Compositions and methods for modulating MYC expression

Abstract

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

Description

Technical field

[0001] Cross-reference of related applications This application is based on U.S. Provisional Patent Application No. 63 / 125,833 filed on December 15, 2020, U.S. Provisional Patent Application No. 63 / 137,097 filed on January 13, 2021, and June 2021. It claims priority to U.S. Provisional Patent Application No. 63 / 212,991, filed on November 21, 2021, and U.S. Provisional Patent Application No. 63 / 281,022, filed on November 18, 2021. , the entire contents of which are hereby incorporated by reference.

[0002] Sequence list This application contains a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy created on December 6, 2021 is named O2057-7029WO_SL.txt and is 624,274 bytes in size. [Background technology]

[0003] Misregulation of gene expression is an underlying cause of many diseases (eg, mammalian, eg, human diseases), such as neoplasia, neurological disorders, metabolic disorders, and obesity. Misregulation 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 a variety of factors, including the lack of a defined ligand binding site and the fact that its physiological function is essential for maintaining normal tissue. Techniques aimed at modulating MYC gene expression provide a viable alternative approach in the treatment of these diseases. New tools, systems, and methods are needed to stably alter, eg, reduce, the expression of disease-associated genes, such as MYC. [Summary of the invention] [Means to solve the problem]

[0004] The present disclosure provides, among other things, expression repressors and expression repressor systems that can be used to modulate, eg, reduce, expression of a target gene, eg, MYC.

[0005] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a target gene promoter, e.g., the MYC promoter, and optionally an effector moiety, wherein the expression repressor comprises: Expression of a target gene, eg MYC, can be reduced.

[0006] In some embodiments, the present disclosure provides expression repressors that include a targeting moiety that binds a target locus, e.g., MYC, and an effector moiety that includes MQ1 or a fragment or variant thereof, wherein said expression A repressor can reduce the expression of a target gene, eg MYC.

[0007] 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, and optionally an effector moiety, wherein the expression repressor is , can reduce the expression of MYC.

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

[0009] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds a regulatory element located in the super-enhancer region of a target gene, e.g., MYC, the targeting moiety comprising a zinc finger domain; Here, the expression repressor can reduce the expression of a target gene, for example, MYC.

[0010] In some embodiments, the present disclosure provides a targeting moiety that binds 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, the targeting moiety binding a regulatory element located in the super-enhancer region of MYC. an effector moiety comprising a pressor (e.g., KRAB or a fragment or variant thereof) or a DNA methyltransferase (e.g., MQ1 or a fragment or variant thereof); MYC expression can be reduced.

[0011] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds a target locus, e.g., MYC, the targeting moiety comprising a zinc finger domain, wherein the expression repressor , the expression of a target gene, e.g. MYC, can be reduced.

[0012] In some embodiments, the present disclosure provides SEQ ID NO. , 80, 81, 84, 85, 86 sequences, wherein , the above expression repressor can reduce the expression of MYC.

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

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

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

[0016] In some embodiments, the present disclosure provides targeting moieties that bind a genomic locus that includes at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, 96, or 108. An expression repressor is provided that includes a targeting moiety that targets the expression.

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

[0018] In some embodiments, the present disclosure provides a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the transcriptional regulatory element ( a first expression repressor that binds, for example, to a promoter or transcription start site (TSS) or to a sequence proximal to a transcriptional regulatory element; and a second expression repressor that binds to a genomic locus located in the super-enhancer region of a target gene, eg, MYC.

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

[0020] In some embodiments, the present disclosure provides a method for binding to a transcriptional regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC, or a sequence proximal to said transcriptional regulatory element. Expression repressors are provided that include a targeting moiety that includes a CRISPR / Cas molecule, eg, a catalytically inactive CRISPR / Cas protein; and an effector moiety that includes MQ1 or a functional variant or fragment thereof.

[0021] In some embodiments, the present disclosure includes a CRISPR / Cas molecule, e.g., a catalytically inactive CRISPR / Cas protein, that binds to a genomic locus located in the super-enhancer region of a target gene, e.g., MYC. Provided is an expression repressor comprising a targeting moiety and an effector moiety comprising KRAB, MQ1, or a functional variant or fragment thereof, wherein the expression repressor reduces expression of a target gene, e.g. MYC. be able to.

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

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

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

[0025] In some embodiments, the present disclosure includes a targeting moiety that includes a zinc finger molecule and KRAB or a functional variant or fragment thereof that binds to a genomic locus located in the super-enhancer region of a target gene, e.g., MYC. and an effector moiety.

[0026] In some embodiments, the present disclosure relates to nucleic acids encoding a first expressed repressor, a second expressed repressor, both, or a component thereof (eg, 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 a vector, nucleic acid, system, or expression repressor described herein. In another aspect, the present disclosure relates to reaction mixtures comprising expression repressors, systems, nucleic acids, vectors, or lipid nanoparticles described herein. In another aspect, the present disclosure relates to pharmaceutical compositions comprising expression repressors, systems, nucleic acids, vectors, lipid nanoparticles, or reaction mixtures described herein.

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

[0029] In some embodiments, the present disclosure relates to methods of treating conditions associated with overexpression of a target gene, e.g., MYC, in a subject, which comprises an expression repressor or system, nucleic acid, or vector described herein. to the subject, thereby treating the condition.

[0030] In some embodiments, the present disclosure relates to a method of treating a condition associated with misregulation of a target gene, e.g., MYC, in a subject, which comprises an expression repressor, system, nucleic acid, or administering the vector to the subject, thereby treating the condition.

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

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

[0033] The present disclosure includes, in part, a) a targeting moiety that binds to a target gene, promoter, e.g., MYC, and an effector moiety that can modulate, e.g., reduce expression of the target gene, e.g., MYC; Further provided is a kit comprising a container comprising a composition comprising an expression repressor, and b) a set of instructions comprising at least one method of modulating expression of a target gene, e.g. MYC, in a cell with said composition.

[0034] The present disclosure describes, in part, a) a targeting moiety that binds to a locus located in a super-enhancer region of a target gene, e.g., MYC, and a method for modulating, e.g., reducing expression of the target gene, e.g., MYC; and b) a set of instructions containing at least one method for modulating the expression of a target gene, e.g. MYC, in a cell with said composition. Further provided is a kit comprising:

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

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

[0037] In some embodiments, the kit includes a set of instructions comprising: b) at least one method for treating a disease or modulating, e.g., reducing expression of a target gene, e.g., MYC, in a cell with said composition. Including further. In some embodiments, the kit may optionally include a delivery vehicle (eg, a lipid nanoparticle) for the composition. Reagents may be provided suspended in the excipient and / or delivery vehicle, or may be provided as separate components that can be later combined with the excipient and / or delivery vehicle. In some embodiments, the kit optionally includes additional therapeutic agents to be co-administered with the composition to affect the regulation of desired target gene expression, e.g., MYC gene expression. You can. Instructional materials typically include, but are not limited to, written or printed materials. Any medium capable of storing such instructions and communicating them to an end user is contemplated in accordance with the present invention. Such media include, but are not limited to, electronic storage media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD ROM), and the like. Such media may include an address to an Internet site that provides such instructional material.

[0038] Other features of any of the aforementioned methods or compositions include one or more of the embodiments listed below.

[0039] Those skilled 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 (eg, sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced herein, eg, in any table herein, are incorporated herein by reference. Unless otherwise specified, sequence accession numbers provided herein, including in all tables herein, refer to database entries as of December 15, 2020. If one gene or protein exhibits multiple sequence accession numbers, all sequence variants are included.

[0041] Enumeration of embodiments 1.A targeting part that binds to the MYC promoter, Optionally, the effector part and An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0042] 2. The targeting moiety according to embodiment 1 binds 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. Expression repressor.

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

[0044] 4. Sequence number 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 75, 76, 78, 79, 80, 81, 84, 85, 86, a targeting moiety that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of a sequence of 190, 191, 192, 200, or 202; Optionally, the effector part and An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0045] 5. a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence SEQ ID NO: 2, 77, 82, 83, 199, or 201; Optionally, the effector part and An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0046] 6. A targeting part that binds the MYC locus, an effector moiety comprising MQ1 or a fragment or variant thereof; An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0047] 7. A targeting part that binds the gene locus in the MYC super enhancer region, optionally an effector moiety, for example an effector moiety comprising a DNA methyltransferase, optionally comprising MQ1 or a fragment or variant thereof; An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0048] 8. A targeting part that binds the gene locus in the MYC super enhancer region, an effector moiety comprising a transcriptional repressor, optionally comprising KRAB or a fragment or variant thereof; An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0049] 9. The targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NO: 96-110, 83, 199, or 201. The expression repressor according to form 7 or 8.

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

[0051] 11. According to any of embodiments 7 to 10, wherein the targeting moiety binds 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. expression repressor.

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

[0053] 13. a targeting moiety that binds a genetic locus, e.g., the MYC locus; a first effector portion comprising EZH2 or a fragment or variant thereof; a second effector moiety comprising KRAB or a fragment or variant thereof; An expression repressor comprising: An expression repressor capable of reducing expression at the above genetic locus, for example reducing expression of MYC.

[0054] 14. The expression repressor of embodiment 13, wherein the targeting moiety binds the MYC promoter, super enhancer region, or anchor sequence.

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

[0056] 16. Expression according to any of embodiments 13 to 15, wherein the first effector moiety is on the N-terminal side of the second effector, or the first effector is on the C-terminal side of the second effector moiety. repressor.

[0057] 17. A targeting moiety that binds the MYC locus and includes a zinc finger domain; Optionally, the effector part and An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0058] 18. Comprising a CRISPR / Cas domain that binds to a transcriptional regulatory element operably linked to the MYC gene (e.g., a promoter, enhancer, super-enhancer, or transcription start site (TSS)) or a sequence proximal to said transcriptional regulatory element. , for example, with a targeting moiety comprising a catalytically inactive CRISPR / Cas protein; an effector portion comprising MQ1 or a functional variant or fragment thereof; Expression repressor containing.

