the target gene, such as MYC, or to a sequence proximal to the anchor sequence.

[0018] In some aspects, the present disclosure provides a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a transcription regulatory element (e.g., a promoter or a transcription start site (TSS)) operably linked to a target gene, such as MYC, or binding to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to a genomic locus located in a super enhancer region of a target gene, such as MYC.

[0019] In some embodiments, the first targeting moiety specifically binds to a first DNA sequence and the second targeting moiety specifically binds to a second DNA sequence different from the first DNA sequence. In some embodiments, the first effector moiety is different from the second effector moiety.

[0020] In some aspects, the present disclosure provides an expression repressor comprising a targeting moiety comprising a CRISPR / Cas molecule, e.g., a catalytically inactive CRISPR / Cas protein, that binds to a transcription regulatory element (e.g., a promoter or a transcription start site (TSS)) operably linked to a target gene, such as MYC, or a sequence proximal to the transcription regulatory element, and an effector moiety comprising MQ1 or a functional variant or fragment thereof.

[0021] In some aspects, the present disclosure provides an expression repressor comprising a targeting moiety comprising a CRISPR / Cas molecule, e.g., a catalytically inactive CRISPR / Cas protein, that binds to a genomic locus located in a super enhancer region of a target gene, such as MYC, and an effector moiety comprising KRAB, MQ1, or a functional variant or fragment thereof, wherein the expression repressor can reduce the expression of a target gene, such as MYC.

[0022] In some embodiments, the present disclosure provides an expression repressor comprising a targeting portion that binds to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising a target gene, such as MYC, or binds to a sequence proximal to the anchor sequence, e.g., a CRISPR / Cas molecule, such as a CRISPR / Cas protein that is catalytically inactive; and an effector portion comprising KRAB or a functional variant or fragment thereof.

[0023] In some embodiments, the present disclosure provides an expression repressor comprising a targeting portion that binds to a transcriptional regulatory element (e.g., a promoter or a transcription start site (TSS)) operably linked to a target gene, such as MYC, or binds to a sequence proximal to the transcriptional regulatory element, e.g., a zinc finger molecule; and an effector portion comprising MQ1 or a functional variant or fragment thereof.

[0024] In some embodiments, the present disclosure provides an expression repressor comprising a targeting portion that binds to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising a target gene, such as MYC, or binds to a sequence proximal to the anchor sequence, e.g., a zinc finger molecule; and an effector portion comprising KRAB or a functional variant or fragment thereof.

[0025] In some embodiments, the present disclosure provides an expression repressor comprising a targeting portion that binds to a genomic locus located in the super enhancer region of a target gene, such as MYC, e.g., a zinc finger molecule; and an effector portion comprising KRAB or a functional variant or fragment thereof.

[0026] In some embodiments, the present disclosure relates to a nucleic acid encoding a first expression repressor, a second expression repressor, both, or a component thereof (e.g., gRNA, mRNA). In some embodiments, the nucleic acid encoding the expression repressor system is a polycistronic sequence. In some embodiments, the polycistronic sequence is a bicistronic sequence.

[0027] In some aspects, the present disclosure relates to vectors comprising the nucleic acids, systems, or expression repressors described herein. In another aspect, the present disclosure relates to lipid nanoparticles comprising the nucleic acids, systems, or expression repressors described herein. In another aspect, the present disclosure relates to reaction mixtures comprising the expression repressors, systems, nucleic acids, vectors, or lipid nanoparticles described herein. In another aspect, the present disclosure relates to pharmaceutical compositions comprising the expression repressors, systems, nucleic acids, vectors, lipid nanoparticles, or reaction mixtures described herein.

[0028] In some aspects, the present disclosure relates to methods of reducing the expression of a target gene, which include providing an expression repressor or expression suppression system described herein, and contacting the target gene and / or one or more operably linked transcriptional control elements with the expression repressor or expression suppression system, thereby reducing the expression of the target gene.

[0029] In some aspects, the present disclosure relates to methods of treating a condition associated with overexpression of a target gene, such as MYC, in a subject, which include administering to the subject an expression repressor or system, nucleic acid, or vector described herein, thereby treating the condition.

[0030] In some aspects, the present disclosure relates to methods of treating a condition associated with dysregulation of a target gene, such as MYC, in a subject, which include administering to the subject an expression repressor, system, nucleic acid, or vector described herein, thereby treating the condition.

[0031] In some aspects, the present disclosure provides a method for reducing the expression of a target gene, such as MYC, in a cell, the method comprising contacting the cell with a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a transcriptional regulatory element (e.g., a promoter or a transcription start site (TSS)) operably linked to the target gene, such as MYC, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising the target gene, such as MYC, or to a sequence proximal to the anchor sequence, thereby reducing the expression of the target gene, such as MYC, in the cell.

[0032] In some aspects, the present disclosure provides a method for reducing the expression of a target gene, such as MYC, in a cell, the method comprising contacting the cell with a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a transcriptional regulatory element (e.g., a promoter or a transcription start site (TSS)) operably linked to the target gene, such as MYC, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to a genomic locus located in the super-enhancer region of the target gene, such as MYC, thereby reducing the expression of the target gene, such as MYC, in the cell.

[0033] The present disclosure further provides, in part, a kit comprising: a) a container comprising a composition comprising an expression repressor comprising a targeting moiety that binds to a target gene, a promoter, such as MYC, and an effector moiety that can regulate, e.g., reduce, the expression of the target gene, such as MYC; and b) a set of instructions comprising at least one method of regulating the expression of a target gene, such as MYC, in a cell with the composition.

[0034] The present disclosure further provides, in part, a kit comprising: a) a container containing a composition comprising an expression repressor comprising a targeting moiety that binds to a locus located in a super enhancer region of a target gene, such as MYC, and an effector moiety that can regulate, such as reduce, the expression of the target gene, such as MYC; and b) a set of instructions comprising at least one method of regulating the expression of a target gene, such as MYC, in a cell with the composition.

[0035] In some embodiments, the kit comprises a container containing a composition comprising a system of two expression repressors: a) a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a transcription regulatory element (such as a promoter or transcription start site (TSS)) operably linked to a target gene, such as MYC, or binding to a sequence proximal to the transcription regulatory element; and b) a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising the target gene, such as MYC, or binding to a sequence proximal to the anchor sequence.

[0036] In some embodiments, the kit comprises a container containing a composition comprising a system of two expression repressors: a) a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a transcription regulatory element (such as a promoter or transcription start site (TSS)) operably linked to a target gene, such as MYC, or binding to a sequence proximal to the transcription regulatory element; and b) a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to a genomic locus located in a super enhancer region of the target gene, such as MYC.

[0037] In some embodiments, the kit further comprises a set of instructions comprising at least one method of treating a disease with the composition or modulating, e.g., reducing, the expression of a target gene in a cell, such as MYC. In some embodiments, the kit may optionally include a delivery vehicle (e.g., lipid nanoparticles) for the composition. The reagent may be provided suspended in an excipient and / or delivery vehicle or as a separate component that can be combined later with an excipient and / or delivery vehicle. In some embodiments, the kit may optionally contain an additional therapeutic agent that is to be co-administered with the composition to affect the regulation of the desired target gene expression, e.g., MYC gene expression. The instructions typically include, but are not limited to, written or printed materials. According to the present invention, any medium capable of storing and transmitting such instructions to an end user is contemplated. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), etc. Such media may include an address to an Internet site that provides such instructions.

[0038] Any other features of any of the methods or compositions described above include one or more of the embodiments listed below.

[0039] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the embodiments listed below.

[0040] All publications, patent applications, patents, and other references (e.g., array database reference numbers) cited in this specification are hereby incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced in this specification, e.g., in any table of this specification, are hereby incorporated by reference. Unless otherwise specified, all sequence accession numbers described in this specification, including all tables of this specification, refer to database entries as of December 15, 2020. Where a gene or protein is indicated by multiple sequence accession numbers, all sequence variants are included.

[0041] Enumeration of Embodiments 1. A composition comprising: (1) A targeting moiety that binds to the MYC promoter, and Optionally, an effector moiety and A first nucleic acid encoding a repressor of expression comprising, Wherein the repressor of expression can reduce the expression of MYC, a first nucleic acid; and (2) A formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of the following (iii) and (iv) (e.g., comprising all of (i)-(iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

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[0042] 2. The composition according to embodiment 1, wherein the targeting moiety binds to a genomic locus that is within 1400, 1200, 1000, 800, 600, 400, or 200 nt upstream or downstream of SEQ ID NO: 4, 199, or 201.

[0043] 3. The composition according to embodiment 1, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, 83, 85, 199, or 201.

[0044] 4. A composition comprising: (1) a targeting moiety that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 75, 76, 78, 79, 80, 81, 84, 85, 86, 190, 191, 192, 200, or 202, and optionally, an effector moiety and a first nucleic acid encoding a transcriptional repressor comprising, wherein the transcriptional repressor is capable of reducing the expression of MYC, the first nucleic acid; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of the following (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

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[0045] 5. A composition comprising: (1) a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 77, 82, 83, 199, or 201, and optionally, an effector moiety and a first nucleic acid encoding a transcriptional repressor comprising, A first nucleic acid capable of reducing the expression of MYC by the above-described expression repressor; and (2) A formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of the following (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i'') and (i'''): (i') Formula (I)

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[0046] 6. A composition comprising: (1) a targeting moiety that binds to the MYC locus, and an effector moiety comprising MQ1 or a fragment or variant thereof a first nucleic acid encoding a transcriptional repressor comprising a first nucleic acid capable of reducing the expression of MYC; and (2) a formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of the following (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) a first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') formula (I)

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[0047] 7. A composition comprising: (1) a targeting moiety that binds to a locus within the MYC super-enhancer region, and Optionally, an effector moiety, for example, an effector moiety comprising a DNA methyltransferase, optionally the effector moiety comprises MQ1 or a fragment or variant thereof and a first nucleic acid encoding a transcriptional repressor comprising the transcriptional repressor capable of reducing the expression of MYC, a first nucleic acid; and (2) A formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i'') and (i'''): (i') Formula (I)

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[0048] 8. A composition comprising: (1) A targeting moiety that binds to a locus within the MYC super-enhancer region, and an effector moiety comprising a transcriptional repressor, optionally wherein the effector moiety is an effector moiety comprising KRAB or a fragment or variant thereof, and a first nucleic acid encoding an expression repressor comprising the above, wherein the expression repressor can reduce the expression of MYC, the first nucleic acid; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of (iii) and (iv) (e.g., comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

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[0049] 9. The composition according to embodiment 7 or 8, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96 - 110, 83, 199, 201.

[0050] 10. The composition according to any one of embodiments 7 - 9, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of GRCh37:chr8:129162465 - 129212140 using the hg19 reference genome.

[0051] 11. The composition according to any one of embodiments 7 - 10, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 96 or 108.

[0052] 12. The composition according to any one of embodiments 7 - 11, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain.

[0053] 13. A composition comprising: (1) A targeting moiety that binds to a locus, e.g., the MYC locus, and A first effector moiety comprising EZH2 or a fragment or variant thereof, and A second effector moiety comprising KRAB or a fragment or variant thereof A first nucleic acid encoding a transcriptional repressor comprising: The transcriptional repressor capable of reducing expression at the locus, e.g., capable of reducing expression of MYC, the first nucleic acid; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of the following (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i'') and (i'''): (i') Formula (I)

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[0054] 14. The composition according to embodiment 13, wherein the targeting moiety binds to a MYC promoter, a super enhancer region, or an anchor sequence.

[0055] 15. The composition according to embodiment 13 or 14, wherein the targeting moiety comprises a TAL effector domain, a CRISPR / Cas domain, or a zinc finger domain.

[0056] 16. The composition according to any one of embodiments 13 to 15, wherein the first effector portion is on the N-terminal side of the second effector, or the first effector is on the C-terminal side of the second effector portion.

[0057] 17. A composition comprising: (1) A targeting portion that binds to the MYC locus, the targeting portion comprising a targeting portion containing a zinc finger domain and, optionally, an effector portion a first nucleic acid encoding a transcriptional repressor comprising, the transcriptional repressor being capable of reducing the expression of MYC; and (2) A formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of the following (iii) and (iv) (e.g., comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i'') and (i'''): (i') Formula (I)

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[0058] 18. A composition comprising: (1) A targeting moiety comprising a CRISPR / Cas domain that binds to a transcription regulatory element (e.g., a promoter, enhancer, super enhancer, or transcription start site (TSS)) operably linked to the MYC gene or a sequence proximal to said transcription regulatory element, for example, a CRISPR / Cas protein that is catalytically inactive; and An effector moiety comprising MQ1 or a functional variant or fragment thereof A first nucleic acid encoding a transcriptional repressor comprising the same; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of (iii) and (iv) (e.g., comprising all of (i)-(iv)): (i) A first compound selected from the group consisting of the following (i'), (i'') and (i'''): (i') Formula (I)

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Chem.

[0059] 19. A composition comprising: (1) a targeting moiety comprising a CRISPR / Cas domain that binds to a transcriptional regulatory element (for example, a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene or a sequence proximal to the transcriptional regulatory element, for example, a CRISPR / Cas protein that is catalytically inactive; and an effector moiety comprising MQ1 or a functional variant or fragment thereof a first nucleic acid encoding a transcriptional repressor comprising; and (2) a formulation comprising: (i) a first compound selected from the group consisting of the following (i'), (i'') and (i'''): (i') Formula (I)

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[0060] 20. A composition comprising: (1) A targeting moiety comprising a CRISPR / Cas domain that binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene or a sequence proximal to the transcriptional regulatory element, e.g., a CRISPR / Cas protein that is catalytically inactive; and an effector moiety comprising KRAB or a functional variant or fragment thereof a first nucleic acid encoding an expression repressor comprising; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of (iii) and (iv) (e.g., comprising all of (i)-(iv)): (i) A first compound selected from the group consisting of the following (i’), (i’’) and (i’’’): (i’) Formula (I) [Chemistry] (In formula (I): L 1and L 2 is, independently of each other, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)O-, or a direct bond; G 1 and G 2 is, independently of each other, unsubstituted C1-C 12 alkylene, or C2-C 12 alkenylene; G 3 is C1-C 24 alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R a is H or C1-C 12 alkyl; R 1 and R 2 are, independently of each other, C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , -NR 11 R 12 , or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; R 5 is H or C1-C6 alkyl; R 11 and R 12 are, independently of each other, C1-C 12 alkyl or -G 4 -OR 5 or R11 and R 12 together with the nitrogen to which they are attached form a 5-, 6- or 7-membered heterocycle; G 4 is C1-C 24 alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; x is 0, 1 or 2) a compound having the general structure of, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof; (i'') Formula (IX):

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[0061] 21. A composition comprising: (1) A targeting moiety comprising a CRISPR / Cas domain that binds to the anchor sequence of an anchor sequence-mediated junction (ASMC) containing the MYC gene or binds to a sequence proximal to the anchor sequence, for example, a CRISPR / Cas protein that is catalytically inactive; An effector moiety comprising KRAB or a functional variant or fragment thereof and a first nucleic acid encoding an expression repressor; and (2) A formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i'') and (i'''): (i') Formula (I)

Chem.