[0059] 19. A CRISPR / Cas domain that binds to a transcriptional regulatory element operably linked to the MYC gene (e.g., a promoter, enhancer, or transcription start site (TSS)) or a sequence proximal to said transcriptional regulatory element, e.g. a targeting moiety comprising a catalytically inactive CRISPR / Cas protein; an effector portion comprising MQ1 or a functional variant or fragment thereof; Expression repressor containing.

[0060] 20. A CRISPR / Cas domain that binds to a transcriptional regulatory element operably linked to the MYC gene (e.g., a promoter, enhancer, or transcription start site (TSS)) or a sequence proximal to said transcriptional regulatory element, e.g. a targeting moiety comprising a catalytically inactive CRISPR / Cas protein; an effector portion comprising KRAB or a functional variant or fragment thereof; Expression repressor containing.

[0061] 21. For example, a catalytically inactive CRISPR / Cas domain containing a CRISPR / Cas domain that binds to the anchor sequence of an anchor sequence mediated junction (ASMC) containing the MYC gene, or that binds to a sequence proximal to said anchor sequence. a targeting moiety containing a Cas protein; an effector portion comprising KRAB or a functional variant or fragment thereof; Expression repressor containing.

[0062] 22. a targeting moiety comprising a zinc finger domain that binds to a transcriptional regulatory element operably linked to the MYC gene (e.g., a promoter, enhancer, or transcription start site (TSS)) or a sequence proximal to said transcriptional regulatory element; an effector portion comprising MQ1 or a functional variant or fragment thereof; Expression repressor containing.

[0063] 23. a targeting moiety comprising a zinc finger domain that binds to a transcriptional regulatory element operably linked to the MYC gene (e.g., a promoter, enhancer, or transcription start site (TSS)) or a sequence proximal to said transcriptional regulatory element; an effector portion comprising KRAB or a functional variant or fragment thereof; Expression repressor containing.

[0064] 24. A targeting moiety that binds a mouse genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 190 to 192; Optionally, the effector part and An expression repressor comprising: An expression repressor that can reduce the expression of MYC.

[0065] 25. An expression repressor according to claim 24, wherein the effector moiety comprises a DNA methyltransferase, such as MQ1 or a fragment or variant thereof.

[0066] 26. The expression repressor of 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 expression repressor according to any of embodiments 24 to 26, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain.

[0068] 28. The expression repressor has an amino acid sequence selected from any of SEQ ID NOs: 160 to 165, or at least 80, 85, 90, 95, 99, or 100% identical to it, or has a difference therefrom. Contains sequences in which a given position does 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 expression repressor according to any one of embodiments 24 to 27.

[0069] 29. The expression repressor has 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 to, or has a difference from, Coded by a sequence in which the position does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 The expression repressor according to any one of embodiments 24 to 28.

[0070] 30. The targeting portion has at least 80, 85, 90, 95, 99, or 100% identity with the amino acid sequence according to any of SEQ ID NOs: 154 to 156, or has a position that differs from it. Embodiments comprising sequences of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than one location. The expression repressor according to any one of 24 to 29.

[0071] 31. The targeting moiety is at least 80, 85, 90, 95, 99, or 100% identical to the nucleic acid sequence according to any of SEQ ID NOs: 157 to 159, or has a position that differs from it. Embodiments comprising sequences of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than one location. The expression repressor according to any one of 24 to 30.

[0072] 32. The expression repressor according to any of embodiments 24 to 31, wherein the effector moiety is a persistent effector moiety.

[0073] 33. The expression repressor according to any one of embodiments 24 to 32, wherein the effector moiety is a transient effector moiety.

[0074] 34. The expression repressor according to any of embodiments 24 to 33, which is a fusion molecule.

[0075] 35. According to any of embodiments 24 to 34, wherein the targeting moiety comprises a zinc finger domain and the effector moiety comprises an epigenetic modification moiety, e.g. a DNA methyltransferase, e.g. MQ1 or a fragment or variant thereof. expression repressor.

[0076] 36. The expression repressor of any of embodiments 18-20, 22, or 23, wherein the regulatory element is part of a cluster of regulatory elements.

[0077] 37. The expression repressor of any of embodiments 18-20, 22, or 23, wherein the regulatory element is located in a non-coding region.

[0078] 38. The expression linkage according to any of embodiments 18-20, 22, or 23, wherein the regulatory element is a distal enhancer, e.g., located at least 1,000 nt from the target gene promoter, e.g. MYC. Presser.

[0079] 39. The expression repressor according to any of embodiments 18-20, 22, 23 or 36-38, wherein the regulatory element increases expression of a target gene, eg MYC.

[0080] 40. The expression repressor of any of embodiments 18-20, 22, 23, or 36-39, wherein said regulatory element has one or more mutations.

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

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

[0083] 43. The expression repressor according to embodiment 42, wherein the enhancer docking site includes a nucleotide sequence according to any one of SEQ ID NOs: 71 to 74.

[0084] 44. A targeting moiety comprising a zinc finger domain that binds to an anchor sequence of an anchor sequence mediated junction (ASMC) comprising the MYC gene, or binds to a sequence proximal to said anchor sequence; an effector portion comprising KRAB or a functional variant or fragment thereof; Expression repressor containing.

[0085] 45. The expression repressor has an amino acid sequence selected from any of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, or 177-186, or at least 80, 85, 90, 95, 99. , or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 44. The expression repressor of any of embodiments 1-23 or 36-43, comprising no more than 4, 3, 2, or 1 sequence.

[0086] 46. ​​The expression repressor has a nucleotide sequence selected from any of SEQ ID NOs: 55-70, 130, 189, or 193-197, or at least 80, 85, 90, 95, 99, or 100% identical thereto. 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or an expression repressor according to any of embodiments 1-23 or 36-45, encoded by no more than one sequence.

[0087] 47. The targeting moiety is at least 80, 85, 90, 95, 99, or 100% identical to, or has at least 80, 85, 90, 95, 99, or 100% identity to the amino acid sequence according to any of SEQ ID NOs: 5 to 16, or 169 to 172, or Sequences in which the number of different positions does 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 expression repressor according to any one of Embodiments 1 to 23 or 36 to 46, comprising:

[0088] 48. The effector moiety has at least 80, 85, 90, 95, 99, or 100% identity with the amino acid sequence according to SEQ ID NO: 18, 19, or 87, or has 20 positions that differ from it. , 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or any preceding implementation containing not more than one sequence The expression repressor according to any of the forms.

[0089] 49. The expression repressor according to any one of Embodiments 1-12, 17-19, 22, 36-42, or 44-47, wherein the effector moiety is a persistent effector moiety.

[0090] 50. The expression repressor according to any one of Embodiments 1 to 23 or 36 to 48, wherein the effector moiety is a transient effector moiety.

[0091] 51. Expression according to any of embodiments 1-12, 17-19, 22, 36-42, or 44-48, wherein the effector moiety comprises a DNA methyltransferase, such as MQ1 or a fragment or variant thereof. repressor.

[0092] 52. The expression repressor according to any of embodiments 1-23, 36-47, or 49, wherein the effector portion comprises a transcriptional repressor, for example, KRAB or a fragment or variant thereof.

[0093] 53. An expression repressor according to any of the preceding embodiments, wherein said targeting moiety comprises a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.

[0094] 54. The CRISPR / Cas domain binds a gRNA, e.g. 54, wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOS: 1 to 4. Presser.

[0095] 55. The CRISPR / Cas domain binds a gRNA, e.g. 54, wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 96 to 110. Presser.

[0096] 56. The expression library according to any of embodiments 53 to 55, wherein the CRISPR / Cas domain comprises a Cas protein or a Cpf1 protein selected from Table 1, or a variant (e.g., a mutant) of either of them. Presser.

[0097] 57. The expression repressor according to any of embodiments 53 to 56, wherein the CRISPR / Cas domain comprises a catalytically inactive CRISPR / Cas protein, such as dCas9.

[0098] 58. The zinc finger domain binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 96-110, for example, the gRNA 54. The expression repressor of embodiment 53, comprising a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 96-110.

[0099] 59. Where the zinc finger domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (and optionally 11, 10, 9, 8, 7, 6, or 58.

[0100] 60.The above zinc finger domains are 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 1~2, 2~10, 2~ 9, 2~8, 2~7, 2~6, 2~5, 2~4, 2~3, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 3~4, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~10, 5~9, 5~8, 5~7, 5~6, 6~ Embodiments 17, 22 comprising 10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. , 26-53, 57, or 58.

[0101] 61. The expression repressor according to any one of embodiments 17, 22, 26-53, or 57-59, wherein the zinc finger domain comprises 3 or 9 zinc fingers.

[0102] 62. An expression repressor according to any of the preceding embodiments, which is a fusion molecule.

[0103] 63. According to any of the preceding embodiments, comprising a linker located between said targeting domain and said effector domain, optionally comprising an amino sequence according to SEQ ID NO: 137 or SEQ ID NO: 138. expression repressor.

[0104] 64. Embodiments 1-17 wherein said targeting moiety comprises a catalytically inactive CRISPR / Cas domain (e.g. dCas9) and said effector moiety comprises a transcriptional repressor, e.g. KRAB or a fragment or variant thereof. , 20, 21, 23, 44-48, 50, or 52-57.

[0105] 65. Embodiments 1-17, 20, 21, 23, 44-48, 50, wherein said targeting moiety comprises a zinc finger domain and said effector moiety comprises a transcriptional repressor, such as KRAB or a fragment or variant thereof. , 52, or the expression repressor according to any one of 53 to 64.

[0106] 66. The expression repressor according to any of embodiments 17, 36-43, 45-47, 53, or 58-63, wherein the targeting moiety comprises a zinc finger domain, and the expression repressor does not comprise an effector moiety. Presser.