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[0062] 22. A composition comprising: (1) a targeting moiety comprising a transcription regulatory element (for example, a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene or a zinc finger domain that binds to a sequence proximal to the transcription regulatory element, and an effector moiety comprising MQ1 or a functional variant or fragment thereof a first nucleic acid encoding a transcriptional repressor comprising; and (2) a formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

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[0063] 23. A composition comprising: (1) a targeting moiety comprising a zinc finger domain that binds to a transcriptional regulatory element (for example, a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene or a sequence proximal to the transcriptional regulatory element; and an effector moiety comprising KRAB or a functional variant or fragment thereof a first nucleic acid encoding an expression repressor comprising; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

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[0064] 24. A composition comprising: (1) a targeting moiety that binds to a mouse genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 190 to 192 and optionally, an effector moiety and an expression repressor comprising, A first nucleic acid encoding the above-described expression repressor, capable of reducing the expression of MYC; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of the following (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

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[0065] 25. The composition according to embodiment 24, wherein the effector moiety comprises a DNA methyltransferase, for example, MQ1 or a fragment or variant thereof.

[0066] 26. The composition according to embodiment 24 or 25, wherein the targeting moiety comprises a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.

[0067] 27. The composition according to any one of embodiments 24 to 26, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain.

[0068] 28. The composition according to any one of embodiments 24 to 27, wherein the expression repressor comprises an amino acid sequence selected from any of SEQ ID NOs: 160 to 165, or is at least 80, 85, 90, 95, 99, or 100% identical thereto, or comprises a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0069] 29. The composition according to any one of embodiments 24 to 28, wherein the expression repressor comprises a nucleotide sequence selected from any of SEQ ID NOs: 166 to 168, or is at least 80, 85, 90, 95, 99, or 100% identical thereto, or is encoded by a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0070] 30. The composition according to any one of embodiments 24 to 29, wherein the targeting moiety comprises an amino acid sequence according to any of SEQ ID NOs: 154 to 156, or is at least 80, 85, 90, 95, 99, or 100% identical thereto, or comprises a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0071] 31. The composition according to any one of Embodiments 24 to 30, wherein the targeting portion is a nucleic acid sequence according to any one of SEQ ID NOs: 157 to 159, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or is encoded by a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0072] 32. The composition according to any one of Embodiments 24 to 31, wherein the effector portion is a persistent effector portion.

[0073] 33. The composition according to any one of Embodiments 24 to 32, wherein the effector portion is a transient effector portion.

[0074] 34. The composition according to any one of Embodiments 24 to 33, which is a fusion molecule.

[0075] 35. The composition according to any one of Embodiments 24 to 34, wherein the targeting portion includes a zinc finger domain, and the effector portion includes an epigenetic modification portion, for example, a DNA methyltransferase, for example, MQ1 or a fragment or mutant thereof.

[0076] 36. The composition according to any one of Embodiments 18 to 20, 22, or 23, wherein the regulatory element is part of a cluster of regulatory elements.

[0077] 37. The composition according to any one of Embodiments 18 to 20, 22, or 23, wherein the regulatory element is located in a non-coding region.

[0078] 38. The composition according to any one of Embodiments 18 to 20, 22, or 23, wherein the regulatory element is a distal enhancer, for example, located at a position at least 1,000 nt away from a target gene promoter, for example, MYC.

[0079] 39. A composition according to any one of embodiments 18 - 20, 22, 23, or 36 - 38, wherein the regulatory element increases the expression of a target gene, such as MYC.

[0080] 40. A composition according to any one of embodiments 18 - 20, 22, 23, or 36 - 39, wherein the regulatory element has one or more mutations.

[0081] 41. A composition according to any one of embodiments 18 - 20, 22, 23, or 36 - 40, wherein the regulatory element has at least one disease - related single - nucleotide polymorphism (SNP).

[0082] 42. A composition according to any one of embodiments 18 - 20, 22, 23, or 36 - 41, wherein the transcriptional regulatory element interacts through a promoter and enhancer docking site of a target gene, such as MYC.

[0083] 43. A composition according to embodiment 42, wherein the enhancer docking site comprises a nucleotide sequence according to any of the nucleic acid sequences of SEQ ID NO: 71, 72, or CCGCCATNTT or AANATGGCGG.

[0084] 44. A composition comprising: (1) A targeting moiety comprising a zinc finger domain that binds to the anchor sequence of an anchor - sequence - mediated junction (ASMC) containing the MYC gene or binds to a sequence proximate to the anchor sequence; and An effector moiety comprising KRAB or a functional variant or fragment thereof; An expression repressor comprising the same; and (2) A formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of (iii) and (iv) (e.g., comprising all of (i) - (iv)): (i) A first compound selected from the group consisting of the following (i’), (i’’), and (i’’’): (i’) Formula (I)

Chemical formula

Chemical formula

Chemical Structure

Chemical formula

[0085] 45. The composition according to any one of embodiments 1 to 44, wherein the first nucleic acid comprises RNA, for example, mRNA.

[0086] 46. The composition according to any one of embodiments 1 to 23 or 36 to 43, wherein the expression repressor is an amino acid sequence selected from any of SEQ ID NOs: 22 to 37, 129, 133, 134, 139 to 149, or 177 to 186, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or comprises a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0087] 47. The composition according to any one of Embodiments 1 to 23 or 36 to 46, wherein the expression repressor is a nucleotide sequence selected from any of SEQ ID NOs: 55 to 70, 130, 189, or 193 to 197, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or is encoded by a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0088] 48. The composition according to any one of Embodiments 1 to 23 or 36 to 47, wherein the targeting moiety is an amino acid sequence according to any of SEQ ID NOs: 5 to 16, or 169 to 172, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0089] 49. The composition according to any of the preceding embodiments, wherein the effector moiety is an amino acid sequence according to SEQ ID NOs: 18, 19, or 87, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0090] 50. The composition according to any one of Embodiments 1 to 12, 17 to 19, 22, 36 to 42, or 44 to 48, wherein the effector moiety is a persistent effector moiety.

[0091] 51. The composition according to any one of Embodiments 1 to 23 or 36 to 49, wherein the effector moiety is a transient effector moiety.

[0092] 52. The composition according to any one of Embodiments 1 to 12, 17 to 19, 22, 36 to 42, or 44 to 49, wherein the effector portion contains a DNA methyltransferase, for example, MQ1 or a fragment or variant thereof.

[0093] 53. The composition according to any one of Embodiments 1 to 23, 36 to 48, or 50, wherein the effector portion contains a transcriptional repressor, for example, KRAB or a fragment or variant thereof.

[0094] 54. The composition according to any of the preceding embodiments, wherein the targeting portion contains a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.

[0095] 55. The composition according to Embodiment 54, wherein the CRISPR / Cas domain binds to a gRNA that binds to a genomic locus containing at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 1 to 4, for example, the gRNA contains a sequence containing at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 1 to 4.

[0096] 56. The composition according to Embodiment 54, wherein the CRISPR / Cas domain binds to a gRNA that binds to a genomic locus containing at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96 to 110, for example, the gRNA contains a sequence containing at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96 to 110.

[0097] 57. The composition according to any one of Embodiments 54 to 56, wherein the CRISPR / Cas domain contains a Cas protein or a Cpf1 protein selected from Table 1 or a variant (e.g., a mutant) thereof.

[0098] 58. The composition according to any one of embodiments 54 to 57, wherein the CRISPR / Cas domain comprises a catalytically inactive CRISPR / Cas protein, such as dCas9.

[0099] 59. The composition according to embodiment 54, wherein the zinc finger domain binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96 to 110, and for example, the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96 to 110.

[0100] 60. The composition according to any one of embodiments 17, 22, 26 to 54, or 58, wherein the zinc finger domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (and optionally not more than 11, 10, 9, 8, 7, 6, or 5 additional zinc fingers).

[0101] 61. The composition according to any one of embodiments 17, 22, 26 to 54, 58, or 59, wherein the zinc finger domain comprises 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 zinc fingers.

[0102] 62. The composition according to any one of embodiments 17, 22, 26 to 54, or 58 to 60, wherein the zinc finger domain comprises 3 or 9 zinc fingers.

[0103] 63. The composition according to any of the preceding embodiments, wherein the expression repressor is a fusion molecule.

[0104] 64. The composition according to any of the preceding embodiments, wherein the expression repressor comprises a linker located between the targeting domain and the effector domain, and optionally the linker comprises the amino acid sequence according to SEQ ID NO: 137 or SEQ ID NO: 138.

[0105] 65. The composition according to any of embodiments 1 to 17, 20, 21, 23, 44 to 49, 51, or 53 to 58, wherein the targeting moiety comprises a catalytically inactive CRISPR / Cas domain (e.g., dCas9), and the effector moiety comprises a transcriptional repressor, such as KRAB or a fragment or variant thereof.

[0106] 66. The composition according to any of embodiments 1 to 17, 20, 21, 23, 44 to 49, 51, 53, or 54 to 65, wherein the targeting moiety comprises a zinc finger domain, and the effector moiety comprises a transcriptional repressor, such as KRAB or a fragment or variant thereof.

[0107] 67. The composition according to any of embodiments 17, 36 to 43, 46 to 48, 54, or 59 to 64, wherein the targeting moiety comprises a zinc finger domain, and the expression repressor does not comprise an effector moiety.

[0108] 68. The composition according to any of embodiments 1 to 12, 18 to 19, 22, 36 to 43, 46 to 50, 52, or 54 to 58, wherein the targeting moiety comprises a catalytically inactive CRISPR / Cas domain (e.g., dCas9), and the effector moiety comprises an epigenetic modification moiety, such as a DNA methyltransferase, such as MQ1 or a fragment or variant thereof.

[0109] 69. The composition according to any one of Embodiments 1 to 12, 17 to 19, 22, 36 to 43, 46 to 50, 52, 54, or 59 to 64, wherein the targeting portion includes a zinc finger domain and the effector portion includes an epigenetic modification portion, for example, a DNA methyltransferase, for example, MQ1 or a fragment or variant thereof.

[0110] 70. The composition according to any one of the preceding embodiments, wherein the expression repressor includes an amino acid sequence of any one of SEQ ID NOs: 22 to 37, 129, 133, 134, 139 to 149, or 177 to 186, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0111] 71. The composition according to any one of the preceding embodiments, wherein the expression repressor (i) includes one or more nuclear localization signal sequences (NLSs), or (ii) does not include an NLS.

[0112] 72. The composition according to any one of the preceding embodiments, wherein the expression repressor includes a first NLS at the N-terminus, for example, the first NLS has the sequence of SEQ ID NO: 88.

[0113] 73. The composition according to any one of the preceding embodiments, wherein the expression repressor includes an NLS at the C-terminus, for example, having the sequence of SEQ ID NO: 89, for example, a second NLS.

[0114] 74. The composition according to any one of the preceding embodiments, wherein the first NLS and the second NLS have the same sequence.

[0115] 75. The composition according to any one of Embodiments 72 to 74, wherein the first NLS and the second NLS have different sequences.

[0116] 76. The composition according to any one of the preceding embodiments, wherein the expression repressor includes an epitope tag.

[0117] 77. The composition according to embodiment 76, wherein the epitope tag is an HA tag.

[0118] 78. The composition according to any one of the preceding embodiments, wherein the anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0119] 79. The composition according to any one of embodiments 1 to 78, wherein the anchor sequence comprises the sequence related to CCGCCATNTT or AANATGGCGG (N is any nucleotide), or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0120] 80. The composition according to any one of the preceding embodiments, wherein the anchor sequence is on the same chromosome as the MYC gene.

[0121] 81. The composition according to any one of the preceding embodiments, wherein the anchor sequence is upstream of the MYC gene (e.g., upstream of the TSS or upstream of the promoter).

[0122] 82. The composition according to any one of the preceding embodiments, wherein the anchor sequence is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene).

[0123] 83. The composition according to any of the preceding embodiments, wherein the anchor array is 0.1 to 0.5, 0.1 to 1, 0.1 to 5, 0.1 to 10, 0.1 to 50, 0.1 to 100, 0.1 to 500, 0.1 to 1000, 0.5 to 1, 0.5 to 5, 0.5 to 10, 0.5 to 50, 0.5 to 100, 0.5 to 500, 0.5 to 1000, 1 to 5, 1 to 10, 1 to 50, 1 to 100, 1 to 500, 1 to 1000, 5 to 10, 5 to 50, 5 to 100, 5 to 500, 5 to 1000, 10 to 50, 10 to 100, 10 to 500, 10 to 1000, 50 to 100, 50 to 500, 50 to 1000, 100 to 500, 100 to 1000, or 500 to 1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene).

[0124] 84. The composition according to any of embodiments 1 to 80, 82, or 82, wherein the target sequence is downstream of the MYC gene (e.g., downstream of the TSS or downstream of the promoter).

[0125] 85. The composition according to any of the preceding embodiments, wherein the targeting portion binds to the sequence at chromosomal coordinates 128746342 to 128746364, 128746321 to 128746343, 128746525 to 128746547, 128748014 to 128748036, 129188878 to 129188900, 129188958 to 129188980, 129188960 to 129188982, 129189067 to 129189089, 129189457 to 129189479, 129189554 to 129189576, 129189679 to 129189701, 129209511 to 129209533, 129209643 to 129209665, 129209658 to 129209680, 129209856 to 129209878, 129189452 to 129189474, 129189190 to 129189212, 129189274 to 129189296, 129189421 to 129189443, 128746405 to 128746425, 128748069 to 128748089, 129188825 to 129188845, or 129188822 to 129188842 or a sequence proximate thereto.

[0126] 86. When the expression repressor binds to a target locus, such as MYC, for example, as measured by ELISA or as described in either Example 7 or 17, methylation at a site in the target locus, such as MYC, is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to methylation in the absence of the expression repressor, and optionally, the site assayed for methylation is chr8:129188693 to 129189048 according to the hg19 reference genome and includes, for example, the sequence according to SEQ ID NO: 123. The composition according to any of the preceding embodiments.

[0127] 87. When the above expression repressor binds to a target locus, such as MYC, for example, as described in Example 17, methylation at a site of the above target locus, such as MYC, increases over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, the composition according to any of the preceding embodiments.

[0128] 88. When the above expression repressor binds to the MYC locus, for example, as measured by ELISA or as described in any of Examples 2-7 or 9, the expression of MYC in cells decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the expression in the absence of the above expression repressor, the composition according to any of the preceding embodiments.

[0129] 89. When the above expression repressor binds to the MYC locus, for example, as measured by ELISA or as described in any of Examples 2-7 or 9, the expression of MYC decreases to a degree that can be observed over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, the composition according to any of the preceding embodiments.

[0130] 90. When the above expression repressor binds to the MYC locus, the expression of MYC decreases to a degree that can be observed 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection, the composition according to any of the preceding embodiments.

[0131] 91. The composition according to any of Embodiments 1-23 or 36-90, wherein the targeting moiety binds to a human genomic locus.

[0132] 92. The composition according to any one of embodiments 24 to 43, 50, 52, 54, 57, 58, 60 to 63, 67 to 69, or 71 to 90, wherein the targeting portion binds to a mouse genomic locus.

[0133] 93. The composition according to any of the preceding embodiments, wherein when the expression repressor binds to the MYC locus, the viability of cells (e.g., cancer cells) containing the MYC locus is reduced.

[0134] 94. The composition according to any of the preceding embodiments, wherein when a plurality of cells are contacted with the composition, the viability of the plurality of cells is reduced.

[0135] 95. For example, when measured by CellTiter Glo or as described in any of Examples 2 to 7, the viability is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the viability in the absence of the first expression repressor. The composition according to any of the preceding embodiments.

[0136] 96. When the composition is administered, apoptosis occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of the target cells (e.g., cancer cells). The composition according to any of the preceding embodiments.

[0137] 97. When the composition is administered, cell death occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, or 80% of the target cells (e.g., cancer cells). The composition according to any of the preceding embodiments.

[0138] 98. The composition according to any of the preceding embodiments, wherein when the composition is administered, the MYC mRNA level in target cells (e.g., cancer cells) decreases to 70%, 60%, 50%, or 40% of the level before administration.

[0139] 99. The composition according to any of the preceding embodiments, wherein the plurality of cells includes a plurality of cancer cells and a plurality of non-cancer cells and / or a plurality of infected cells and a plurality of non-infected cells.

[0140] 100. The composition according to any of the preceding embodiments, wherein when the plurality of cells is contacted with the composition, the viability of the plurality of cancer cells decreases more significantly than the decrease in the viability of the plurality of non-cancer cells thereby.

[0141] 101. The composition according to any of the preceding embodiments, wherein when the plurality of cells is contacted with the composition, the viability of the plurality of cancer cells decreases by 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× more than the decrease in the viability of the plurality of non-cancer cells thereby.

[0142] 102. The composition according to any of embodiments 93 to 100, wherein the cancer cells are gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or liver cancer cells.

[0143] 103. The composition according to any of embodiments 93 to 102, wherein the cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, adenocarcinoma, large cell (undifferentiated) carcinoma, triple negative breast cancer, gastric adenocarcinoma, endometrial cancer, or pancreatic cancer.

[0144] 104. The composition according to any of the preceding embodiments, wherein when contacted with a plurality of infected cells and a plurality of non-infected cells, it reduces the viability of the plurality of infected cells more significantly than it reduces the viability of the plurality of non-infected cells.

[0145] 105. The composition according to any one of the preceding embodiments, wherein the infection is a viral infection.

[0146] 106. The composition according to embodiment 105, wherein the viral infection is hepatitis, for example, hepatitis B.

[0147] 107. The composition according to any one of embodiments 93 to 106, wherein the infected cells are human hepatocytes.

[0148] 108. An assay regarding the viability of cancer cells (e.g., HCC cells) using LNP delivery of the mRNA encoding the expression repressor, for example, when tested in the assay according to Example 12, having an EC50 of 0.04 - 0.4, 0.04 - 0.1, 0.1 - 0.2, 0.2 - 0.3, or 0.3 - 0.4 μg / mL, the composition according to any one of the preceding embodiments.

[0149] 109. The composition according to any one of embodiments 1 to 107, having an EC50 of 0.1 - 2.5, 0.5 - 2.2, 1.0 - 1.5, 1.2 - 2 μg / mL when tested in an assay regarding the viability of cancer cells.

[0150] 110. An assay regarding the decrease in MYC mRNA level of cancer cells (e.g., HCC cells) using LNP delivery of the mRNA encoding the expression repressor, for example, when tested in the assay according to Example 12, having an EC50 of 0.004 - 0.08, 0.004 - 0.01, 0.01 - 0.02, 0.02 - 0.04, or 0.04 - 0.08 μg / mL, the composition according to any one of the preceding embodiments.

[0151] 111. The composition according to any one of the preceding embodiments, having an EC50 of 0.04 - 0.1, 0.04 - 0.09, 0.05 - 0.09, or 0.06 - 0.8 μg / mL when tested in an assay regarding the decrease in MYC mRNA level of cancer cells.

[0152] 112. The composition according to any of the preceding embodiments, which reduces the level of a target gene in a cell, for example, a protein encoded by MYC, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the protein level of an untreated cell.

[0153] 113. The composition according to any of the preceding embodiments, which can reduce the tumor volume of, for example, a human subject or a mammalian model.

[0154] 114. The composition according to any of the preceding embodiments, which can reduce the tumor volume to the same extent as or more highly than a chemotherapeutic agent in a mammalian model, for example, when measured on the 20th day after the start of treatment such that the composition is administered at a dose of 3 mg / kg every 5 days.

[0155] 115. The composition according to any of the preceding embodiments, which can reduce the tumor volume in a mammalian model compared to a PBS control, for example, when measured on the 20th day after the start of treatment such that the composition is administered at 1 mg / kg, 1.5 mg / kg, or 3 mg / kg for 4 doses every 5 days, followed by 3 doses every 3 days.

[0156] 116. The composition according to any of the preceding embodiments, wherein the tumor volume is reduced by at least about 10%, 20%, 30%, or 40% compared to a control treated with PBS on the 20th day after the start of treatment.

[0157] 117. The composition according to any of embodiments 114 - 116, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0158] 118. The composition according to any of the preceding embodiments, which can reduce the tumor volume to the same extent as or more highly than a small molecule MYC inhibitor.

[0159] 119. The composition according to embodiment 118, wherein the small molecule MYC inhibitor is MYCi975, and optionally the tumor volume is reduced by at least about 10%, 20%, 30%, or 40% compared to a control treated with MYCi975, for example, on the 20th day after the start of treatment.

[0160] 120. The composition according to any of the preceding embodiments, wherein no weight loss occurs or the weight loss that occurs is less than 3%, 2%, or 1% compared to the start of treatment.

[0161] 121. The composition according to any of the preceding embodiments, further comprising a second expression repressor, for example, the second expression repressor described herein, for example, the second expression repressor according to any of the preceding embodiments.

[0162] 122. A composition comprising: (1) A first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first nucleic acid encoding a first expression repressor that binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene or binds to a sequence proximal to the transcriptional regulatory element; (2) A formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of the following (iii) and (iv) (e.g., comprising all of (i)-(iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

Chemical formula

Chemical formula

Chemical formula

[0163] 123. The transcription regulatory element comprises a promoter, and the anchor sequence contains a CTCF binding motif, the composition according to embodiment 121 or 122.

[0164] 124. The second expression repressor binds to a downstream region adjacent to the CTCF binding motif, the composition according to any one of embodiments 121 to 123.

[0165] 125. The second expression repressor binds to an upstream region adjacent to the CTCF binding motif, the composition according to any one of embodiments 121 to 123.

[0166] 126. The above-described first expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the nucleic acid sequence of SEQ ID NO: 2, 3, 4, 71, 72, 75-86, or 200-206 or CCGCCATNTT or AANATGGCGG (where N is any nucleotide); and The composition according to any one of embodiments 121-125, wherein the above-described second expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the nucleic acid sequence of SEQ ID NO: 2, 3, 4, 71, 72, 74-86, or 200-206 or CCGCCATNTT or AANATGGCGG (where N is any nucleotide).

[0167] 127. The above-described first expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96-110, The composition according to any one of embodiments 121-126.

[0168] 128. The above-described first expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 83; and The above-described second expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 77. The composition according to any one of embodiments 121-127.

[0169] 129. A composition comprising: (1) A first nucleic acid encoding a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a promoter operably linked to the MYC gene or binding to a sequence proximal to the promoter, (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of the following (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0170] 130. The above first expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 204, and The above-described second expression repressor includes a targeting moiety that binds to a genomic locus containing at least 16, 17, 18, 19, or 20 nucleotides of either the sequence of SEQ ID NO: 199 or 201. The composition according to Embodiment 129.

[0171] A composition for reducing MYC expression, the composition comprising: (1) A first nucleic acid, i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and a first nucleic acid encoding a first expression repressor, comprising (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

Chemical formula

Chemical formula

Chemical Formula

[0172] 132. The composition according to embodiment 131, wherein the first expression repressor further comprises a first nuclear localization signal, such as an SV40 NLS, for example, the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the N-terminal side of the first targeting portion.

[0173] 133. The composition according to embodiment 131 or 132, wherein the first expression repressor further comprises a second nuclear localization signal, such as a nucleoplasmin NLS, for example, the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the C-terminal side of the first effector portion.

[0174] 134. The composition according to any one of embodiments 131 to 133, wherein the second expression repressor further comprises a first nuclear localization signal, such as an SV40 NLS, for example, the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the N-terminal side of the second targeting portion.

[0175] 135. The composition according to any one of embodiments 131 to 134, wherein the second expression repressor further comprises a second nuclear localization signal, such as a nucleoplasmin NLS, for example, the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the C-terminal side of the second effector portion.

[0176] 136. The composition according to any one of embodiments 131 to 135, wherein the first expression repressor further comprises a first linker located between the first targeting portion and the first effector portion, and optionally the first linker has the amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0177] 137. The above-described second expression repressor further includes a second linker located between the above-described second targeting portion and the above-described second effector portion, and optionally, the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 131 to 136.

[0178] 138. The above-described first expression repressor further includes an amino acid sequence on the C-terminal side of the above-described first effector portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, for example, a sequence according to SEQ ID NO: 126 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 131 to 137.

[0179] 139. The above-described second expression repressor further includes, for example, an amino acid sequence on the N-terminal side of the above-described second targeting portion, for example, up to 30, 25, 20, or 18 amino acids, for example, the sequence of amino acid P. The system according to any one of Embodiments 131 to 135.

[0180] 140. The above-described first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 131 to 139.

[0181] 141. The above-described second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 131 to 140.

[0182] 142. The above-described second targeting portion includes an amino acid sequence according to SEQ ID NO: 169, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 131 to 140.

[0183] 143. The composition according to any one of Embodiments 131 to 140, wherein the second targeting portion comprises the amino acid sequence according to SEQ ID NO: 171, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0184] 144. The composition according to any one of Embodiments 131 to 143, wherein the second expression repressor has the amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0185] 145. The composition according to any one of Embodiments 131 to 143, wherein the second expression repressor has the amino acid sequence according to SEQ ID NO: 179, 185, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0186] 146. The composition according to any one of Embodiments 121 to 145, wherein the first nucleic acid and the second nucleic acid are contained within the same nucleic acid molecule.

[0187] 147. The composition according to any one of Embodiments 121 to 145, wherein the first nucleic acid and the second nucleic acid are contained within different nucleic acid molecules.

[0188] 148. The composition according to any one of Embodiments 121 to 147, wherein the first nucleic acid and the second nucleic acid are contained within the same LNP.

[0189] 149. The composition according to any one of Embodiments 121 to 145 or 147, wherein the first nucleic acid and the second nucleic acid are contained within different LNPs.

[0190] 150. A composition for reducing MYC expression, the composition comprising: (1) The following: a) A first region encoding a first expression repressor, wherein the first expression repressor is i) a first targeting moiety having the amino acid sequence according to SEQ ID NO: 13 or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position; ii) a first effector moiety having the amino acid sequence according to SEQ ID NO: 19 or 87 or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position; comprising a first region and b) a second region encoding a second expression repressor, wherein the second expression repressor i) has the amino acid sequence according to SEQ ID NO: 7 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or has a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position; ii) has the amino acid sequence according to SEQ ID NO: 18 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or has a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position; comprising a second region; a nucleic acid comprising; and (2) a formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) a first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0191] 151. The composition according to embodiment 150, wherein the first region is on the 5'-side of the second region.

[0192] 152. The composition according to embodiment 150, wherein the first region is on the 3'-side of the second region.

[0193] 153. The composition according to embodiment 151 or 152, further comprising a nucleotide sequence encoding a first nuclear localization signal, for example, the SV40 NLS, for example, the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, wherein the first region is located, for example, on the N-terminal side of the first targeting moiety.

[0194] 154. The composition according to any one of embodiments 151 to 153, wherein the first region further comprises a second nuclear localization signal, for example, located on the C-terminal side of the first effector portion, such as the nucleoplasmin NLS, for example, a nucleotide sequence encoding the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0195] 155. The composition according to any one of embodiments 151 to 154, wherein the second region further comprises a first nuclear localization signal, for example, located on the N-terminal side of the second targeting portion, such as the SV40 NLS, for example, a nucleotide sequence encoding the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0196] 156. The composition according to any one of embodiments 151 to 155, wherein the second region further comprises a second nuclear localization signal, for example, located on the C-terminal side of the second effector portion, such as the nucleoplasmin NLS, for example, a nucleotide sequence encoding the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0197] 157. The composition according to any one of embodiments 151 to 156, wherein the first region is a first linker located between the first targeting portion and the first effector portion, and optionally further comprises a nucleotide sequence encoding a first linker having the amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0198] 158. The composition according to any one of embodiments 151 to 157, wherein the second region is a second linker located between the second targeting portion and the second effector portion, and optionally further comprises a nucleotide sequence encoding a second linker having the amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0199] 159. The composition according to any one of Embodiments 151 to 158, wherein the first region further comprises a nucleotide sequence encoding an amino acid sequence located on the C-terminal side of the first effector portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, for example, the sequence according to SEQ ID NO: 126 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0200] 160. The composition according to any one of Embodiments 151 to 159, wherein the second region further comprises an amino acid sequence located on the N-terminal side of the second targeting portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, or a nucleotide sequence encoding amino acid P.