[0107] 67. The targeting moiety comprises a catalytically inactive CRISPR / Cas domain (e.g. dCas9) and the effector moiety comprises an epigenetic modification moiety, e.g. a DNA methyltransferase, e.g. MQ1 or a fragment or variant thereof. The expression repressor according to any one of Embodiments 1 to 12, 18 to 19, 22, 36 to 43, 45 to 49, 51, or 53 to 57, comprising:

[0108] 68. Embodiments 1-12, 17-19, wherein said targeting moiety comprises a zinc finger domain and said effector moiety comprises an epigenetic modification moiety, e.g. a DNA methyltransferase, e.g. MQ1 or a fragment or variant thereof; The expression repressor according to any one of 22, 36-43, 45-49, 51, 53, or 58-63.

[0109] 69. Any of the amino acid sequences of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, or 177-186, or at least 80%, 85%, 90%, 95%, 96%, 97% thereof, An expression repressor according to any of the preceding embodiments, comprising sequences with 98%, 99% identity.

[0110] 70. An expression repressor according to any of the preceding embodiments, (i) comprising one or more nuclear localization signal sequences (NLS), or (ii) not comprising a NLS.

[0111] 71. An expression repressor according to any of the preceding embodiments, comprising a first NLS at the N-terminus, eg having a sequence of SEQ ID NO: 88.

[0112] 72. An expression repressor according to any of the preceding embodiments, comprising an NLS, eg a second NLS, at the C-terminus, eg having the sequence of SEQ ID NO: 89.

[0113] 73. The expression repressor according to any of the preceding embodiments, wherein said first NLS and said second NLS have the same sequence.

[0114] 74. The expression repressor according to any one of embodiments 71 to 73, wherein the first NLS and the second NLS have different sequences.

[0115] 75. An expression repressor according to any of the preceding embodiments, comprising an epitope tag.

[0116] 76. The expression repressor of embodiment 75, wherein said epitope tag is an HA tag.

[0117] 77. of the preceding embodiments, wherein the anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto. The expression repressor according to any one of the above.

[0118] 78. Embodiments 1 to 7, wherein the anchor sequence comprises the sequence according to SEQ ID NO: 73 or 74, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto. 77. The expression repressor according to any one of 77.

[0119] 79. An expression repressor according to any of the preceding embodiments, wherein said anchor sequence is on the same chromosome as the MYC gene.

[0120] 80. An expression repressor according to any of the preceding embodiments, wherein the anchor sequence is upstream of the MYC gene (eg upstream of the TSS or upstream of the promoter).

[0121] 81. According to any of the preceding embodiments, 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). Expression repressor.

[0122] 82. The above anchor sequence is from the MYC gene (for example, from the TSS or promoter of the MYC gene) 0.1~0.5, 0.1~1, 0.1~5, 0.1~10, 0.1~50, 0.1~100, 0.1~500, 0.1 ~1000, 0.5~1, 0.5~5, 0.5~10, 0.5~50, 0.5~100, 0.5~500, 0.5~1000, 1~5, 1~10, 1~50, 1~100, 1~500 , 1~1000, 5~10, 5~50, 5~100, 5~500, 5~1000, 10~50, 10~100, 10~500, 10~1000, 50~100, 50~500, 50 The expression repressors of any of the preceding embodiments are ~1000, 100-500, 100-1000, or 500-1000 kilobases apart.

[0123] 83. The expression repressor according to any of embodiments 1 to 79 or 81 to 82, wherein the target sequence is downstream of the MYC gene (for example, downstream of the TSS or downstream of the promoter).

[0124] 84.The above targeting part has chromosome coordinates 128746342~128746364, 128746321~128746343, 128746525~128746547, 128748014~128748036, 129188878~129188900, 129188958~1 29188980, 129188960~129188982, 129189067~129189089, 129189457~129189479, 129189554~129189576, 129189679 ~129189701, 129209511~129209533, 129209643~129209665, 129209658~129209680, 129209856~129209878, 129189452~129189474, 129189190~129 189212, 129189274~129189296, 129189421~129189443, 128746405~128746425, 128748069~128748089, 129188825~129188845, or 129188822~ 129188842 or a sequence adjacent thereto.

[0125] 85. Binding of said expression repressor to a target locus, e.g. Optionally, methylation at one site is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to methylation in the absence of the expression repressor. , wherein the site assayed for methylation is chr8:129188693-129189048 according to the hg19 reference genome, and includes, for example, the sequence according to SEQ ID NO: 123.

[0126] 86. When the expression repressor binds to a target locus, e.g. MYC, methylation at one site of the target locus, e.g. , 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.

[0127] 87. When the expression repressor binds to the MYC locus, the expression of MYC in the cell, as measured, for example, by ELISA or as described in any of Examples 2 to 7 or 9, The expression repressor of any of the preceding embodiments is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of.

[0128] 88. When the expression repressor binds to the MYC locus, the expression of MYC decreases by 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.

[0129] 89. When the above expression repressor binds to the MYC locus, 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.

[0130] 90. The expression repressor according to any of embodiments 1 to 23 or 36 to 89, wherein the targeting moiety binds to a human genomic locus.

[0131] 91. The expression repressor according to any one of embodiments 24-43, 49, 51, 53, 56-57, 59-62, 66-68, 70-89, wherein the targeting moiety binds to a mouse genomic locus.

[0132] 92. An expression repressor according to any of the preceding embodiments, wherein binding of said expression repressor to the MYC locus reduces the viability of a cell (eg, a cancer cell) comprising the MYC locus.

[0133] 93. An expressed repressor according to any of the preceding embodiments, wherein contacting a plurality of cells with said expressed repressor or a nucleic acid encoding said expressed repressor reduces the viability of said plurality of cells.

[0134] 94. For example, if the viability, as measured by CellTiter Glo or as described in any of Examples 2-7, is 10 compared to the viability in the absence of the first expressed repressor, The expression repressor of any of the preceding embodiments is reduced by 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0135] 95. Administration of the expression repressor described above results in at least 5%, 6%, 7%, 8%, 9% 10%, 12%, 15%, 17% 20%, 25% of target cells (e.g. cancer cells) The expressed repressor according to any of the preceding embodiments, wherein 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% apoptosis occurs.

[0136] 96. The expressed repressor of any preceding embodiment, wherein the plurality of cells comprises a plurality of cancer cells and a plurality of non-cancer cells and / or a plurality of infected cells and a plurality of uninfected cells.

[0137] 97. When the plurality of cells are contacted with the expression repressor or a nucleic acid encoding the expression repressor, the viability of the plurality of cancer cells is reduced more than the decrease in the viability of the plurality of non-cancer cells. The expression repressor according to any of the preceding embodiments.

[0138] 98. When the plurality of cells are contacted with the expressed repressor or a nucleic acid encoding the expressed repressor, the viability of the plurality of cancer cells decreases by 1.05×( i.e. 1.05x), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x , 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100×.

[0139] 99. The expression repressor according to any one of embodiments 92 to 97, wherein the cancer cell is a lung cancer cell, a gastric cancer cell, a gastrointestinal cancer cell, a colorectal cancer cell, a pancreatic cancer cell, or a liver cancer cell.

[0140] 100.The above cancers include hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, and small cell lung cancer (SCLC). , large cell (undifferentiated) cancer, triple negative breast cancer, gastric adenocarcinoma, endometrial cancer, or pancreatic cancer.

[0141] 101. The preceding practice, which reduces the viability of the plurality of infected cells when brought into contact with the plurality of infected cells and the plurality of uninfected cells to a greater extent than it reduces the viability of the plurality of infected cells. The expression repressor according to any of the forms.

[0142] 102. An expression repressor according to any of the preceding embodiments, wherein said infection is a viral infection.

[0143] 103. The expression repressor according to embodiment 102, wherein the viral infection is hepatitis, eg, hepatitis B.

[0144] 104. The expression repressor according to any one of embodiments 92 to 103, wherein the infected cell is a human hepatocyte.

[0145] 105. Assay for the viability of cancer cells (e.g. HCC cells) using LNP delivery of mRNA encoding the expression repressor, e.g. 0.04-0.4, 0.04-0.1 when tested in the assay according to Example 12 , 0.1-0.2, 0.2-0.3, or 0.3-0.4 μg / mL.

[0146] 106. Assay for the viability of cancer cells (e.g., lung cancer cells) using LNP delivery of mRNA encoding the expression repressor, e.g., 0.1-2.5, 0.5-2.2 when tested in the assay according to Example 18. , 1.0 to 1.5, and 1.2 to 2 μg / mL, the expression repressor according to any one of embodiments 1 to 104.

[0147] 107. Assay for reduction of MYC mRNA levels in cancer cells (e.g. HCC cells) using LNP delivery of mRNA encoding the expression repressor, e.g. 0.004 to 0.08 when tested in the assay according to Example 12; The expression repressor according to any of the preceding embodiments, having an EC50 of 0.004-0.01, 0.01-0.02, 0.02-0.04, or 0.04-0.08 μg / mL.

[0148] 108. Assay for reduction in MYC mRNA levels in cancer cells (e.g., lung cancer cells) using LNP delivery of mRNA encoding the expression repressor, e.g., 0.04 to 0.1 when tested in the assay according to Example 18; The expression repressor according to any of the preceding embodiments, having an EC50 of 0.04-0.09, 0.05-0.09, or 0.06-0.8 μg / mL.

[0149] 109. increasing the level of a protein encoded by a target gene, e.g. MYC, in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% compared to the protein level in untreated cells; A repressor of expression according to any of the preceding embodiments, which reduces expression by at least 60%, at least 70%, at least 80%, or at least 90%.

[0150] 110. An expression repressor according to any of the preceding embodiments, which is capable of reducing tumor volume, eg, in a human subject or in a mammalian model.

[0151] 111. For example, in a mammalian model, tumor volume is compared to chemotherapeutic agents when measured, for example, on day 20 after the start of treatment, where the expressed repressor is administered at a dose of 3 mg / kg every 5 days. An expression repressor according to any of the preceding embodiments, wherein the expression repressor is capable of being reduced to the same extent or to a higher degree.

[0152] 112. For example, on the 20th day after the start of treatment, the expression repressor 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. An expression repressor according to any of the preceding embodiments, which is capable of reducing tumor volume, eg, when measured, eg, in a mammalian model, compared to a PBS control.

[0153] 113. Any of the preceding embodiments, wherein the tumor volume is reduced by at least about 10%, 20%, 30%, or 40%, e.g., 20 days after initiation of treatment, compared to a PBS-treated control. An expression repressor described in Crab.

[0154] 114. The expression repressor according to any one of embodiments 111 to 113, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0155] 115. An expression repressor according to any of the preceding embodiments, wherein the system is capable of reducing tumor volume to a similar or higher degree compared to a small molecule MYC inhibitor.

[0156] 116. The small molecule MYC inhibitor is MYCi975, and optionally the tumor volume is at least about 10%, 20%, 30% compared to MYCi975-treated controls, e.g., 20 days after initiation of treatment. , or 40%.

[0157] 117. The expression repressor of any of the preceding embodiments, wherein no weight loss occurs or less than 3%, 2%, or 1% weight loss occurs compared to the time treatment was initiated.

[0158] 118. A first expression repressor according to any of the preceding embodiments; a second expressed repressor, e.g. a second expressed repressor as described herein, e.g. a second expressed repressor as described in any of the preceding embodiments; system containing.

[0159] 119. A first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor comprising a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS) operably linked to the MYC gene. )) or a first expression repressor that binds to a sequence proximal to the transcriptional regulatory element; a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to or proximate to the anchor sequence of an anchor sequence mediated junction (ASMC) comprising the MYC gene; a second expression repressor that binds to the system containing.

[0160] 120. The transcriptional regulatory element comprises a promoter, and the anchor sequence contains a CTCF binding motif; 119. The system according to embodiment 118 or 119.

[0161] 121. The system according to any of embodiments 118 to 120, wherein the second expression repressor binds to a downstream region adjacent to the CTCF binding motif.

[0162] 122. The system according to any of embodiments 118 to 120, wherein the second expression repressor binds to an upstream region adjacent to the CTCF binding motif.

[0163] 123. Targeting wherein the first expression repressor binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence SEQ ID NO: 2, 3, 4, 71-86, or 200-206. including parts; and The second expression repressor has a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 3, 4, 71-86, or 200-206. include, The system according to any of embodiments 118-122.

[0164] 124. The first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 96-110. The system according to any of embodiments 118-123.

[0165] 125. said first expression repressor comprises a targeting moiety that binds 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; as well as the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 77; The system according to any of embodiments 118-124.

[0166] 126. A first expression repressor comprising a first targeting moiety and optionally a first effector moiety, which binds to a promoter operably linked to the MYC gene or binds to a sequence proximal to said promoter. a first expression repressor; a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to an enhancer (e.g., a super enhancer) of the MYC gene; system containing.

[0167] 127. said first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 204, and the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of either SEQ ID NO: 199 or 201; 127. The system according to embodiment 126.

[0168] 128. A system for reducing MYC expression, the system comprising: a) a first expression repressor, i) The amino acid sequence according to SEQ ID NO: 13 or at least 80, 85, 90, 95, 99, or 100% identical to it, or 20, 19, 18, 17, 16, 15 positions differing therefrom; , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first targeting moiety having no more than one sequence; ii) 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 20, 19, 18, 17, 16 positions differing therefrom; , 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first effector portion having no more than one sequence; a first expression repressor comprising; b) a second expression repressor, i) Amino acid sequence according to SEQ ID NO: 7 169 or 171 or at least 80, 85, 90, 95, 99, or 100% identical to it, or 20, 19, 18, 17 positions differing therefrom , 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a second targeting moiety having no more than one sequence; ii) the amino acid sequence according to SEQ ID NO: 18, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a second effector portion having a sequence of no more than 1 position; a second expression repressor comprising, and system containing.

[0169] 129. Said first expression repressor comprises a first nuclear localization signal, e.g. an SV40 NLS, e.g. a sequence according to SEQ ID NO: 135 or at least 80 , 85, 90, 95, 99, or 100% identity.

[0170] 130. The first expression repressor comprises a second nuclear localization signal, for example, a nucleoplasmin NLS, for example, a sequence according to SEQ ID NO: 136, or at least a second nuclear localization signal located on the C-terminal side of the first effector moiety. 129. The system of embodiment 128 or 129, further comprising sequences of 80, 85, 90, 95, 99, or 100% identity.

[0171] 131. Said second expression repressor comprises a first nuclear localization signal, e.g. an SV40 NLS, e.g. a sequence according to SEQ ID NO: 135 or at least 80 , 85, 90, 95, 99, or 100% identity.

[0172] 132. The second expression repressor comprises a second nuclear localization signal, e.g., nucleoplasmin NLS, e.g., the sequence according to SEQ ID NO: 136, or at least a second nuclear localization signal located on the C-terminal side of the second effector moiety. 132. The system of any of embodiments 128-131, further comprising sequences of 80, 85, 90, 95, 99, or 100% identity.

[0173] 133. Said first expression repressor further comprises a first linker located between said first targeting moiety and said first effector moiety, optionally said first linker comprising an amino acid sequence according to SEQ ID NO: 137. or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto.

[0174] 134. Said second expression repressor further comprises a second linker located between said second targeting moiety and said second effector moiety, optionally said second linker comprising an amino acid sequence according to SEQ ID NO: 138. or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto.

[0175] 135. The first expression repressor comprises an amino acid sequence C-terminal to the first effector moiety, such as a sequence of up to 30, 25, 20, or 18 amino acids, such as the sequence according to SEQ ID NO: 126, or at least 135. The system of any of embodiments 128-134, further comprising sequences of 80, 85, 90, 95, 99, or 100% identity.

[0176] 136. The second expression repressor comprises an amino acid sequence N-terminal to the second targeting moiety, such as a sequence of up to 30, 25, 20, or 18 amino acids, such as the sequence according to SEQ ID NO: 128, or at least 133. The system of any of embodiments 128-132, further comprising sequences of 80, 85, 90, 95, 99, or 100% identity.

[0177] 137. The first expression repressor of embodiments 128 to 136, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The system described in any of the above.

[0178] 138. Any of embodiments 128 to 137, wherein the second expression repressor has the amino acid sequence according to SEQ ID NO: 24, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. system described in.

[0179] 139. Any of embodiments 128 to 137, wherein the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 169, or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto. The system described.

[0180] 140. Any of embodiments 128 to 137, wherein the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 171, or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto. The system described.

[0181] 141. Any of embodiments 128 to 140, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto. The system described in Crab.

[0182] 142. The second expression repressor of embodiments 128 to 140, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 179, 185, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The system described in any of the above.

[0183] 143. A nucleic acid encoding the first expression repressor and second repressor of the system according to any one of embodiments 128 to 142.

[0184] 144. A nucleic acid encoding a system for reducing MYC expression, comprising: a) A first region encoding a first expression repressor, wherein the first expression repressor is i) The amino acid sequence according to SEQ ID NO: 13 or at least 80, 85, 90, 95, 99, or 100% identical to it, or 20, 19, 18, 17, 16, 15 positions differing therefrom; , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first targeting moiety having no more than one sequence; ii) 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 20, 19, 18, 17, 16 positions differing therefrom; , 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first effector portion having no more than one sequence; a first area including; b) a second region encoding a second expression repressor, wherein the second expression repressor is i) Amino acid sequence according to SEQ ID NO: 7 or at least 80, 85, 90, 95, 99, or 100% identical to it, or 20, 19, 18, 17, 16, 15 positions differing therefrom; , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a second targeting moiety having no more than one sequence; ii) the amino acid sequence according to SEQ ID NO: 18, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a second effector portion having a sequence of no more than 1 position; the second area, including Nucleic acid containing.

[0185] 145. The nucleic acid of embodiment 144, wherein said first region is 5' of said second region.

[0186] 146. The nucleic acid of embodiment 144, wherein said first region is 3' of said second region.

[0187] 147. The first region comprises a first nuclear localization signal, e.g. an SV40 NLS, e.g. a sequence according to SEQ ID NO: 135 or at least 80,85 , 90, 95, 99, or 100% identity.

[0188] 148. The first region comprises a second nuclear localization signal, e.g. nucleoplasmin NLS, e.g. a sequence according to SEQ ID NO: 136 or at least 80 sequences thereof, e.g. located C-terminal to the first effector moiety; 148. The nucleic acid of any of embodiments 145-147, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0189] 149. The second region comprises a first nuclear localization signal, e.g. an SV40 NLS, e.g. a sequence according to SEQ ID NO: 135 or at least 80,85 , 90, 95, 99, or 100% identity.

[0190] 150. The second region comprises a second nuclear localization signal, e.g. nucleoplasmin NLS, e.g. a sequence according to SEQ ID NO: 136 or at least 80 sequences thereof, e.g. located C-terminal to the second effector moiety. 149. The nucleic acid according to any of embodiments 145-149, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0191] 151. said first region is a first linker located between said first targeting moiety and said first effector moiety, optionally comprising an amino acid sequence according to SEQ ID NO: 137 or at least 80, 85, 151. The nucleic acid of any of embodiments 145-150, further comprising a nucleotide sequence encoding a first linker having 90, 95, 99, or 100% sequence identity.

[0192] 152. said second region is a second linker located between said second targeting moiety and said second effector moiety, optionally comprising an amino acid sequence according to SEQ ID NO: 138 or at least 80, 85, 152. The nucleic acid of any of embodiments 145-151, further comprising a nucleotide sequence encoding a second linker having 90, 95, 99, or 100% sequence identity.

[0193] 153. The first region comprises an amino acid sequence C-terminal to the first effector portion, such as a sequence of up to 30, 25, 20, or 18 amino acids, such as a sequence according to SEQ ID NO: 126 or at least 80 amino acids, 153. The nucleic acid according to any of embodiments 145-152, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0194] 154. The second region comprises an amino acid sequence N-terminal to the second targeting moiety, such as a sequence of up to 30, 25, 20, or 18 amino acids, such as a sequence according to SEQ ID NO: 128 or at least 80 amino acids, 154. The nucleic acid according to any of embodiments 145-153, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0195] 155. The first expression repressor of embodiments 145 to 154, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any of the above.

[0196] 156. Any of embodiments 145 to 155, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. Nucleic acid described in.