[0201] 161. The composition according to any one of Embodiments 151 to 160, wherein the first expression repressor has the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0202] 162. The composition according to any one of Embodiments 151 to 161, wherein the second expression repressor has the amino acid sequence according to SEQ ID NO: 24, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0203] 163. The composition according to any one of Embodiments 151 to 162, wherein the first region comprises a nucleotide sequence encoding the first targeting portion, and the nucleotide sequence encoding the first targeting portion is the sequence according to SEQ ID NO: 46 or 131 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or comprises a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0204] 164. The above-mentioned first region contains a nucleotide sequence encoding the above-mentioned first effector portion, and the nucleotide sequence encoding the above-mentioned first effector portion is the sequence according to SEQ ID NO: 52 or 132, or has at least 80, 85, 90, 95, 99, or 100% identity with it, or a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, the composition according to any one of Embodiments 151 to 163.

[0205] 165. The above-mentioned second region contains a nucleotide sequence encoding the above-mentioned second targeting portion, and the nucleotide sequence encoding the above-mentioned second targeting portion is the sequence according to SEQ ID NO: 40 or has at least 80, 85, 90, 95, 99, or 100% identity with it, or a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, the composition according to any one of Embodiments 151 to 164.

[0206] 166. The above-mentioned first region contains a nucleotide sequence encoding the above-mentioned first effector portion, and the nucleotide sequence encoding the above-mentioned first effector portion is the sequence according to SEQ ID NO: 51 or has at least 80, 85, 90, 95, 99, or 100% identity with it, or a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, the composition according to any one of Embodiments 151 to 165.

[0207] 167. The above-mentioned first region contains the nucleotide sequence according to SEQ ID NO: 63 or 130, or has at least 80, 85, 90, 95, 99, or 100% identity with it, or a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the polyA sequence is optional, the composition according to any one of Embodiments 151 to 166.

[0208] 168. The composition according to any one of embodiments 151 to 167, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 57, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the number of positions different from it does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and the polyA sequence is optional.

[0209] 169. A composition for reducing MYC expression, the composition comprising: (1) The following: a) A first region encoding a first expression repressor, wherein the first expression repressor has i) a first targeting portion having an amino acid sequence according to SEQ ID NO: 13 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the number of positions different from it does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and ii) a first effector portion having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the number of positions different from it does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and the first region comprising and b) A second region encoding a second expression repressor, wherein the second expression repressor has i) a second targeting portion having an amino acid sequence according to SEQ ID NO: 169 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the number of positions different from it does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, and (ii) the amino acid sequence according to SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions different therefrom, as a second effector moiety comprising a second region a nucleic acid comprising; and (2) a formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of (iii) and (iv) (e.g., comprising all of (i)-(iv)): (i) a first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') formula (I) [Chemical formula] (in formula (I): L 1 and L 2 each independently is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)O-, or a direct bond; G 1 and G 2 each independently is unsubstituted C1-C 12 alkylene, or C2-C 12 alkenylene; G 3 is C1-C 24 alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R a is H or C1-C 12 alkyl; R1 and R 2 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , -NR 11 R 12 , or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; R 5 is H or C1-C6 alkyl; R 11 and R 12 each independently is C1-C 12 alkyl or -G 4 -OR 5 , or R 11 and R 12 together with the nitrogen to which they are attached form a 5-, 6- or 7-membered heterocycle; G 4 is C1-C 24 alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; x is 0, 1 or 2) a compound having the general structure of, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof; (i'') formula (IX):

Chemical formula

Chemical Formula

Chemical formula

[0210] A composition for reducing MYC expression, the composition comprising: (1) The following: a) A first region encoding a first expression repressor, wherein the first expression repressor has i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom and including the first region and b) A second region encoding a second expression repressor, wherein the second expression repressor has i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 171 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom and including the second region a nucleic acid comprising; and (2) A formulation comprising one or both of the following (i) and (ii), and optionally further comprising one or both of the following (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) A first compound selected from the group consisting of the following (i’), (i’’), and (i’’’): (i’) Formula (I)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0211] 171. The composition according to embodiment 169 or 170, wherein the first region is on the 5'-side of the second region.

[0212] 172. The composition according to embodiment 169 or 170, wherein the first region is on the 3'-side of the second region.

[0213] 173. The composition according to any one of embodiments 169 to 172, wherein the first region further comprises a first nuclear localization signal, such as an SV40 NLS, such as the sequence according to SEQ ID NO: 135 or a nucleotide sequence encoding a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the N-terminal side of the first targeting portion.

[0214] 174. The composition according to any one of embodiments 169 to 173, wherein the first region further comprises a second nuclear localization signal, such as a nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 or a nucleotide sequence encoding a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the C-terminal side of the first effector portion.

[0215] 175. The composition according to any one of embodiments 169 to 174, wherein the second region further comprises a first nuclear localization signal, such as an SV40 NLS, such as the sequence according to SEQ ID NO: 135 or a nucleotide sequence encoding a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the N-terminal side of the second targeting portion.

[0216] 176. The composition according to any one of embodiments 169 to 175, wherein the second region further comprises a second nuclear localization signal, such as a nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 or a nucleotide sequence encoding a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, which is located, for example, on the C-terminal side of the second effector portion.

[0217] 177. The composition according to any one of embodiments 169 to 176, wherein the first region is a first linker located between the first targeting portion and the first effector portion, and optionally further comprises a nucleotide sequence encoding a first linker having the amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0218] 178. The second region is a second linker located between the second targeting portion and the second effector portion, and optionally further comprises a nucleotide sequence encoding a second linker having the amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 169 to 177.

[0219] 179. The first region is an amino acid sequence on the C-terminal side of the first effector portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, for example, the sequence according to SEQ ID NO: 126 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 169 to 177.

[0220] 180. The second region is an amino acid sequence on the N-terminal side of the second targeting portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, or a nucleotide sequence encoding amino acid P. The composition according to any one of Embodiments 169 to 179.

[0221] 181. The first expression repressor has the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 169 to 180.

[0222] 182. The second expression repressor has the amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The composition according to any one of Embodiments 150 to 181.

[0223] 183. The composition according to any one of Embodiments 150 to 182, wherein the second expression repressor has the amino acid sequence according to SEQ ID NO: 179 or 185, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0224] 184. The composition according to any one of Embodiments 150 to 183, wherein the first expression repressor includes the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, and the second expression repressor has the amino acid sequence according to SEQ ID NO: 24, 141, 177, 179, 183, or 185, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0225] 185. The composition according to any one of Embodiments 150 to 184, wherein the first region includes the nucleotide sequence encoding the first targeting portion, and the nucleotide sequence encoding the first targeting portion is the sequence according to SEQ ID NO: 46 or 131, or has at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0226] 186. The composition according to any one of Embodiments 150 to 185, wherein the first region includes the nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion is the sequence according to SEQ ID NO: 52 or 132, or has at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0227] 187. The composition according to any one of embodiments 150 to 186, wherein the second region is the nucleotide sequence according to SEQ ID NO: 173 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the polyA sequence is optional.

[0228] 188. The composition according to any one of embodiments 150 to 187, wherein the second region is the nucleotide sequence according to SEQ ID NO: 175 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the polyA sequence is optional.

[0229] 189. The composition according to any one of embodiments 150 to 188, wherein the second region includes the nucleotide sequence encoding the second effector portion, and the nucleotide sequence encoding the second effector portion is the sequence according to SEQ ID NO: 51 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0230] 190. The composition according to any one of embodiments 150 to 189, wherein the first region is the nucleotide sequence according to SEQ ID NO: 63 or 130, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the polyA sequence is optional.

[0231] 191. The composition according to any one of Embodiments 150 to 190, wherein the second region comprises the nucleotide sequence according to SEQ ID NO: 189, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the polyA sequence is optional.

[0232] 192. The composition according to any one of Embodiments 150 to 191, wherein the second region comprises the nucleotide sequence according to SEQ ID NO: 194, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the polyA sequence is optional.

[0233] 193. The composition according to any one of Embodiments 150 to 192, wherein the first region comprises the nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion is the sequence according to SEQ ID NO: 52 or 132, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0234] 194. The composition according to any one of Embodiments 150 to 193, wherein the first region comprises the nucleotide sequence encoding the first targeting portion, and the nucleotide sequence encoding the first targeting portion is the sequence according to SEQ ID NO: 46 or 131, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0235] 195. The above-mentioned second region contains a nucleotide sequence encoding the above-mentioned second effector portion, and the nucleotide sequence encoding the above-mentioned second effector portion is the sequence according to SEQ ID NO: 51 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the positions different from it exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position does not exceed, the composition according to any one of embodiments 150 to 194.

[0236] 196. The above-mentioned second region is the nucleotide sequence according to SEQ ID NO: 189, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the positions different from it exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position does not exceed, the composition according to any one of embodiments 150 to 195.

[0237] 197. The above-mentioned second region is the nucleotide sequence according to SEQ ID NO: 194, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the positions different from it exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position does not exceed, the composition according to any one of embodiments 150 to 196.

[0238] 198. The nucleotide sequence according to SEQ ID NO: 93, 112, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the positions different from it exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position does not exceed, the composition according to any one of embodiments 150 to 197.

[0239] 199. The composition according to any one of Embodiments 150 to 198, having a nucleotide sequence according to SEQ ID NO: 196 or 197, or having a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto, or having a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0240] 200. The composition according to any one of Embodiments 121 to 199, wherein the first expression repressor comprises the first effector portion.

[0241] 201. The composition according to any one of Embodiments 121 to 200, wherein the second expression repressor comprises the second effector portion.

[0242] 202. The composition according to any one of Embodiments 121 to 201, wherein the first effector portion has an amino acid sequence different from that of the second effector portion.

[0243] 203. The composition according to any one of Embodiments 121 to 202, wherein the first effector portion is a persistent effector portion.

[0244] 204. The composition according to any one of Embodiments 121 to 128 or 150 to 203, wherein the first effector portion is a transient effector portion.

[0245] 205. The composition according to any one of Embodiments 121 to 204, wherein the first effector portion is an epigenetic modification portion.

[0246] 206. The composition according to any one of Embodiments 121 to 149, 169 to 203, or 205, wherein the first effector portion comprises a histone methyltransferase.

[0247] 207. The composition according to embodiment 206, wherein the first effector portion comprises SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any of these, for example, a protein selected from any of the SET domains of these.

[0248] 208. The composition according to any one of embodiments 121 - 149, 169 - 203, or 205, wherein the first effector portion comprises a histone demethylase (e.g., lysine demethylase).

[0249] 209. The composition according to embodiment 208, wherein the first effector portion comprises a protein selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or fragment of any of these).

[0250] 210. The composition according to any one of embodiments 121 - 149, 169 - 203, or 205, wherein the first effector portion comprises a histone deacetylase.

[0251] 211. The composition according to embodiment 210, wherein the first effector portion comprises a protein selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any of these.

[0252] 212. The composition according to any one of embodiments 121 - 203 or 206, wherein the first effector portion comprises a DNA methyltransferase.

[0253] 213. The composition according to embodiment 212, wherein the first effector portion comprises a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any of these.

[0254] 214. The composition according to any one of embodiments 121-149, 166-202, or 204, wherein the first effector portion is a transcriptional repressor portion, for example, comprising a transcriptional repressor.

[0255] 215. The composition according to embodiment 204 or 205, wherein the first effector portion comprises a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these.

[0256] 216. The composition according to any one of embodiments 121-215, wherein the first effector portion promotes epigenetic modification of the transcriptional regulatory element or a sequence adjacent thereto.

[0257] 217. The composition according to any one of embodiments 121-216, wherein the first effector portion catalyzes epigenetic modification of the transcriptional regulatory element or a sequence adjacent thereto.

[0258] 218. The composition according to any one of embodiments 121-128, 200, or 203-217, wherein the second expression repressor does not comprise an effector portion.

[0259] 219. The composition according to any one of embodiments 121-218, wherein the second effector portion is a transient effector portion.

[0260] 220. The composition according to any one of embodiments 121-128 or 200-217, wherein the second effector portion is a persistent effector portion.

[0261] 221. The composition according to any one of Embodiments 121 to 217 or 220, wherein the second effector part is an epigenetic modification part.

[0262] 222. The composition according to any one of Embodiments 121 to 128, 200 to 217, 220, or 221, wherein the second effector part contains a histone methyltransferase.

[0263] 223. The composition according to Embodiment 222, wherein the second effector part contains a protein selected from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any of these, for example, a protein selected from the SET domain of any of these.

[0264] 224. The composition according to any one of Embodiments 121 to 128, 200 to 217, 220, or 221, wherein the second effector part contains a histone demethylase (e.g., lysine demethylase).

[0265] 225. The composition according to Embodiment 224, wherein the second effector part contains a protein selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional variant or fragment of any of these.

[0266] 226. The composition according to any one of Embodiments 121 to 128, 200 to 217, 220, or 221, wherein the second effector part contains a histone deacetylase.

[0267] 227. The composition according to embodiment 226, wherein the second effector portion comprises a protein selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any of these.

[0268] 228. The composition according to any one of embodiments 121 to 128, 200 to 217, 220, or 221, wherein the second effector portion comprises a DNA methyltransferase.

[0269] 229. The composition according to embodiment 228, wherein the second effector portion comprises a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any of these.

[0270] 230. The composition according to any one of embodiments 121 to 217 or 219, wherein the second effector portion is a transcriptional repressor portion.

[0271] 231. The composition according to embodiment 230, wherein the second effector portion promotes epigenetic modification of the anchor sequence or a sequence adjacent thereto.

[0272] 232. The composition according to embodiment 229 or 230, wherein the second effector portion binds to one or more endogenous epigenetic modification proteins or one or more endogenous transcriptional modification proteins.