[0197] 157. The first region comprises a nucleotide sequence encoding the first targeting moiety, and the nucleotide sequence encoding the first targeting moiety comprises the sequence according to SEQ ID NO: 46 or 131, or at least 80, 85, 90 , 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 157. The nucleic acid according to any of embodiments 145-156, comprising no more than 6, 5, 4, 3, 2, or 1 sequence.

[0198] 158. The first region comprises a nucleotide sequence encoding the first effector moiety, and the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132, or at least 80, 85, 90, 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 , 6, 5, 4, 3, 2, or 1 of the nucleic acids according to any of embodiments 145-157.

[0199] 159. The second region comprises a nucleotide sequence encoding the second targeting moiety, and the nucleotide sequence encoding the second targeting moiety comprises the sequence according to SEQ ID NO: 40 or at least 80, 85, 90, 95 , 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 159. The nucleic acid according to any of embodiments 145-158, comprising no more than 5, 4, 3, 2, or 1 sequence.

[0200] 160. The first region comprises a nucleotide sequence encoding the first effector moiety, and the nucleotide sequence encoding the first effector moiety is a sequence according to SEQ ID NO: 51, or at least 80, 85, 90, 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 , 5, 4, 3, 2, or 1 of the nucleic acids according to any of embodiments 145-159.

[0201] 161. The first region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 63 or 130, or has 20 positions that differ from it, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence, and the polyA sequence is Optionally, the nucleic acid according to any of embodiments 145-160.

[0202] 162. The second region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 57, or has 20, 19, Contains no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or one polyA sequence, optionally The nucleic acid according to any one of embodiments 145 to 161.

[0203] 163. A nucleic acid encoding a system for reducing MYC expression, comprising: a) A first region encoding a first expression repressor, wherein the first expression repressor is i) The amino acid sequence according to SEQ ID NO: 13 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or 20, 19, 18, 17, 16, 15 positions differing therefrom; , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first targeting moiety having no more than one sequence; ii) 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 20, 19, 18, 17, 16 positions differing therefrom; , 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first effector portion having no more than one sequence; a first area including; b) a second region encoding a second expression repressor, wherein the second expression repressor is i) Amino acid sequence according to SEQ ID NO: 169 or at least 80, 85, 90, 95, 99, or 100% identical to it, or 20, 19, 18, 17, 16, 15 positions differing therefrom; , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a second targeting moiety having no more than one sequence; ii) the amino acid sequence according to SEQ ID NO: 18, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a second effector portion having a sequence of no more than 1 position; the second area, including Nucleic acid containing.

[0204] 164. A nucleic acid encoding a system for reducing MYC expression, comprising: a) A first region encoding a first expression repressor, wherein the first expression repressor is i) The amino acid sequence according to SEQ ID NO: 13 or at least 80, 85, 90, 95, 99, or 100% identical to it, or 20, 19, 18, 17, 16, 15 positions differing therefrom; , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first targeting moiety having no more than one sequence; ii) 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 20, 19, 18, 17, 16 positions differing therefrom; , 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a first effector portion having no more than one sequence; a first area including; b) a second region encoding a second expression repressor, wherein the second expression repressor is i) Amino acid sequence according to SEQ ID NO: 171 or at least 80, 85, 90, 95, 99, or 100% identical to it, or 20, 19, 18, 17, 16, 15 positions differing therefrom , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a second targeting moiety having no more than one sequence; ii) the amino acid sequence according to SEQ ID NO: 18, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or 20, 19, 18, 17, 16, a second effector moiety having a sequence of not more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; the second area, including Nucleic acid containing.

[0205] 165. The nucleic acid of embodiment 163 or 164, wherein the first region is 5' of the second region.

[0206] 166. The nucleic acid according to embodiment 163 or 164, wherein the first region is 3' of the second region.

[0207] 167. The first region comprises a first nuclear localization signal, e.g. an SV40 NLS, e.g. a sequence according to SEQ ID NO: 135 or at least 80,85 , 90, 95, 99, or 100% identity.

[0208] 168. The first region comprises a second nuclear localization signal, e.g. nucleoplasmin NLS, e.g. a sequence according to SEQ ID NO: 136 or at least 80 sequences thereof, e.g. located C-terminal to the first effector moiety; 168. The nucleic acid of any of embodiments 163-167, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0209] 169. The second region comprises a first nuclear localization signal, e.g. SV40 NLS, e.g. a sequence according to SEQ ID NO: 135 or at least 80, 85 , 90, 95, 99, or 100% identity.

[0210] 170. The second region comprises a second nuclear localization signal, e.g. nucleoplasmin NLS, e.g. a sequence according to SEQ ID NO: 136 or at least 80 sequences thereof, e.g. located C-terminal to the second effector moiety. 169. The nucleic acid according to any of embodiments 163-169, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0211] 171. said first region is a first linker located between said first targeting moiety and said first effector moiety, optionally comprising an amino acid sequence according to SEQ ID NO: 137 or at least 80, 85, 171. The nucleic acid of any of embodiments 163-170, further comprising a nucleotide sequence encoding a first linker having a sequence identity of 90, 95, 99, or 100%.

[0212] 172. Said second region is a second linker located between said second targeting moiety and said second effector moiety, optionally comprising an amino acid sequence according to SEQ ID NO: 138 or at least 80, 85, 172. The nucleic acid of any of embodiments 163-171, further comprising a nucleotide sequence encoding a second linker having a sequence identity of 90, 95, 99, or 100%.

[0213] 173. The first region comprises an amino acid sequence C-terminal to the first effector portion, such as a sequence of up to 30, 25, 20, or 18 amino acids, such as a sequence according to SEQ ID NO: 126 or at least 80 amino acids, 172. The nucleic acid of any of embodiments 163-171, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0214] 174. The second region comprises an amino acid sequence N-terminal to the second targeting moiety, such as a sequence of up to 30, 25, 20, or 18 amino acids, such as a sequence according to SEQ ID NO: 128 or at least 80 amino acids, 174. The nucleic acid according to any of embodiments 163-173, further comprising a nucleotide sequence encoding a sequence of 85, 90, 95, 99, or 100% identity.

[0215] 175. The first expression repressor of embodiments 163 to 174, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any of the above.

[0216] 176. The second expression repressor of embodiments 144 to 175, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any of the above.

[0217] 177. Embodiments 144 to 176, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 179 or 185, or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto. The nucleic acid according to any one of.

[0218] 178. The 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, 99, or 100% identity thereto, and the second expression repressor described above has an amino acid sequence according to SEQ ID NO: 24, 141, 177, 179, 183, or 185, or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto. The nucleic acid according to any one of.

[0219] 179. The first region comprises a nucleotide sequence encoding the first targeting moiety, and the nucleotide sequence encoding the first targeting moiety comprises the sequence according to SEQ ID NO: 46 or 131, or at least 80, 85, 90 , 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 179. The nucleic acid according to any of embodiments 144-178, comprising no more than 6, 5, 4, 3, 2, or 1 sequence.

[0220] 180. The first region comprises a nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion comprises a sequence according to SEQ ID NO: 52 or 132, or at least 80, 85, 90, 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 , 6, 5, 4, 3, 2, or 1 of the nucleic acids according to any of embodiments 144-179.

[0221] 181. The second region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 173, or has 20, 19, 18 positions that differ from it. , 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence, with optional polyA sequences 180. The nucleic acid according to any one of embodiments 144 to 180.

[0222] 182. The second region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence of SEQ ID NO: 175, or has 20, 19, 18 positions that differ from it. , 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence, with optional polyA sequences 182. The nucleic acid according to any one of embodiments 144 to 181.

[0223] 183. The second region comprises a nucleotide sequence encoding the second effector moiety, and the nucleotide sequence encoding the second effector moiety is a sequence according to SEQ ID NO: 51, or at least 80, 85, 90, 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 , 5, 4, 3, 2, or 1 of the nucleic acids according to any of embodiments 144-182.

[0224] 184. The first region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 63 or 130, or has 20 positions that differ from it, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence, and the polyA sequence is Optionally, the nucleic acid according to any of embodiments 144-183.

[0225] 185. The second region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 189, or has 20, 19, Contains no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or one polyA sequence, optionally The nucleic acid according to any one of embodiments 144 to 184.

[0226] 186. The second region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 194, or has 20, 19, Contains 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one polyA sequence, optionally The nucleic acid according to any one of embodiments 144 to 185,

[0227] 187. The first region comprises a nucleotide sequence encoding the first effector moiety, and the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132, or at least 80, 85, 90 sequences thereof. , 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 187. The nucleic acid according to any of embodiments 144-186, comprising no more than 6, 5, 4, 3, 2, or 1 sequence.

[0228] 188. The first region comprises a nucleotide sequence encoding the first targeting moiety, and the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 46 or 131, or at least 80, 85, 90, 95, 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 , 6, 5, 4, 3, 2, or 1 of the nucleic acids according to any of embodiments 144-187.

[0229] 189. The second region comprises a nucleotide sequence encoding the second effector moiety, and the nucleotide sequence encoding the second effector moiety comprises the sequence according to SEQ ID NO: 51 or at least 80, 85, 90, 95 , 99, or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 189. The nucleic acid according to any of embodiments 144-188, comprising no more than 5, 4, 3, 2, or 1 sequence.

[0230] 190. The second region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 189, or has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than one sequence of embodiments 144-189. The nucleic acid according to any of the above.

[0231] 191. The second region has at least 80, 85, 90, 95, 99, or 100% identity with the nucleotide sequence according to SEQ ID NO: 194, or has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or more than one sequence of embodiments 144-190. The nucleic acid according to any of the above.

[0232] 192. The nucleotide sequence according to SEQ ID NO: 93, 112, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than one sequence according to any of embodiments 144-191. of nucleic acids.

[0233] 193. A nucleotide sequence according to SEQ ID NO: 196 or 197, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than one sequence according to any of embodiments 144-192. of nucleic acids.

[0234] 194. The system or nucleic acid according to any of embodiments 118-193, wherein the first expression repressor comprises the first effector moiety.

[0235] 195. The system or nucleic acid according to any of embodiments 118-194, wherein said second expression repressor comprises said second effector moiety.

[0236] 196. The system or nucleic acid according to any of embodiments 118-195, wherein the first effector moiety has a different amino acid sequence than the second effector moiety.

[0237] 197. The system or nucleic acid according to any of embodiments 118-196, wherein the first effector moiety is a persistent effector moiety.

[0238] 198. The system or nucleic acid according to any of embodiments 118-125 or 144-197, wherein the first effector moiety is a transient effector moiety.

[0239] 199. The system or nucleic acid according to any of embodiments 118-198, wherein the first effector moiety is an epigenetic modification moiety.

[0240] 200. The system or nucleic acid according to any of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone methyltransferase.

[0241] 201. The first effector portion is SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional mutation of any of these. 201. A system or a nucleic acid according to embodiment 200, comprising a protein selected from a body or a fragment, e.g., a SET domain of any of these.

[0242] 202. The system or nucleic acid of any of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone demethylase (eg, a lysine demethylase).

[0243] 203. The first effector moiety is KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or 203. The system or nucleic acid of embodiment 202, comprising a protein selected from (fragments).

[0244] 204. The system or nucleic acid according to any of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone deacetylase.

[0245] 205. The first effector part is HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or 205. The system or nucleic acid of embodiment 204, comprising a protein selected from a functional variant or fragment of any of these.

[0246] 206. The system or nucleic acid according to any of embodiments 118-197 or 200, wherein the first effector moiety comprises a DNA methyltransferase.

[0247] 207. The first effector moiety 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. , the system or nucleic acid of embodiment 206.

[0248] 208. The system or nucleic acid according to any of embodiments 118-143, 160-196, or 198, wherein the first effector moiety is a transcriptional repressor moiety, eg, comprising a transcriptional repressor.

[0249] 209. According to embodiment 198 or 199, the first effector moiety comprises a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these. system or nucleic acid.

[0250] 210. The system or nucleic acid according to any of embodiments 118-209, wherein the first effector moiety promotes epigenetic modification of the transcriptional regulatory element or sequences proximal thereto.

[0251] 211. The system or nucleic acid according to any of embodiments 118-210, wherein the first effector moiety catalyzes epigenetic modification of the transcriptional regulatory element or sequences proximal thereto.

[0252] 212. The system or nucleic acid according to any of embodiments 118-125, 194, or 197-211, wherein the second expression repressor does not include an effector moiety.

[0253] 213. The system or nucleic acid according to any of embodiments 118-212, wherein the second effector moiety is a transient effector moiety.

[0254] 214. The system or nucleic acid according to any of embodiments 118-125 or 194-211, wherein the second effector moiety is a persistent effector moiety.

[0255] 215. The system or nucleic acid according to any of embodiments 118-211 or 214, wherein the second effector moiety is an epigenetic modification moiety.

[0256] 216. The system or nucleic acid according to any of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone methyltransferase.

[0257] 217. The second effector moiety is SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional mutation of any of these. 217. The system or nucleic acid of embodiment 216, comprising a protein selected from a body or fragment, e.g., a SET domain of any of these.

[0258] 218. The system or nucleic acid according to any of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone demethylase (eg, lysine demethylase).

[0259] 219. Where the second effector moiety is KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant of any of these or 219. The system or nucleic acid of embodiment 218, comprising a protein selected from a fragment.

[0260] 220. The system or nucleic acid according to any of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone deacetylase.

[0261] 221. The second effector part is HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or 221. The system or nucleic acid of embodiment 220, comprising a protein selected from a functional variant or fragment of any of these.

[0262] 222. The system or nucleic acid according to any of embodiments 118-125, 194-211, or 214-215, wherein the second effector portion comprises a DNA methyltransferase.

[0263] 223. The second effector moiety 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. , the system or nucleic acid of embodiment 222.

[0264] 224. The system or nucleic acid according to any of embodiments 118-211 or 213, wherein the second effector moiety is a transcriptional repressor moiety.

[0265] 225. The system or nucleic acid of embodiment 224, wherein said second effector moiety promotes epigenetic modification of said anchor sequence or sequences adjacent thereto.

[0266] 226. The system or nucleic acid of embodiment 223 or 224, wherein the second effector moiety binds to one or more endogenous epigenetic modification proteins or to one or more endogenous transcriptional modification proteins.

[0267] 227. The system of any of embodiments 223-226, wherein the second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these. Or nucleic acid.

[0268] 228. The first effector portion is a persistent effector portion, and the second effector part is a temporary effector part; The system or nucleic acid according to any one of embodiments 118-197, 199-207, 210-211, 213, or 224-227.

[0269] 229. The system or nucleic acid of embodiment 228, wherein the first effector moiety is an epigenetic modification moiety.

[0270] 230. The system or nucleic acid of embodiment 227 or 228, wherein the second effector moiety is a transcriptional repressor moiety.

[0271] 231. The first effector moiety comprises a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any of these, or any combination thereof, and the second effector portion comprises a transcriptional repressor or a functional variant or fragment of any of these; The system or nucleic acid according to any of embodiments 227-230.

[0272] 232. The first effector moiety comprises a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any of these, or any combination thereof, and the second expressed repressor does not contain a second effector part; The system or nucleic acid according to any of embodiments 118-125, 194, 197, 199-207, 210-212, or 190.

[0273] 233. The first effector portion is 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, a functional variant or fragment of any of these, or any of these. including a combination of; and the second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, a functional variant or fragment of any of these, or a combination of any of these; The system or nucleic acid according to any one of embodiments 118-125, 199-207, 210-211, 213-214, or 224-231.

[0274] 234. The first effector moiety comprises a DNA methyltransferase, and the second effector portion includes a transcriptional repressor; The system or nucleic acid according to any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, or 233.

[0275] 235. The first effector moiety comprises a DNA methyltransferase, and the second expressed repressor does not contain a second effector part; The system or nucleic acid according to any of embodiments 118-125, 194, 197, 200, 206-207, 210-212, or 232.

[0276] 236. Embodiments 118~, wherein the first effector moiety comprises MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any of these. The system or nucleic acid according to any one of 125, 200, 206 to 207, and 210 to 235.

[0277] 237. Embodiments 118-211, 214, 224-234, wherein the second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these. or the system or nucleic acid according to any of 236.

[0278] 238. The first effector moiety comprises MQ1 or a functional variant or fragment of any of these, and the second effector comprises KRAB or a functional variant or fragment of any of these; The system or nucleic acid according to any one of embodiments 118-211, 199, 206-207, 210-211, 213, 224-234, or 236-237.

[0279] 239. The first effector moiety comprises MQ1 or a functional variant or fragment of any of these, and the second expressed repressor does not contain a second effector part; The system or nucleic acid according to any of embodiments 118-125, 194, 197, 199-207, or 210-212, 229, 232, 235, or 236.

[0280] 240. The first expression repressor has an amino acid sequence selected from any of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, 177-180, or 183-186, or at least 80, 85 , 90, 95, or 99% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 , 5, 4, 3, 2, or one sequence.

[0281] 241. The second expression repressor has an amino acid sequence selected from any of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, 177-180, or 183-186, or at least 80, 85 , 90, 95, or 99% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 . system or nucleic acid.

[0282] 242. The first expression repressor has the amino acid sequence of SEQ ID NO: 30, 129, 133, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20 positions differing therefrom, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence, and The expressed repressor has the amino acid sequence of SEQ ID NO: 24, 134, 141, 177, 179, 183, or 185, or a sequence that is at least 80, 85, 90, 95, or 99% identical thereto, or differs therefrom. 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or a sequence that does not exceed 1 position; The system or nucleic acid according to any one of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240-241.

[0283] 243. The first expression repressor has the first nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20 positions differing therefrom, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence, and 2. The expressed repressor has a second nucleotide sequence of SEQ ID NO: 57, 189, or 194, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19 positions differing therefrom. , 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence. The system or nucleic acid according to any one of ~198, 200, 206-211, 213-216, 222-223, 236-237, or 240-242.

[0284] 244. The first and second repressors are the nucleic acid sequence of SEQ ID NO: 93, 94, 112, 113, 196, or 197, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto. , or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 location that differs from that. The system or nucleic acid of any of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240-243, encoded by a sequence that does not exceed.

[0285] 245. The amino acid sequence of SEQ ID NO: 91, 92, 121, 122, 181, 182, 187, or 188, or a sequence at least 80, 85, 90, 95, or 99% identical thereto, or a position that differs therefrom. 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence, The system or nucleic acid according to Form 244.

[0286] 246.The first expression repressor described above is from the N-terminus to the C-terminus: (i) a first nuclear localization signal, e.g. SV40 NLS; e.g. the sequence according to SEQ ID NO: 135; (ii) a first targeting moiety, e.g. a zinc finger binding domain, e.g. ZF9; e.g. a sequence according to SEQ ID NO: 13; (iii) a first effector moiety, e.g. a DNA methyltransferase, e.g. MQ1; e.g. a sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, e.g. nucleoplasmin NLS; e.g. the sequence according to SEQ ID NO: 136; including; and the above-mentioned second expression repressor from the N-terminus to the C-terminus: (v) a third nuclear localization signal, for example SV40NLS; for example the sequence according to SEQ ID NO: 135; (vi) a second targeting moiety, e.g. a zinc finger binding domain, e.g. ZF3; e.g. a sequence according to SEQ ID NO: 7; (vii) a second effector moiety, e.g. KRAB, e.g. the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, e.g. nucleoplasmin NLS, e.g. the sequence according to SEQ ID NO: 136; The system or nucleic acid according to any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, comprising:

[0287] 247.The first expression repressor described above is from the N-terminus to the C-terminus: (i) a first nuclear localization signal, e.g. SV40 NLS; e.g. the sequence according to SEQ ID NO: 135; (ii) a first targeting moiety, e.g. a zinc finger binding domain, e.g. ZF9; e.g. a sequence according to SEQ ID NO: 13; (iii) a first effector moiety, e.g. a DNA methyltransferase, e.g. MQ1; e.g. a sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, e.g. nucleoplasmin NLS; e.g. the sequence according to SEQ ID NO: 136; including; and the above-mentioned second expression repressor from the N-terminus to the C-terminus: (v) a third nuclear localization signal, for example SV40NLS; for example the sequence according to SEQ ID NO: 135; (vi) a second targeting moiety, e.g. a zinc finger binding domain, e.g. ZF54; e.g. a sequence according to SEQ ID NO: 169; (vii) a second effector moiety, e.g. KRAB, e.g. the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, e.g. nucleoplasmin NLS, e.g. the sequence according to SEQ ID NO: 136; The system or nucleic acid according to any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, comprising:

[0288] 248.The first expression repressor described above is from the N-terminus to the C-terminus: (i) a first nuclear localization signal, e.g. SV40 NLS; e.g. the sequence according to SEQ ID NO: 135; (ii) a first targeting moiety, e.g. a zinc finger binding domain, e.g. ZF9; e.g. a sequence according to SEQ ID NO: 13; (iii) a first effector moiety, e.g. a DNA methyltransferase, e.g. MQ1; e.g. a sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, e.g. nucleoplasmin NLS; e.g. the sequence according to SEQ ID NO: 136; including; and the above-mentioned second expression repressor from the N-terminus to the C-terminus: (v) a third nuclear localization signal, for example SV40NLS; for example the sequence according to SEQ ID NO: 135; (vi) a second targeting moiety, e.g. a zinc finger binding domain, e.g. ZF67; e.g. a sequence according to SEQ ID NO: 171; (vii) a second effector moiety, e.g. KRAB, e.g. the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, e.g. nucleoplasmin NLS, e.g. the sequence according to SEQ ID NO: 136; The system or nucleic acid according to any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, comprising:

[0289] 249. The system according to any one of embodiments 118 to 248, which is capable of reducing MYC expression to a higher degree than the first expression repressor alone or the second expression repressor alone.

[0290] 250. Embodiments 128-194, which are capable of reducing the expression of MYC to a higher degree than any of the expression repressors of SEQ ID NOs: 22, 23, 25-29, 31-37, alone or in combination, or The system described in any of 242 to 249.

[0291] 251. The system of any of embodiments 118-250, which is capable of reducing tumor volume, eg, in a human subject or in a mammalian model.

[0292] 252. For example, in a model system as described in Example 15, when measured, for example, on the 20th day after the start of treatment, where the expressed repressor is administered at a dose of 3 mg / kg every 5 days, For example, the system of any of embodiments 128-193 or 242-209 is capable of reducing tumor volume to a similar or higher degree compared to chemotherapeutic agents in a mammalian model.

[0293] 253. For example, in a model system as described in Example 14, when measured, for example, on day 15 after initiation of treatment, where the expressed repressor is administered at a dose of 6 mg / kg every 5 days, For example, the system of any of embodiments 128-193 or 242-252 is capable of reducing tumor volume to a higher degree than chemotherapeutic agents in a mammalian model.

[0294] 254. For example, at day 20 after initiation of treatment, the tumor volume is reduced by at least about 10%, 20%, 30%, 40%, 50%, or 60% compared to controls treated with PBS. The system according to any one of embodiments 128-193 or 242-253.

[0295] 255. The system of embodiment 254, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0296] 256. The system of any of embodiments 128-193 or 242-253, which is capable of reducing tumor volume to a similar or higher degree than a small molecule MYC inhibitor.

[0297] 257. The small molecule MYC inhibitor is MYCi975, and optionally the tumor volume is at least about 10%, 20%, 30% compared to the MYCi975-treated control, e.g., 20 days after initiation of treatment. %, or 40%.

[0298] 258. The system of any of embodiments 118-257, wherein there is no weight loss or less than 3%, 2%, or 1% weight loss compared to when treatment began.

[0299] 259. The system or nucleic acid of any of embodiments 118-258, wherein said 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. .

[0300] 260. The system or nucleic acid of any of embodiments 118-260, wherein said 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. .

[0301] 261. The system or nucleic acid of any of embodiments 118-260, wherein the first targeting moiety comprises a CRISPR / Cas domain (eg, a first CRISPR / Cas domain).

[0302] 262. The system or nucleic acid of any of embodiments 118-261, wherein the second targeting moiety comprises a second CRISPR / Cas domain (eg, a second CRISPR / Cas domain).

[0303] 263.i) said first CRISPR / Cas domain binds a first guide RNA, ii) said second CRISPR / Cas domain binds a second guide RNA, or iii) (i) and (ii) 263. The system or nucleic acid of embodiment 262, which is both.

[0304] 264. said first CRISPR / Cas domain does not bind said second guide RNA, or has a K of at least 10, 20, 50, 100, 1000, or 10,000 nM Dand said second CRISPR / Cas domain does not bind said first guide RNA, or has a K of at least 10, 20, 50, 100, 1000, or 10,000 nM. D 264. The system or nucleic acid of embodiment 262 or 263, which binds with.

[0305] 265. The system or nucleic acid according to any of embodiments 260 to 264, wherein the first CRISPR / Cas domain comprises a different amino acid sequence than the second CRISPR / Cas domain.

[0306] 266. Embodiments 260~, wherein the first or second CRISPR / Cas domain comprises an amino acid sequence of a Cas protein or a Cpf1 protein selected from Table 1, or a variant (e.g., a mutant) of either of them. 265. The system or nucleic acid according to any of 265.

[0307] 267. Said first CRISPR / Cas domain comprises an amino acid sequence of a Cas protein selected from Table 1 or a Cpf1 protein, or a variant (e.g., a mutant) thereof, and said second CRISPR / Cas domain comprises The system or nucleic acid according to any of embodiments 260-266, wherein the Cas domain comprises an amino acid sequence of a different Cas protein or Cpf1 protein selected from Table 1, or a variant (e.g., a mutant) of either. .

[0308] 268. The system or nucleic acid of any of embodiments 118-260, wherein the first targeting moiety comprises a zinc finger domain (eg, a first zinc finger domain).

[0309] 269. The system or nucleic acid of any of embodiments 118-260 or 268, wherein the second targeting moiety comprises a zinc finger domain (eg, a second zinc finger domain).

[0310] 270. The system or nucleic acid according to any of embodiments 118-261 or 268-269, wherein the first targeting moiety comprises a first zinc finger domain, and the second targeting moiety comprises a second zinc finger domain.

[0311] 271. The system or nucleic acid according to any of embodiments 268 to 270, wherein the first zinc finger domain and the second zinc finger domain bind the same genomic locus and have, for example, the same amino acid sequence.

[0312] 272. The system or nucleic acid according to any of embodiments 268 to 271, wherein the first zinc finger domain and the second zinc finger domain have different amino acid sequences or bind different genomic loci.

[0313] 273. The first zinc finger molecule comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally 11, 10, 9, 8, 7, 6, or no more than 5 zinc fingers).

[0314] 274. The first zinc finger molecule is 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, 2~9, 2~8, 2~7, 2~6, 2~5, 2~4, 2~3, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~ 5, 3~4, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~10, 5~9, 5~8, 5~7, 5~6, Embodiment 267, comprising 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. The system or nucleic acid according to any of ~273.

[0315] 275. The system or nucleic acid according to any of embodiments 268-274, wherein the first zinc finger domain comprises 3 or 9 zinc fingers.

[0316] 276. The second zinc finger domain comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally 11, 10, 9, 8, 7, 6, or no more than 5 zinc fingers).

[0317] 277. The second zinc finger domain is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2~9, 2~8, 2~7, 2~6, 2~5, 2~4, 2~3, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~ 5, 3~4, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~10, 5~9, 5~8, 5~7, 5~6, Embodiment 268 comprising 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. The system or nucleic acid according to any of ~276.

[0318] 278. The system or nucleic acid according to any of embodiments 268-277, wherein the second zinc finger domain comprises 3 or 9 zinc fingers.

[0319] 279. The system or nucleic acid of any of embodiments 118-278, wherein the first targeting moiety comprises a TAL effector domain (eg, a first TAL effector domain).

[0320] 280. The system or nucleic acid of any of embodiments 118-260 or 279, wherein the second targeting moiety comprises a TAL effector domain (eg, a second TAL effector domain).

[0321] 281. The first TAL effector domain is 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).

[0322] 282. The first TAL effector domain is 2-40, 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 2-35, 5-35 , 10~35, 15~35, 20~35, 25~35, 30~35, 2~30, 5~30, 10~30, 15~30, 20~30, 25~30, 2~25, 5 ~25, 10~25, 15~25, 20~25, 2~20, 5~20, 10~20, 15~20, 2~15, 5~15, 10~15, 2~10, 5~10 , or 2 to 5 central repeats.

[0323] 283. The second TAL effector domain is 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). Or nucleic acid.

[0324] 284. The second TAL effector domain is 2-40, 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 2-35, 5-35 , 10~35, 15~35, 20~35, 25~35, 30~35, 2~30, 5~30, 10~30, 15~30, 20~30, 25~30, 2~25, 5 ~25, 10~25, 15~25, 20~25, 2~20, 5~20, 10~20, 15~20, 2~15, 5~15, 10~15, 2~10, 5~10 , or 2 to 5 central repeats.

[0325] 285. The system or nucleic acid of any of embodiments 118-284, wherein the first targeting moiety comprises a nucleic acid (eg, a first nucleic acid).

[0326] 286. The system of any of embodiments 129-285, wherein the second targeting moiety comprises a nucleic acid (eg, a second nucleic acid).

[0327] 287. The system or nucleic acid of any of embodiments 129-286, wherein the first targeting moiety comprises a polypeptide (eg, a first polypeptide).

[0328] 288. The system or nucleic acid of any of embodiments 129-287, wherein the second targeting moiety comprises a polypeptide (eg, a second polypeptide).

[0329] 289. The system of embodiment 287 or 288, wherein said nucleic acid is covalently linked to said polypeptide.

[0330] 290. The system of embodiment 288 or 289, wherein said nucleic acid is non-covalently associated with said polypeptide.