[0273] 233. The composition according to any one of embodiments 229 to 232, wherein the second effector portion comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these.

[0274] 234. The first effector portion is a persistent effector portion, and the second effector portion is a transient effector portion, The composition according to any one of Embodiments 121 to 203, 205 to 213, 216, 217, 219, or 230 to 233.

[0275] 235. The composition according to Embodiment 234, wherein the first effector portion is an epigenetic modification portion.

[0276] 236. The composition according to Embodiment 233 or 234, wherein the second effector portion is a transcriptional repressor portion.

[0277] 237. The first effector portion includes a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any of these, or a combination of any of these, and the second effector portion includes a transcriptional repressor or a functional variant or fragment of any of these, The composition according to any one of Embodiments 233 to 236.

[0278] 238. The first effector portion includes a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any of these, or a combination of any of these, and the second expression repressor does not include the second effector portion, The composition according to any one of Embodiments 212 to 128, 196, 200, 203, 205 to 213, or 216 to 218.

[0279] 239. The first effector portion includes SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, any functional variant or fragment thereof, or any combination thereof, and The second effector portion includes KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, any functional variant or fragment thereof, or any combination thereof, The composition according to any one of Embodiments 121 to 128, 203 to 213, 216, 217, 219, 220, or 230 to 236.

[0280] 240. The first effector portion includes a DNA methyltransferase, and The second effector portion includes a transcriptional repressor, The composition according to any one of Embodiments 121 to 203, 205, 212, 213, 216, 217, 219, 221, 230 to 237, or 239.

[0281] 241. The first effector portion includes a DNA methyltransferase, and The second expression repressor does not include the second effector portion, The composition according to any one of Embodiments 121 to 128, 200, 203, 206, 212, 213, 216 to 218, or 238.

[0282] 242. The composition according to any one of Embodiments 121 to 128, 206, 212, 213, or 216 to 241, wherein the first effector portion comprises MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof.

[0283] 243. The composition according to any one of Embodiments 121 to 217, 220, 230 to 240, or 242, wherein the second effector portion comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or any functional variant or fragment thereof.

[0284] 244. The first effector portion comprises MQ1 or any functional variant or fragment thereof, and the second effector comprises KRAB or any functional variant or fragment thereof. The composition according to any one of Embodiments 121 to 217, 212, 213, 219, 230 to 240, 242, or 243.

[0285] 245. The first effector portion comprises MQ1 or any functional variant or fragment thereof, and the second expression repressor does not comprise the second effector portion. The composition according to any one of Embodiments 121 to 128, 200, 203, 205 to 213, 216 to 218, 235, 238, 241, or 242.

[0286] 246. The composition according to any one of Embodiments 121 to 206, wherein the first expression repressor comprises an amino acid sequence selected from any one of SEQ ID NOs: 22 to 37, 129, 133, 134, 139 to 149, 177 to 180, or 183 to 186, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0287] 247. The composition according to any one of Embodiments 121 to 204, 206, 212, 213, 219 to 222, 228 to 229, 242, 243, or 246, wherein the second expression repressor comprises an amino acid sequence selected from any one of SEQ ID NOs: 22 to 37, 129, 133, 134, 139 to 149, 177 to 180, or 183 to 186, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0288] 248. The composition according to any one of Embodiments 121 to 204, 206, 212, 213, 219 to 222, 228 to 229, 242, 243, 246, or 247, wherein the first expression repressor comprises an amino acid sequence of SEQ ID NOs: 30, 129, 133, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second expression repressor comprises an amino acid sequence of SEQ ID NOs: 24, 134, 141, 177, 179, 183, or 185, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0289] 249. The composition according to any one of Embodiments 121 to 204, 206, 212, 213, 219 to 222, 228 to 229, 242, 243, or 246 to 248, wherein the first expression repressor is encoded by the first nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second expression repressor is encoded by the second nucleotide sequence of SEQ ID NO: 57, 189, or 194, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0290] 250. The composition according to any one of Embodiments 121 to 204, 206, 212, 213, 219 to 222, 228 to 229, 242, 243, or 246 to 249, wherein the first and second repressors are encoded by the nucleic acid sequence of SEQ ID NO: 93, 94, 112, 113, 196, or 197, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0291] 251. The composition according to Embodiment 250, wherein the expression repressor comprises the amino acid sequence of SEQ ID NO: 91, 92, 121, 122, 181, 182, 187, or 188, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0292] 252. The first expression repressor, from the N-terminus to the C-terminus: (i) a first nuclear localization signal, such as the SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (ii) a first targeting moiety, such as a zinc finger binding domain, such as ZF9; for example, the sequence according to SEQ ID NO: 13; (iii) a first effector moiety, such as a DNA methyltransferase, such as MQ1; for example, the sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, such as the nucleoplasmin NLS; for example, the sequence according to SEQ ID NO: 136 comprising; and the second expression repressor is, from the N-terminus to the C-terminus: (v) a third nuclear localization signal, such as the SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (vi) a second targeting moiety, such as a zinc finger binding domain, such as ZF3; for example, the sequence according to SEQ ID NO: 7; (vii) a second effector moiety, such as KRAB, such as the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, such as the nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 A composition according to any one of embodiments 121-203, 205, 212, 213, 216, 217, 219, 221, 230-237, 239, 240, 242, 243, or 246-250, comprising.

[0293] 253. The first expression repressor is, from the N-terminus to the C-terminus: (i) a first nuclear localization signal, such as the SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (ii) a first targeting moiety, such as a zinc finger binding domain, such as ZF9; for example, the sequence according to SEQ ID NO: 13; (iii) a first effector moiety, such as a DNA methyltransferase, such as MQ1; for example, the sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, such as the nucleoplasmin NLS; for example, the sequence according to SEQ ID NO: 136 comprising; and said second expression repressor, from the N-terminus to the C-terminus: (v) a third nuclear localization signal, such as SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (vi) a second targeting moiety, such as a zinc finger binding domain, such as ZF54; for example, the sequence according to SEQ ID NO: 169; (vii) a second effector moiety, such as KRAB, such as the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, such as nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 A composition according to any one of Embodiments 121-203, 205, 212, 213, 216, 217, 219, 221, 230-237, 239, 240, 242, 243, or 246-250, comprising.

[0294] 254. Said first expression repressor, from the N-terminus to the C-terminus: (i) a first nuclear localization signal, such as SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (ii) a first targeting moiety, such as a zinc finger binding domain, such as ZF9; for example, the sequence according to SEQ ID NO: 13; (iii) a first effector moiety, such as DNA methyltransferase, such as MQ1; for example, the sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, such as nucleoplasmin NLS; such as the sequence according to SEQ ID NO: 136 comprising; and said second expression repressor, from the N-terminus to the C-terminus: (v) a third nuclear localization signal, such as SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (vi) a second targeting moiety, such as a zinc finger binding domain, such as ZF67; for example, the sequence according to SEQ ID NO: 171; (vii) a second effector moiety, such as KRAB, such as the sequence according to SEQ ID NO: 18; and (viii) A fourth nuclear localization signal, such as the nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 The composition according to any one of Embodiments 121 to 203, 205, 212, 213, 216, 217, 219, 221, 230 to 237, 239, 240, 242, 243, or 246 to 250, comprising

[0295] 255. The composition according to any one of Embodiments 121 to 254, which can reduce the expression of MYC more highly as compared with the above-described first expression repressor alone or the above-described second expression repressor alone.

[0296] 256. The composition according to any one of Embodiments 131 to 200 or 248 to 255, which can reduce the expression of MYC more highly as compared with any of the expression repressors of SEQ ID NOs: 22, 23, 25 to 29, 31 to 37 alone or in combination.

[0297] 257. The composition according to any one of Embodiments 121 to 256, which can reduce the tumor volume of, for example, a human subject or a mammalian model.

[0298] 258. The composition according to any one of Embodiments 131 to 199 or 248 to 255, which can reduce the tumor volume to the same extent as or more highly than a chemotherapeutic agent, for example, when measured, for example, on the 20th day after the start of treatment such that the above-described expression repressor is administered at a dose of 3 mg / kg every 5 days in a model composition as described in Example 15, for example, in a mammalian model.

[0299] 259. The composition according to any one of Embodiments 131 to 199 or 248 to 258, which can reduce the tumor volume more highly than a chemotherapeutic agent, for example, when measured, for example, on the 15th day after the start of treatment such that the above-described composition is administered at a dose of 6 mg / kg every 5 days in a model composition as described in Example 14, for example, in a mammalian model.

[0300] 260. For example, on the 20th day after the start of treatment, the composition according to any one of Embodiments 131-199 or 248-259, wherein the tumor volume is reduced by at least about 10%, 20%, 30%, 40%, 50%, or 60% compared to the control treated with PBS.

[0301] 261. The composition according to Embodiment 260, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0302] 262. The composition according to any one of Embodiments 131-199 or 248-259, which can reduce the tumor volume to the same extent as or more highly than a small molecule MYC inhibitor.

[0303] 263. The composition according to Embodiment 262, wherein the small molecule MYC inhibitor is MYCi975, and optionally the tumor volume is reduced by at least about 10%, 20%, 30%, or 40% compared to the control treated with the above MYCi975, for example, on the 20th day after the start of treatment.

[0304] 264. The composition according to any one of Embodiments 121-263, wherein there is no weight loss or the weight loss that occurs is less than 3%, 2%, or 1% compared to the start of treatment.

[0305] 265. The composition according to any one of Embodiments 121-264, wherein the first targeting moiety is selected from a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0306] 266. The composition according to any one of Embodiments 121-265, wherein the second targeting moiety is selected from a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0307] 267. The composition according to any one of embodiments 121 to 266, wherein the first targeting moiety comprises a CRISPR / Cas domain (for example, a first CRISPR / Cas domain).

[0308] 268. The composition according to any one of embodiments 121 to 267, wherein the second targeting moiety comprises a second CRISPR / Cas domain (for example, a second CRISPR / Cas domain).

[0309] 269. The composition according to embodiment 268, wherein (i) the first CRISPR / Cas domain binds to a first guide RNA, (ii) the second CRISPR / Cas domain binds to a second guide RNA, or (iii) both (i) and (ii).

[0310] 270. The first CRISPR / Cas domain does not bind to the second guide RNA, or binds with a K of at least 10, 20, 50, 100, 1000, or 10,000 nM, and the second CRISPR / Cas domain does not bind to the first guide RNA, or binds with a K of at least 10, 20, 50, 100, 1000, or 10,000 nM. The composition according to embodiment 268 or 269. D and binds, and the second CRISPR / Cas domain does not bind to the first guide RNA, or binds with a K of at least 10, 20, 50, 100, 1000, or 10,000 nM. D The composition according to embodiment 268 or 269.

[0311] 271. The composition according to any one of embodiments 266 to 270, wherein the first CRISPR / Cas domain comprises an amino acid sequence different from that of the second CRISPR / Cas domain.

[0312] 272. The composition according to any one of embodiments 266 to 271, wherein the first or second CRISPR / Cas domain comprises the amino acid sequence of a Cas protein or a Cpf1 protein selected from Table 1 or a variant thereof (for example, a mutant).

[0313] 273. The composition according to any one of embodiments 266 to 272, wherein the first CRISPR / Cas domain comprises the amino acid sequence of a Cas protein or Cpf1 protein selected from Table 1 or a variant thereof (e.g., a mutant), and the second CRISPR / Cas domain comprises the amino acid sequence of a different Cas protein or Cpf1 protein selected from Table 1 or a variant thereof (e.g., a mutant).

[0314] 274. The composition according to any one of embodiments 121 to 266, wherein the first targeting moiety comprises a zinc finger domain (e.g., a first zinc finger domain).

[0315] 275. The composition according to any one of embodiments 121 to 266 or 274, wherein the second targeting moiety comprises a zinc finger domain (e.g., a second zinc finger domain).

[0316] 276. The composition according to any one of embodiments 121 to 267, 274, or 275, wherein the first targeting moiety comprises a first zinc finger domain and the second targeting moiety comprises a second zinc finger domain.

[0317] 277. The composition according to any one of embodiments 274 to 276, wherein the first zinc finger domain and the second zinc finger domain bind to the same genomic locus, e.g., have the same amino acid sequence.

[0318] 278. The composition according to any one of embodiments 274 to 277, wherein the first zinc finger domain and the second zinc finger domain have different amino acid sequences or bind to different genomic loci.

[0319] 279. The composition according to any one of Embodiments 121 to 267 or 273 to 278, wherein the first zinc finger molecule comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally not more than 11, 10, 9, 8, 7, 6, or 5 additional zinc fingers).

[0320] 280. The composition according to any one of Embodiments 273 to 279, wherein the first zinc finger molecule comprises 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 zinc fingers.

[0321] 281. The composition according to any one of Embodiments 274 to 280, wherein the first zinc finger domain comprises 3 or 9 zinc fingers.

[0322] 282. The composition according to any one of Embodiments 274 to 281, wherein the second zinc finger domain comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally not more than 11, 10, 9, 8, 7, 6, or 5 additional zinc fingers).

[0323] 283. The composition according to any one of Embodiments 274 to 282, wherein the second zinc finger domain comprises 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 zinc fingers.

[0324] 284. The composition according to any one of embodiments 274 to 283, wherein the second zinc finger domain contains 3 or 9 zinc fingers.

[0325] 285. The composition according to any one of embodiments 121 to 284, wherein the first targeting moiety contains a TAL effector domain (e.g., a first TAL effector domain).

[0326] 286. The composition according to any one of embodiments 121 to 266 or 285, wherein the second targeting moiety contains a TAL effector domain (e.g., a second TAL effector domain).

[0327] 287. The composition according to embodiment 285 or 286, wherein the first TAL effector domain contains at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, no more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).

[0328] 288. The composition according to any one of embodiments 285 to 287, wherein the first TAL effector domain contains 2 to 40, 5 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 2 to 35, 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, 30 to 35, 2 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 2 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 2 to 20, 5 to 20, 10 to 20, 15 to 20, 2 to 15, 5 to 15, 10 to 15, 2 to 10, 5 to 10, or 2 to 5 central repeats.

[0329] 289. The composition according to any one of embodiments 285 to 288, wherein the second TAL effector domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, not more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).

[0330] 290. The composition according to any one of embodiments 285 to 289, wherein the second TAL effector domain comprises 2 to 40, 5 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 2 to 35, 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, 30 to 35, 2 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 2 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 2 to 20, 5 to 20, 10 to 20, 15 to 20, 2 to 15, 5 to 15, 10 to 15, 2 to 10, 5 to 10, or 2 to 5 central repeats.

[0331] 291. The composition according to any one of embodiments 121 to 290, wherein the first targeting moiety comprises a nucleic acid (e.g., a first nucleic acid).

[0332] 292. The composition according to any one of embodiments 131 to 291, wherein the second targeting moiety comprises a nucleic acid (e.g., a second nucleic acid).

[0333] 293. The composition according to any one of embodiments 131 to 292, wherein the first targeting moiety comprises a polypeptide (e.g., a first polypeptide).

[0334] 294. The composition according to any one of embodiments 131 to 293, wherein the second targeting moiety comprises a polypeptide (e.g., a second polypeptide).

[0335] 295. The composition according to embodiment 293 or 294, wherein the nucleic acid is covalently bound to the polypeptide.

[0336] The composition according to embodiment 294 or 295, wherein the nucleic acid is non-covalently bound to the polypeptide.

[0337] 297. The composition according to any one of embodiments 281 to 296, wherein the nucleic acid comprises a sequence complementary to the transcription regulatory element or a sequence proximate thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatch compared to the transcription regulatory element or a sequence proximate thereto.

[0338] 298. The composition according to any one of embodiments 281 to 297, wherein the nucleic acid comprises a sequence complementary to the anchor sequence or a sequence proximate thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatch compared to the anchor sequence or a sequence proximate thereto.

[0339] 299. The composition according to any one of embodiments 281 to 298, wherein the nucleic acid comprises DNA, peptide nucleic acid (PNA), peptide-oligonucleotide conjugate, locked nucleic acid (LNA), bridged nucleic acid (BNA), polyamide, triple-stranded forming oligonucleotide, antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecule.

[0340] 300. The composition according to any one of embodiments 281 to 299, wherein the nucleic acid comprises gRNA.

[0341] 301. The composition according to any one of embodiments 281 to 300, wherein the nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity to any one of SEQ ID NOs: 1 to 4, or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions different therefrom.

[0342] 302. The composition according to any one of Embodiments 281 to 301, wherein the first nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 1 to 4, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the second nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 1 to 4, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0343] 303. The composition according to any one of Embodiments 281 to 301, wherein the first nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 96 to 110, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the second nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 96 to 110, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0344] 304. The composition according to any one of Embodiments 121 to 303, wherein the transcription regulatory element comprises a promoter.

[0345] 305. The composition according to any one of Embodiments 121 to 304, wherein the transcription regulatory element comprises an enhancer; for example, a super enhancer.

[0346] 306. The composition according to any one of Embodiments 121 to 305, wherein the anchor sequence comprises a CTCF binding motif.

[0347] 307. The composition according to any one of Embodiments 121 to 306, wherein the anchor sequence comprises a YY1 binding motif.

[0348] The composition according to any one of Embodiments 121 to 307, wherein the anchor array comprises the sequence of SEQ ID NO: 71 or 72, or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0349] The composition according to any one of Embodiments 121 to 308, wherein the anchor array comprises a sequence related to the nucleic acid sequence of CCGCCATNTT or AANATGGCGG (N is any nucleotide), or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0350] The composition according to any one of Embodiments 121 to 309, wherein the anchor array is on the same chromosome as the MYC gene.

[0351] The composition according to any one of Embodiments 121 to 310, wherein the anchor array is upstream of the MYC gene (for example, upstream of the TSS or upstream of the promoter).

[0352] The composition according to any one of Embodiments 121 to 311, wherein the anchor array is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (for example, from the TSS or promoter of the MYC gene).

[0353] The composition according to any one of Embodiments 121 to 312, wherein the anchor array is 0.1 to 0.5, 0.1 to 1, 0.1 to 5, 0.1 to 10, 0.1 to 50, 0.1 to 100, 0.1 to 500, 0.1 to 1000, 0.5 to 1, 0.5 to 5, 0.5 to 10, 0.5 to 50, 0.5 to 100, 0.5 to 500, 0.5 to 1000, 1 to 5, 1 to 10, 1 to 50, 1 to 100, 1 to 500, 1 to 1000, 5 to 10, 5 to 50, 5 to 100, 5 to 500, 5 to 1000, 10 to 50, 10 to 100, 10 to 500, 10 to 1000, 50 to 100, 50 to 500, 50 to 1000, 100 to 500, 100 to 1000, or 500 to 1000 kilobases away from the MYC gene (for example, from the TSS or promoter of the MYC gene).

[0354] 314. The composition according to any one of embodiments 121 - 309 or 311 - 313, wherein the anchor array is on a chromosome different from the MYC gene.

[0355] 315. The composition according to any one of embodiments 121 - 314, wherein the second targeting moiety binds to the anchor array or a sequence proximate to the anchor array with an affinity sufficient to compete for binding with an endogenous polypeptide (e.g., CTCF or YY1).

[0356] 316. The composition according to any one of embodiments 121 - 315, wherein the first targeting moiety binds to a sequence at chromosomal coordinates 128746342 - 128746364, 128746321 - 128746343, or 128746525 - 128746547, or a sequence proximate thereto.

[0357] 317. The composition according to any one of embodiments 121 - 315, wherein the first targeting moiety binds to a sequence at chromosomal coordinates 128746405 - 128746425, 128748069 - 128748089, 129188825 - 129188845, or 129188822 - 129188842, or a sequence proximate thereto.

[0358] 318. The composition according to any one of embodiments 121 - 317, wherein the second targeting moiety binds to a sequence at chromosomal coordinates 128748014 - 128748036, or a sequence proximate thereto.

[0359] 319. The composition according to any one of embodiments 121 - 317, wherein the second targeting moiety binds to a sequence at chromosomal coordinates 128746405 - 128746425, 128748069 - 128748089, 129188825 - 129188845, or 129188822 - 129188842, or a sequence proximate thereto.

[0360] 320. The composition according to any one of embodiments 121 to 319, wherein the first expression repressor is a fusion molecule.

[0361] 321. The composition according to any one of embodiments 121 to 319, wherein the second expression repressor is a fusion molecule.

[0362] 322. The composition according to any one of embodiments 121 to 321, wherein the first expression repressor comprises a linker.

[0363] 323. The composition according to any one of embodiments 121 to 322, wherein the second expression repressor comprises a linker.

[0364] 324. The first expression repressor includes a targeting portion containing a first CRISPR / Cas molecule, for example, a CRISPR / Cas protein that is inactive as a first catalyst, and an effector portion containing an epigenetic modification portion; and The second expression repressor includes a targeting portion containing a second CRISPR / Cas molecule, for example, a CRISPR / Cas protein that is inactive as a second catalyst, and an effector portion optionally containing a transcriptional repressor, The composition according to any one of embodiments 121 to 173 or 291 to 323.

[0365] 325. The first expression repressor includes a targeting portion containing a first zinc finger domain and an effector portion containing an epigenetic modification portion; and The second expression repressor includes a targeting portion containing a second zinc finger domain and an effector portion optionally containing a transcriptional repressor, The composition according to any one of embodiments 121 to 166, 274 to 184, or 281 to 323.

[0366] 326. The first expression repressor includes a targeting portion containing a CRISPR / Cas molecule, for example, a CRISPR / Cas protein that is inactive as a catalyst, and an effector portion containing an epigenetic modification portion; and The second expression repressor includes a targeting portion containing a zinc finger domain and, optionally, an effector portion containing a transcriptional repressor. The composition according to any one of Embodiments 121 to 123, 268, 274, or 181 to 324.

[0367] 327. The first expression repressor includes a targeting portion containing a zinc finger domain and an effector portion containing an epigenetic modification portion; and The second expression repressor includes a targeting portion containing a CRISPR / Cas domain, for example, a CRISPR / Cas protein that is inactive as a catalyst, and, optionally, an effector portion containing a transcriptional repressor. The composition according to any one of Embodiments 121 to 266, 274, or 281 to 324.

[0368] 328. The zinc finger domain (for example, the first or second zinc finger domain) includes 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 zinc fingers, for example, 3 or 9 zinc fingers. The composition according to any one of Embodiments 266 or 274 to 324.

[0369] 329. The epigenetic modification portion includes a DNA methyltransferase. The composition according to any one of Embodiments 328.

[0370] The composition according to any one of Embodiments 121 to 328, wherein the epigenetic modification portion contains MQ1 or a functional variant or fragment thereof.

[0371] The composition according to any one of Embodiments 121 to 330, wherein the second expression repressor contains an effector portion containing a transcriptional repressor.

[0372] The composition according to any one of Embodiments 121 to 329, wherein the transcriptional repressor contains KRAB or a functional variant or fragment thereof.

[0373] The composition according to any one of Embodiments 121 to 332, wherein the first expression repressor contains an amino acid sequence of any one of SEQ ID NOs: 28 to 33 or 35 to 37, 145 to 149, 151, 152, or is at least 80, 85, 90, 95, 99, or 100% identical thereto, or contains a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0374] The composition according to any one of Embodiments 121 to 333, wherein the second expression repressor contains an amino acid sequence of any one of SEQ ID NOs: 22 to 27, 34, 139 to 144, 150, 177 to 180, 183 to 186, or is at least 80, 85, 90, 95, 99, or 100% identical thereto, or contains a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0375] The composition according to any one of Embodiments 121 to 334, wherein when the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto, the expression of MYC in cells decreases.

[0376] 336. For example, when measured by QPCR or ELISA, the composition according to embodiment 333, wherein the expression is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% as compared to the expression in the absence of the first expression repressor.

[0377] 337. When the first expression repressor binds to the transcription regulatory element, for example, when measured by QPCR or ELISA, the expression of MYC is decreased such that it can be observed over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, in the composition according to embodiment 332 or 333.

[0378] 338. When the first expression repressor binds to the transcription regulatory element, the expression of MYC is decreased such that it can be observed at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection, in the composition according to any one of embodiments 335 - 338.

[0379] 339. When the second expression repressor binds to the anchor sequence or a sequence in proximity thereto, the expression of MYC in the cell is decreased, in the composition according to any one of embodiments 334 - 338.

[0380] 340. For example, when measured by QPCR or ELISA, the composition according to embodiment 339, wherein the expression is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% as compared to the expression in the absence of the second expression repressor.

[0381] When the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC decreases such that it can be observed over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, as measured by, for example, QPCR or ELISA. The composition according to embodiment 339 or 340.

[0382] When the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC decreases such that it can be observed at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection. The composition according to embodiment 340 or 341.

[0383] When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto, and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC in the cell decreases. The composition according to any one of embodiments 335 to 342.

[0384] When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto, and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC decreases such that it can be observed at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection. The composition according to any one of embodiments 335 to 342.

[0385] 345. For example, when measured by QPCR or ELISA, the composition according to embodiment 343 or 344, wherein the expression is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% as compared to the expression in the absence of the first and second expression repressors.

[0386] 346. When the first expression repressor binds to the transcription regulatory element or a sequence proximate thereto, and the second expression repressor binds to the anchor sequence or a sequence proximate thereto, for example, when measured by QPCR or ELISA, the expression of MYC is reduced such that it can be observed over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions. The composition according to any one of embodiments 335 - 345.

[0387] 347. The decrease in expression caused by the first expression repressor binding to the transcription regulatory element or a sequence proximate thereto and the second expression repressor binding to the anchor sequence or a sequence proximate thereto is greater than the decrease in expression caused individually by the first expression repressor binding to the transcription regulatory element or a sequence proximate thereto or the second expression repressor binding to the anchor sequence or a sequence proximate thereto. The composition according to any one of embodiments 335 - 346.

[0388] When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression, as measured by, for example, QPCR or ELISA, is 1.05× (i.e., 1.05-fold), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× lower than when either the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto or the second expression repressor binds to the anchor sequence or a sequence adjacent thereto alone. The composition according to Embodiment 347.

[0389] The decrease in expression caused by the first expression repressor binding to the transcription regulatory element or a sequence adjacent thereto and the second expression repressor binding to the anchor sequence or a sequence adjacent thereto persists over a longer period (e.g., over a greater number of hours, days, or cell divisions) compared to the decrease in expression caused by either the first expression repressor binding to the transcription regulatory element or a sequence adjacent thereto or the second expression repressor binding to the anchor sequence or a sequence adjacent thereto alone. The composition according to any one of Embodiments 335 to 348.

[0390] When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression is 1.05× (i.e., 1.05-fold), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× longer (e.g., when measured in terms of number of hours, number of days, or number of cell divisions) compared to either the binding of the first expression repressor to the transcription regulatory element or a sequence adjacent thereto or the binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto, the composition according to Embodiment 349.

[0391] 351. When the first expression repressor binds to the promoter or a sequence adjacent thereto and the second expression repressor binds to the super enhancer or a sequence adjacent thereto, the expression of MYC decreases to an extent that can be observed over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, as measured by, for example, QPCR or ELISA, the composition according to any one of Embodiments 335 to 350.

[0392] 352. The decrease in expression caused by the binding of the first expression repressor to the promoter or a sequence adjacent thereto and the binding of the second expression repressor to the super enhancer or a sequence adjacent thereto is greater than the decrease in expression caused by either the binding of the first expression repressor to the promoter or a sequence adjacent thereto or the binding of the second expression repressor to the super enhancer or a sequence adjacent thereto, the composition according to any one of Embodiments 335 to 351.

[0393] 353. When the first expression repressor binds to the promoter or a sequence proximate thereto, and the second expression repressor binds to the super-enhancer or a sequence proximate thereto, the expression, as measured by, for example, QPCR or ELISA, is 1.05× (i.e., 1.05-fold), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× lower than when either the first expression repressor binds to the promoter or a sequence proximate thereto or the second expression repressor binds to the super-enhancer or a sequence proximate thereto alone. The composition according to Embodiment 352.

[0394] 354. The decrease in expression resulting from the first expression repressor binding to the promoter or a sequence proximate thereto and the second expression repressor binding to the super-enhancer or a sequence proximate thereto persists over a longer period of time (e.g., over a greater number of hours, days, or cell divisions) compared to the decrease in expression resulting from either the first expression repressor binding to the promoter or a sequence proximate thereto or the second expression repressor binding to the super-enhancer or a sequence proximate thereto alone. The composition according to any one of Embodiments 335 to 353.

[0395] When the first expression repressor binds to the promoter or a sequence adjacent thereto and the second expression repressor binds to the super enhancer or a sequence adjacent thereto, for example, when measured by QPCR or ELISA, the expression is 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× longer (e.g., when measured in hours, days, or number of cell divisions) than when either the first expression repressor binds to the promoter or a sequence adjacent thereto or the second expression repressor binds to the super enhancer or a sequence adjacent thereto alone, the composition according to embodiment 354.

[0396] 356. The composition according to any one of embodiments 335 to 355, wherein the expression decreases to such an extent that it can be recognized infinitely (e.g., over a period longer than can be experimentally measured).

[0397] 357. The composition according to any one of embodiments 335 to 356, wherein when the first expression repressor binds to the transcriptional regulatory element or a sequence adjacent thereto, the viability of the cells containing the transcriptional regulatory element or a sequence adjacent thereto decreases.

[0398] 358. When a plurality of cells are contacted with the first expression repressor or a nucleic acid encoding the first expression repressor, the viability of the plurality of cells decreases, and optionally, the plurality of cells includes cancerous and non-cancerous cells and / or infected and non-infected cells, the composition according to any one of embodiments 335 to 357.

[0399] 359. The composition according to embodiment 358, wherein the viability decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% as compared to the viability in the absence of the first expression repressor, for example, when measured by CellTiter Glo.

[0400] 360. By administering the above-described first expression repressor, apoptosis occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells). The composition according to any one of Embodiments 335 to 359.

[0401] 361. When the above-described second expression repressor binds to the above-described anchor sequence or a sequence adjacent thereto, the viability of cells containing the above-described anchor sequence or a sequence adjacent thereto decreases. The composition according to any one of Embodiments 335 to 360.

[0402] 362. When a plurality of cells are contacted with the above-described second expression repressor or a nucleic acid encoding the above-described second expression repressor, the viability of the plurality of cells decreases. The composition according to any one of Embodiments 335 to 361.

[0403] 363. When the above-described second expression repressor binds to the above-described super enhancer or a sequence adjacent thereto, the viability of cells containing the above-described transcription regulatory element or a sequence adjacent thereto decreases. The composition according to any one of Embodiments 335 to 362.

[0404] 364. When a plurality of cells are contacted with the above-described second expression repressor or a nucleic acid encoding the above-described first expression repressor, the viability of the plurality of cells decreases, and optionally, the plurality of cells include cancerous and non-cancerous cells and / or infected and non-infected cells. The composition according to any one of Embodiments 335 to 363.

[0405] 365. For example, when measured by CellTiter Glo, the viability decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the viability in the absence of the above-described second expression repressor. The composition according to Embodiment 364.

[0406] 366. A composition according to any one of embodiments 335 - 365, wherein administration of the second expression repressor causes apoptosis in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

[0407] 367. A composition according to any one of embodiments 335 - 366, wherein when the first expression repressor binds to the transcription regulatory element or a sequence proximate thereto, and the second expression repressor binds to the anchor sequence or a sequence proximate thereto, the viability of cells containing the anchor sequence or a sequence proximate thereto is decreased.

[0408] 368. A composition according to any one of embodiments 335 - 367, wherein when the first expression repressor binds to the promoter or a sequence proximate thereto, and the second expression repressor binds to the super - enhancer or a sequence proximate thereto, the viability of the cells is decreased.

[0409] 369. A composition according to any one of embodiments 335 - 368, wherein when a plurality of cells are contacted with the composition, the viability of the plurality of cells is decreased.

[0410] 370. A composition according to any one of embodiments 335 - 369, wherein, for example, when measured by CellTiter Glo, the viability is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the viability in the absence of the composition.

[0411] The decrease in viability resulting from the binding of the first expression repressor to the transcription regulatory element or a sequence adjacent thereto and the binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto is greater than the decrease in viability resulting individually from the binding of the first expression repressor to the transcription regulatory element or a sequence adjacent thereto or from the binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto, the composition according to any of embodiments 335 to 370.

[0412] The decrease in viability resulting from the binding of the first expression repressor to the promoter or a sequence adjacent thereto and the binding of the second expression repressor to the super enhancer or a sequence adjacent thereto is greater than the decrease in viability resulting individually from the binding of the first expression repressor to the promoter or a sequence adjacent thereto or from the binding of the second expression repressor to the super enhancer or a sequence adjacent thereto, the composition according to any of embodiments 335 to 371.

[0413] When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the viability decreases by 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× as compared to either the binding of the first expression repressor to the transcription regulatory element or a sequence adjacent thereto or the binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto, when measured, for example, by CellTiter Glo, the composition according to embodiment 372.

[0414] When the first expression repressor binds to the promoter or an array adjacent thereto, and the second expression repressor binds to the super enhancer or an array adjacent thereto, for example, when measured by CellTiter Glo, the viability is 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× greater than the decrease when the first expression repressor binds to the promoter or an array adjacent thereto or the second expression repressor binds to the super enhancer or an array adjacent thereto individually. The composition according to Embodiment 372 or 373.

[0415] By administering the first expression repressor and the second expression repressor, apoptosis occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells). The composition according to any one of Embodiments 335 to 374.

[0416] The composition according to any one of Embodiments 335 to 375, wherein the plurality of cells includes a plurality of cancer cells and a plurality of non-cancer cells.

[0417] When the plurality of cells is contacted with the composition, the viability of the plurality of cancer cells is decreased more than the decrease in the viability of the plurality of non-cancer cells thereby. The composition according to Embodiment 376.

[0418] When the plurality of cells are contacted with the composition, the viability of the plurality of cancer cells is 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× greater than the decrease in the viability of the plurality of non-cancer cells thereby, the composition according to embodiment 376 or 377.

[0419] 379. For example, when assayed according to Example 18, the viability of non-cancer cells (e.g., primary hepatocytes) does not decrease by more than 5, 10, 15, or 20%, the composition according to any of the preceding embodiments.

[0420] 380. The viability is assayed 72 hours after contacting the cells with the composition, the composition according to embodiment 379.

[0421] 381. The assay includes contacting the non-cancer cells with the composition at 2.5, 2, 1.25, 1, 0.6, or 0.5 μg / ml, the composition according to embodiment 380.

[0422] 382. When contacted with a plurality of infected cells and a plurality of non-infected cells, it reduces the viability of the plurality of infected cells more than it reduces the viability of the plurality of non-infected cells, and / or it reduces the viability of the plurality of cancerous cells more than it reduces the viability of the plurality of non-cancerous cells, the composition according to any of embodiments 358 - 381.

[0423] 383. The cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, adenocarcinoma, large cell (undifferentiated) carcinoma, triple negative breast cancer, gastric adenocarcinoma, endometrial cancer, or pancreatic cancer, the composition according to any of embodiments 358 - 382.

[0424] The composition according to any one of Embodiments 358 to 383, wherein the cancer cells are gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or liver cancer cells.

[0425] The composition according to any one of Embodiments 358 to 384, wherein the infection is a viral infection.

[0426] The composition according to Embodiment 385, wherein the viral infection is hepatitis, for example, hepatitis B.

[0427] The composition according to any one of Embodiments 384 to 386, wherein the infected cells are human hepatocytes.

[0428] The composition according to any one of Embodiments 358 to 387, wherein the viral infection is a chronic infection.

[0429] A composition comprising: (1) a first amino acid region comprising a sequence encoding the first expression repressor of the composition according to any one of Embodiments 121 to 388; and a second amino acid region comprising a sequence encoding the second expression repressor described in the composition according to any one of Embodiments 121 to 388 a first nucleic acid encoding a fusion protein comprising; and (2) a formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of (iii) and (iv) (for example, comprising all of (i) to (iv)): (i) a first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') Formula (I)

Chemical formula

Chemical formula

Chemical formula

[0430] 390. The composition according to embodiment 389, wherein the fusion protein comprises a third amino acid region located between the first amino acid region and the second amino acid region.

[0431] 391. The composition according to embodiment 390, wherein the third amino acid region comprises a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a T2A self-cleaving peptide sequence, for example, the sequence according to SEQ ID NO: 120.

[0432] 392. The composition according to embodiment 391, wherein the third amino acid region comprises a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a tandem 2A peptide sequence, for example, a tPT2A sequence, for example, the sequence according to SEQ ID NO: 124.

[0433] 393. The composition according to embodiment 390, wherein the peptide sequence comprises a T2A peptide sequence and a P2A peptide sequence.

[0434] 394. The above-mentioned first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and the above-mentioned second expression repressor comprises the amino acid sequence according to SEQ ID NO: 24 or 142, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The composition according to any one of embodiments 389 to 393.

[0435] 395. The above-mentioned first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and the above-mentioned second expression repressor comprises the amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The composition according to any one of embodiments 389 to 393.

[0436] 396. The above-mentioned first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and the above-mentioned second expression repressor comprises the amino acid sequence according to SEQ ID NO: 179 or 185, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The composition according to any one of embodiments 389 to 393.

[0437] 397. The above-mentioned fusion protein comprises the amino acid sequence of SEQ ID NO: 91, 92, 121, or 122, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and is the composition according to any one of embodiments 389 to 396.

[0438] 398. The composition according to any one of embodiments 389 to 397, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 181, 182, 187, or 188, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 difference from it.

[0439] 399. A first region comprising a sequence encoding the first expression repressor of the composition according to any one of embodiments 121 to 398; A second region comprising a sequence encoding the second expression repressor of the composition according to any one of embodiments 121 to 398 The composition according to any one of embodiments 121 to 398, comprising a nucleic acid comprising the above.

[0440] 400. The composition according to embodiment 399, wherein the nucleic acid comprises a third region, and the third region is located between the first region and the second region.

[0441] 401. The composition according to embodiment 399 or 400, wherein the third region encodes a ribosome skipping sequence.

[0442] 402. The composition according to embodiment 400 or 401, wherein the third region encodes a tPT2A peptide sequence, for example, the sequence according to SEQ ID NO: 124.

[0443] 403. The composition according to any one of embodiments 400 to 402, wherein the third region encodes a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a T2A self-cleaving peptide sequence, for example, the sequence according to SEQ ID NO: 95.

[0444] 404. The composition according to any one of embodiments 400 to 403, wherein the third region encodes a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a tandem 2A peptide sequence, for example, a tPT2A peptide sequence, for example, the sequence according to SEQ ID NO: 124.

[0445] 405. The above first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30, 129 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and The above second expression repressor comprises the amino acid sequence according to SEQ ID NO: 24, 142, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The composition according to any one of Embodiments 399 to 404.

[0446] 406. The above first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30, 129 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and The above second expression repressor comprises the amino acid sequence according to SEQ ID NO: 177, 179, 183, or 185 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The composition according to any one of Embodiments 399 to 404.

[0447] 407. The above nucleic acid encodes the amino acid sequence of SEQ ID NO: 91, 92, 121, 122 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the composition according to any one of Embodiments 399 to 406.

[0448] 408. The above nucleic acid encodes the amino acid sequence of SEQ ID NO: 181, 182, 187, 188, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the composition according to any one of Embodiments 399 to 407.

[0449] 409. The composition according to any one of Embodiments 399 to 408, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 93, 94, 112, or 113, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0450] 410. The composition according to any one of Embodiments 399 to 409, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 196, 197, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0451] 411. The composition according to any one of Embodiments 46 to 410, wherein the nucleic acid comprises RNA, for example, mRNA.

[0452] 412. The composition according to any one of Embodiments 399 to 411, wherein the nucleic acid comprises, for example, N7-methylated guanosine linked, for example, by an inverted 5'→5' triphosphate bond, to the 5' end of the RNA.

[0453] 413. The composition according to any one of Embodiments 399 to 412, wherein the nucleic acid comprises a 5'UTR.

[0454] 414. The composition according to any one of Embodiments 399 to 413, wherein the nucleic acid comprises, for example, a Kozak sequence between the 5'UTR and the sequence encoding the expression repressor.

[0455] 415. The composition according to any one of Embodiments 121 to 398, wherein the first nucleic acid has the nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second nucleic acid has the nucleotide sequence of SEQ ID NO: 57, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0456] 416. The composition according to Embodiment 414, wherein the first nucleic acid has the nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second nucleic acid has the nucleotide sequence of SEQ ID NO: 189 or 194, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0457] 417. The first nucleic acid has the nucleotide sequence of SEQ ID NO: 189, 194, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second nucleic acid has the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the composition according to embodiment 416.

[0458] 418. The composition according to any one of embodiments 399 to 417, wherein the nucleic acid contains mRNA.

[0459] 419. The composition according to any one of the preceding embodiments, containing lipid nanoparticles.

[0460] 420. The first compound is of formula (III):

Chemical formula

[0461] 421. The above first compound is of formula (IV):

Chemical formula

[0462] 422. The above first compound is as follows:

Chemical formula

Chem.

Chem.

Chem.

[0463] 423. The first compound is as follows:

Chem.

[0464] 424. The first compound is of formula (IX):

Chem.

[0465] 425. The first compound is of formula (XI):

Chemical formula

[0466] 426. The second compound is as follows:

Chemical formula

[0467] 427. The second compound is as follows:

Chemical formula

[0468] 428. The first compound is as follows:

Chemical formula

Chemical formula

[0469] 429. The first compound is as follows:

Chemical formula

Chemical formula

[0470] 430. The first compound is as follows:

Chemical formula

Chemical formula

[0471] 431. As follows:

Chemical formula

Chemical formula

[0472] 432. The following:

Chem.

Chem.

[0473] 433. The neutral lipid (e.g., phosphatidylcholine) is distearoylphosphatidylcholine (DSPC) (Formula V):

Chem.

[0474] 434. The steroid (e.g., sterol, e.g., cholesterol) is cholesterol (Formula VI):

Chem.

[0475] 435. The first compound

Chem.

Chem.

Chemical formula

Chemical formula

[0476]

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0477] 437. The above first compound is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0478] 438. The following:

Chemical formula

Chemical formula

Chem.

Chem.

[0479] 439. The following:

Chem.

Chem.

Chem.

Chem.

[0480] 440. A lipid nanoparticle or composition according to any of the preceding embodiments, wherein the molar ratio of (i):(ii):(iii):(iv) is (47.5 ± 20%):(2.5 ± 20%):(10 ± 20%):(40 ± 20%).

[0481] 441. A lipid nanoparticle or composition according to any of the preceding embodiments, wherein the above molar ratio of (i):(ii):(iii):(iv) is (47.5 ± 10%):(2.5 ± 10%):(10 ± 10%):(40 ± 10%).

[0482] 442. A lipid nanoparticle or composition according to any of the preceding embodiments, wherein the above molar ratio of (i):(ii):(iii):(iv) is (47.5 ± 5%):(2.5 ± 5%):(10 ± 5%):(40 ± 5%).

[0483] 443. The above molar ratio of (i):(ii):(iii):(iv) is as follows: (38 to 57):(2 to 3):(8 to 12):(32 to 48); (42.75 to 52.25):(2.25 to 2.75):(9 to 11):(36 to 44); (45.125 to 49.875):(2.375 to 2.625):(9.5 to 10.5):(38 to 42); or 47.5:2.5:10:40 A lipid nanoparticle or composition according to any of the preceding embodiments, wherein it is any of the above.

[0484] 444. A lipid nanoparticle or composition according to any of the preceding embodiments, wherein the molar ratio of (i):(ii):(iii):(iv) is about 47.5:2.5:10:40.

[0485] 445. As follows: The above first compound contains about 47.5 mol% of the sum of (i), (ii), (iii), and (iv); The above second compound contains about 2.5 mol% of the sum of (i), (ii), (iii), and (iv); The above neutral lipid, such as phosphatidylcholine, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, contains about 10 mol% of the sum of (i), (ii), (iii), and (iv); and The above steroid (e.g., sterol, e.g., cholesterol), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, contains about 40 mol% of the sum of (i), (ii), (iii), and (iv) is one or more of the above, wherein the above first compound, the above second compound, the above neutral lipid, such as phosphatidylcholine, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, and the above steroid (e.g., sterol, e.g., cholesterol) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, are lipid nanoparticles or compositions according to any of the preceding embodiments, containing 100 mol% of the sum of (i), (ii), (iii), and (iv).

[0486] 446. The lipid nanoparticles or composition according to any of the preceding embodiments, wherein the above nucleic acid is encapsulated within the LNP.

[0487] 447. A reaction mixture comprising the composition or lipid nanoparticles according to any of the preceding embodiments.

[0488] 448. The reaction mixture according to embodiment 447, further comprising cells.

[0489] 449. A pharmaceutical composition comprising the composition or lipid nanoparticles or reaction mixture according to any of the preceding embodiments.

[0490] 450. A method for reducing the expression of the MYC gene in cells, comprising contacting the above cells (e.g., cancer cells) with the composition, reaction mixture, or pharmaceutical composition according to any one of Embodiments 1 to 449 comprising a method for reducing the expression of the MYC gene in the above cells.

[0491] 451. A method for treating cancer in a subject in need thereof, comprising administering to the subject the composition, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 446 comprising a method for treating the above cancer in the above subject thereby.

[0492] 452. A method for reducing tumor growth in a subject in need thereof, comprising administering to the subject the composition, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 449 comprising a method for reducing the tumor size in the above subject thereby.

[0493] 453. The method according to Embodiment 452, wherein the above reduction in tumor growth comprises a reduction in tumor volume compared to the tumor volume at the start of treatment.

[0494] 454. The method according to Embodiment 453, wherein the above reduction in tumor growth in the above subject is greater compared to an untreated subject.

[0495] 455. A method for delivering an expression repressor to the liver of a subject, the method comprising delivering to the subject the composition or pharmaceutical composition according to any one of Embodiments 1 to 449, thereby delivering the above expression repressor to the liver of the above subject.

[0496] 456. The method according to Embodiment 455, wherein the above administration is parenteral administration, for example, intravenous administration.

[0497] A method for increasing or restoring cancer sensitivity to a kinase inhibitor, such as sorafenib, comprising administering to a subject having said cancer a composition described herein.

[0498] 458. When the above composition is administered, for example, in a cancer cell viability assay, such as the assay according to Example 26, the IC of the above kinase inhibitor 50 decreases by 10%, 20%, 30%, or 40%, the method according to embodiment 457.

[0499] 459. The method according to embodiment 457 or 458, wherein the kinase inhibitor inhibits one or more (e.g., all) of VEGFR, PDGFR, or RAF kinase.

[0500] 460. A method for increasing or restoring cancer sensitivity to a bromodomain inhibitor, such as a BET inhibitor, such as JQ1, comprising administering to a subject having said cancer a composition described herein (e.g., any of embodiments 1 - 448), and optionally, when the above composition is administered, for example, in a cancer cell viability assay, such as the assay according to Example 29, the IC of the above bromodomain inhibitor 50 decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, the method.

[0501] 461. The method according to embodiment 460, wherein the bromodomain inhibitor is JQ1, BET672, or vilablociclib, or comprises the same.

[0502] 462. A method for increasing or restoring cancer sensitivity to a MEK inhibitor, such as trametinib, comprising administering to a subject having said cancer a composition described herein (e.g., any of embodiments 1 - 448), and optionally, when the above composition is administered, for example, in a cancer cell viability assay, the IC of the above MEK inhibitor 50A method of decreasing by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0503] 463. The method according to any one of embodiments 452 - 462, wherein the decrease in tumor growth of the subject is greater than or equal to the decrease in tumor size when the subject is treated with a chemotherapeutic agent or a small molecule MYC inhibitor.

[0504] 464. The method according to embodiment 463, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0505] 465. The method according to embodiment 464, wherein the small molecule MYC inhibitor is MYCi975.

[0506] 466. A method of decreasing the tumor size of a subject in need thereof, comprising administering to the subject a composition, lipid nanoparticle, or pharmaceutical composition according to 1 - 448, wherein the decrease in tumor size is greater than or equal to the decrease in tumor size when the subject is treated with a chemotherapeutic agent. 1 - 448, and the decrease in tumor size is greater than or equal to the decrease in tumor size when the subject is treated with a chemotherapeutic agent.

[0507] 467. The method according to 466, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0508] 468. The method according to any one of the preceding embodiments, wherein no significant side effects occur in the subject as compared to when treated with a chemotherapeutic agent or a small molecule MYC inhibitor.

[0509] 469. The method according to any one of embodiments 463 - 468, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0510] 470. The method according to embodiment 469, wherein the small molecule MYC inhibitor is MYCi975.

[0511] The method according to any one of embodiments 451 to 470, wherein the cancer is stage I, stage II, stage III, or stage IV cancer.

[0512] 472. The method according to any one of the preceding embodiments, wherein the weight of the subject remains substantially the same before and after treatment.

[0513] 473. The method according to any one of the preceding embodiments, wherein the subject does not experience weight loss, or the weight loss that occurs in the subject is less than 3%, 2%, or 1% compared to the start of treatment.

[0514] 474. The method according to any one of the preceding embodiments, wherein there is no decrease or increase in the weight of the subject after treatment compared to the weight of the subject before treatment.

[0515] 475. A method for treating a liver disease of a subject in need thereof, comprising administering a composition to the subject, the composition comprising: (1) a first nucleic acid encoding an expression repressor; and (2) a formulation comprising one or both of the following (i) and (ii) and optionally further comprising one or both of the following (iii) and (iv) (e.g., comprising all of (i) to (iv)): (i) a first compound selected from the group consisting of the following (i'), (i''), and (i'''): (i') formula (I)

Chemical formula

Chemical Structure

Chemical formula

Chemical formula

[0516] 476. Further comprising administering to said subject a second nucleic acid encoding a second expression repressor comprising a targeting moiety that binds to the anchor sequence of an anchor sequence-mediated junction (ASMC) containing a target gene, e.g., MYC, and optionally a second effector moiety, e.g., an effector moiety described herein; e.g., KRAB; The method according to embodiment 475, thereby treating the liver disease of said subject.

[0517] 477. The method according to embodiment 475 or 476, wherein said first nucleic acid and said second nucleic acid are located within the same nucleic acid molecule.

[0518] 478. A method of treating a liver disease of a subject in need thereof, Administering to the subject the composition, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 448 comprising a method for treating the liver disease of the subject thereby.

[0519] 479. The method according to Embodiment 478, wherein the liver di...

Claims

1. (1) A nucleic acid (e.g., RNA, e.g., mRNA) that encodes an expression repressor, (a) (i) a targeting portion that binds to the MYC promoter, or (ii) a genomic locus containing at least 16, 17, 18, 19, or 20 nucleotides of any sequence 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, (b) Optionally, the effects section and The expression repressor includes nucleic acids that can reduce the expression of MYC; (2) (i) Equation (I) 【Chemistry 229】 (wherein, L １ and L ２ are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -, -NR ａ C(=O)O-, or a direct bond; G １ and G ２ Each of these is independently of the non-substituted C １ ~C １２ Alkylene, or C ２ ~C １２ It is alkenylene; G ３ C １ ~C ２４ Alkylene, C ２ ~C ２４ Alkenylene, C ３ ~C ８ Cycloalkylene, C ３ ~C ８ It is a cycloalkenylene; R ａ is H or C １ ~C １２ It is alkyl; R １ and R ２ Each of them is independent of C ６ ~C ２４ Alkyl or C ６ ~C ２４ It is an alkenil; R ３ H, OR ５ , CN, -C(=O)OR ４ -OC(=O)R ４ , -NR １１ R １２ , or -NR ５ C(=O)R ４ And; R ４ C １ ~C １２ It is alkyl; R ５ is H or C １ ~C ６ It is alkyl; R １１ and R １２ Each of them is independent of C １ ~C １２ Alkyl or -G ４ -OR ５ is or R １１ and R １２ Together with the nitrogen atoms to which they are bonded, they form a 5, 6, or 7-membered heterocycle; G ４ C １ ~C ２４ Alkylene, C ２ ~C ２４ Alkenylene, C ３ ~C ８ Cycloalkylene, C ３ ~C ８ It is a cycloalkenylene; x is 0, 1, or 2. A first compound having the general structure of; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof; and (ii) Formula (II): 【Chemistry 230】 (In the formula, R ６ and R ７ Each is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally separated by one or more ester bonds; (y has an average value in the range of 30 to 60.) A second compound having the general structure of; or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. A formulation comprising one or both of the above, and a composition comprising the above.

2. (iii) Neutral lipids such as phosphatidylcholine, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof; and / or (iv) Sterols such as cholesterol, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof. The composition according to claim 1, comprising:

3. The first compound is of formula (III): 【Chemical 233】 (In the formula, L １ and L ２ Each of these is independently -(C=O)O- or -O(C=O)-; R １ and R ２ Each of them is independent of C ６ ~C ２４ Alkyl or C ６ ~C ２４ It is an alkenil; R ３ It appears once, H, OR ５ , CN, -C(=O)OR ４ -OC(=O)R ４ or -NR ５ C(=O)R ４ And; R ４ C １ ~C １２ It is alkyl; R ５ is H or C １ ~C ６ It is alkyl; R ８ It appears n times, and each time it appears, independently of H, OH, or C １ ~C ２４ It is alkyl; n is an integer between 1 and 15; (m and l are each independent integers between 1 and 12.) The composition according to claim 1, having the general structure of; or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

4. The first compound is of formula (IV): 【Chemistry 234】 (In the formula, R １ and R ２ are each independently C ６ to C ２４ alkyl or C ６ to C ２４ alkenyl; R ３ is H, OR ５ , CN, -C(=O)OR ４ , -OC(=O)R ４ or -NR ５ C(=O)R ４ ; and R ４ C １ ~C １２ It is alkyl; R ５ is H or C １ ~C ６ It is alkyl; R ８ is H, OH, or C １ ~C ２４ It is alkyl; n is an integer between 1 and 15; (m and l are each independent integers between 1 and 12.) The composition according to claim 1, having the general structure of; or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

5. The first compound is as follows: 【Chemistry 256】 【Chemistry 257】 【Chemistry 258】 【Chemistry 259】 【Chemistry 260】 The composition according to claim 1, comprising any of the above, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

6. The second compound is as follows: 【Chemistry 242】 The composition according to claim 1, comprising any of the following (wherein n has an average value in the range of 30 to 60), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

7. The phosphatidylcholine mentioned above is distearoylphosphatidylcholine (DSPC) (formula V): 【Chemistry 246】 ; or the composition according to claim 2, comprising a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

8. The aforementioned sterols are cholesterol (formula VI): 【Chemistry 247】 ; or the composition according to claim 2, comprising a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

9. The molar ratio of (i):(ii):(iii):(iv) is as follows: （３８～５７）：（２～３）：（８～１２）：（３２～４８）； （４２．７５～５２．２５）：（２．２５～２．７５）：（９～１１）：（３６～４４）； (45.125–49.875):(2.375–2.625):(9.5–10.5):(38–42); or ４７．５：２．５：１０：４０ The composition according to claim 2, which is any one of the following.

10. The composition according to claim 1, wherein the formulation forms lipid nanoparticles (LNPs), and the nucleic acid is encapsulated within the LNPs.

11. The composition according to claim 1, having an N:P ratio of 3-22, 4-12, 4-8, 5-9, 5-7, 5.5-6.5, 6-9, 7-9, 6-8, about 5, about 6, about 7, or about 8.

12. The first targeting portion binds to a genomic locus containing at least 16, 17, 18, 19, or 20 nucleotides of the sequence of Sequence ID No. 83, and The composition according to claim 1, wherein the expression repressor includes the first effector portion, and the first effector portion includes DNA methyltransferase.

13. The composition according to claim 12, wherein the first targeting portion comprises a zinc finger domain, and / or the first effector portion comprises MQ1 or a functional variant or fragment thereof.

14. The composition according to claim 12, wherein the first targeting portion includes the amino acid sequence relating to Sequence ID No. 13, or a sequence in which the number of positions that are at least 80, 85, 90, 95, 99, or 100% identical thereto, or differ therefrom, does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

15. The composition according to claim 12, wherein the first effector portion includes the sequence of sequence numbers 19, 87, or 129, or the sequence in which there are at least 80, 85, 90, 95, 99, or 100% identity thereto, or where the number of positions that differ therefrom is not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

16. The RNA further encodes a second expression repressor, and the second expression repressor, The second targeting region binds to the second genome locus, The second effects pedal section and The composition according to claim 12, comprising:

17. The composition according to claim 16, wherein when multiple cells are brought into contact with the composition, the viability of the multiple cells is reduced.

18. A composition according to any one of claims 1 to 19 for use in the treatment of cancer of a specific target.

19. The composition for use according to claim 18, wherein the cancer is hepatocellular carcinoma (HCC), fibrous hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, or secondary liver cancer.

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