[0331] 291. The nucleic acid comprises a sequence complementary to the transcriptional regulatory element or a sequence adjacent thereto, or 10, 9, 8, 7, 6, 5, 4 compared to the transcriptional regulatory element or a sequence adjacent thereto. , 3, 2, or 1 mismatch.

[0332] 292. The nucleic acid comprises a sequence complementary to the anchor sequence or a sequence adjacent thereto, or 10, 9, 8, 7, 6, 5, 4, 3 compared to the anchor sequence or a sequence adjacent thereto. , 2, or 1 mismatch.

[0333] 293. The above nucleic acid is DNA, peptide nucleic acid (PNA), peptide-oligonucleotide conjugate, locked nucleic acid (LNA), cross-linked nucleic acid (BNA), polyamide, triple-stranded oligonucleotide, antisense oligonucleotide, tRNA, 293. The system of any of embodiments 275-292, comprising mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecules.

[0334] 294. The system according to any of embodiments 275-293, wherein the nucleic acid comprises gRNA.

[0335] 295. The above nucleic acid contains a sequence with at least 80, 85, 90, 95, 99, or 100% identity with any of SEQ ID NOs: 1 to 4, or has 1, 2, 3, 295. The system of any of embodiments 275-294, having no more than 4, 5, 6, 7, 8, 9, or 10 locations.

[0336] 296. The first nucleic acid contains a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any of SEQ ID NOs: 1 to 4, or has 1, 2, or 1 position that differs therefrom. 3, 4, 5, 6, 7, 8, 9, or 10 positions, and the second nucleic acid has at least 80, 85, 90, 95, 99, or 100% of any of SEQ ID NOs: 1 to 4. in any of embodiments 275-295, comprising a sequence of identity or differing from no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions. The system described.

[0337] 297. The first nucleic acid contains a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any of SEQ ID NOs: 96 to 110, or has 1, 2, or 3, 4, 5, 6, 7, 8, 9, or 10 positions, and the second nucleic acid has at least 80, 85, 90, 95, 99, or 100% of any of SEQ ID NOs: 96 to 110. in any of embodiments 275-295, comprising a sequence of identity or differing from no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions. The system described.

[0338] 298. The system according to any of embodiments 118-297, wherein the transcriptional regulatory element comprises a promoter.

[0339] 299. The system according to any of embodiments 118-298, wherein the transcriptional regulatory element comprises an enhancer; eg, a super-enhancer.

[0340] 300. The system according to any of embodiments 118-299, wherein the anchor sequence comprises a CTCF binding motif.

[0341] 301. The system according to any of embodiments 118-300, wherein the anchor sequence comprises a YY1 binding motif.

[0342] 302. Embodiments 118-301, wherein the anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto. The system described in any of the above.

[0343] 303. Embodiments 118~, wherein the anchor sequence comprises the sequence according to SEQ ID NO: 73 or 74, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto. Any system described in 302.

[0344] 304. The system according to any of embodiments 118 to 303, wherein the anchor sequence is on the same chromosome as the MYC gene.

[0345] 305. The system according to any of embodiments 118-304, wherein the anchor sequence is upstream of the MYC gene (eg, upstream of the TSS or upstream of the promoter).

[0346] 306. According to any of embodiments 118-305, 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). system.

[0347] 307. The above anchor sequence is from the MYC gene (for example, from the TSS or promoter of the MYC gene) 0.1~0.5, 0.1~1, 0.1~5, 0.1~10, 0.1~50, 0.1~100, 0.1~500, 0.1 ~1000, 0.5~1, 0.5~5, 0.5~10, 0.5~50, 0.5~100, 0.5~500, 0.5~1000, 1~5, 1~10, 1~50, 1~100, 1~500 , 1~1000, 5~10, 5~50, 5~100, 5~500, 5~1000, 10~50, 10~100, 10~500, 10~1000, 50~100, 50~500, 50 307. The system of any of embodiments 118-306, wherein the system is ~1000, 100-500, 100-1000, or 500-1000 kilobases apart.

[0348] 308. The system according to any one of embodiments 118-303 or 305-307, wherein the anchor sequence is on a different chromosome than the MYC gene.

[0349] 309. Embodiment 118, wherein the second targeting moiety binds to the anchor sequence or a sequence proximal to the anchor sequence with sufficient affinity to compete for binding with an endogenous polypeptide (e.g., CTCF or YY1). Any system listed in ~308.

[0350] 310. The system according to any of embodiments 118-309, wherein the first targeting moiety binds to a sequence at or near chromosomal coordinates 128746342-128746364, 128746321-128746343, or 128746525-128746547.

[0351] 311. The system according to any of embodiments 118-309, wherein the first targeting moiety binds to a sequence at or near chromosomal coordinates 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842. .

[0352] 312. The system according to any one of embodiments 118 to 311, wherein the second targeting moiety binds to a sequence at chromosomal coordinates 128748014 to 128748036, or a sequence adjacent thereto.

[0353] 313. The second targeting moiety binds to a sequence at or near chromosomal coordinates 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842, according to any of embodiments 118-311. system.

[0354] 314. The system according to any of embodiments 118-314, wherein the first expression repressor is a fusion molecule.

[0355] 315. The system according to any of embodiments 118-314, wherein the second expression repressor is a fusion molecule.

[0356] 316. The system according to any of embodiments 118-315, wherein the first expression repressor comprises a linker.

[0357] 317. The system according to any of embodiments 118-316, wherein the second expression repressor comprises a linker.

[0358] 318. The first expression repressor comprises a first CRISPR / Cas molecule, for example, a targeting portion comprising an inactive CRISPR / Cas protein as a first catalyst, and an effector portion comprising an epigenetic modification portion. including; and The second expression repressor comprises a targeting moiety comprising a second CRISPR / Cas molecule, e.g. a second catalytically inactive CRISPR / Cas protein, and optionally an effector moiety comprising a transcriptional repressor. including, The system according to any one of embodiments 118-267 or 285-317.

[0359] 319. The first expression repressor comprises a targeting moiety comprising a first zinc finger domain and an effector moiety comprising an epigenetic modification moiety; and the second expression repressor comprises a targeting moiety comprising a second zinc finger domain and optionally an effector moiety comprising a transcriptional repressor; The system according to any one of embodiments 118-260, 268-278, or 285-317.

[0360] 320. The first expression repressor comprises a targeting moiety comprising a CRISPR / Cas molecule, e.g. a catalytically inactive CRISPR / Cas protein, and an effector moiety comprising an epigenetic modification moiety; and the second expression repressor comprises a targeting moiety comprising a zinc finger domain and optionally an effector moiety comprising a transcriptional repressor; The system according to any of embodiments 118-120, 262, 268, or 275-318.

[0361] 321. The first expression repressor comprises a targeting moiety comprising a zinc finger domain and an effector moiety comprising an epigenetic modification moiety; and the second expression repressor comprises a targeting moiety comprising a CRISPR / Cas domain, e.g. a catalytically inactive CRISPR / Cas protein, and optionally an effector moiety comprising a transcriptional repressor; The system according to any one of embodiments 118-260, 268, or 275-318.

[0362] 322. The zinc finger domain (e.g., the first or second zinc finger domain) is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 ~3, 1~2, 2~10, 2~9, 2~8, 2~7, 2~6, 2~5, 2~4, 2~3, 3~10, 3~9, 3~8 , 3~7, 3~6, ​​3~5, 3~4, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~10, 5~9, 5 ~8, 5~7, 5~6, 6~10, 6~9, 6~8, 6~7, 7~10, 7~9, 7~8, 8~10, 8~9, or 9~ The system of any of embodiments 260, 268-278, or 275-318, comprising 10 zinc fingers, such as 3 or 9 zinc fingers.

[0363] 323. The system of any of embodiments 322, wherein the epigenetic modification moiety comprises a DNA methyltransferase.

[0364] 324. The system according to any of embodiments 118 to 323, wherein the epigenetic modification moiety comprises MQ1 or a functional variant or fragment thereof.

[0365] 325. The system according to any of embodiments 118-324, wherein the second expression repressor includes an effector moiety that includes a transcriptional repressor.

[0366] 326. The system according to any of embodiments 118-323, wherein the transcriptional repressor comprises KRAB or a functional variant or fragment thereof.

[0367] 327. The first expression repressor has the amino acid sequence of SEQ ID NO: 28-33 or 35-37, 145-149, 151, 152, or at least 80, 85, 90, 95, 99, or 100% thereof. 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 327. The system of any of embodiments 118-326, comprising no more than 2, or 1 sequence.

[0368] 328. The second expression repressor has an amino acid sequence of any of SEQ ID NOs: 22-27, 34, 139-144, 150, 177-180, 183-186, or at least 80, 85, 90, 95, 99. , or 100% identity, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 328. The system of any of embodiments 118-327, comprising no more than 4, 3, 2, or 1 sequence.

[0369] 329. The system according to any of embodiments 118 to 328, wherein binding of the first expression repressor to the transcriptional regulatory element or sequences adjacent thereto reduces cellular expression of MYC.

[0370] 330. For example, when the expression is 10, 20, 30, 40, 50, 60, 70, 80, 90, or 328. The system of embodiment 327, wherein the system is reduced by 100%.

[0371] 331. When said first expression repressor binds to said transcriptional regulatory element, the expression of MYC is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, as measured by, for example, QPCR or ELISA. 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, 328. The system of embodiment 326 or 327, which appreciably decreases over a period of 8, 9, or 10 cell divisions.

[0372] 332. When the first expression repressor binds to the transcriptional regulatory element, the expression of MYC decreases after transfection. , 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours. system.

[0373] 333. The system of any of embodiments 328-332, wherein binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto decreases cellular expression of MYC.

[0374] 334. Expression is 10, 20, 30, 40, 50, 60, 70, 80, 90, compared to expression in the absence of said second expression repressor, as measured by e.g. QPCR or ELISA. or 100%. The system of embodiment 333.

[0375] 335. When the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC is at least 1, 2, 3, 4, 5, 6, 7, as measured by, for example, QPCR or ELISA. 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, 335. The system of embodiment 333 or 334, which appreciably decreases over a period of 6, 7, 8, 9, or 10 cell divisions.

[0376] 336. When the second expression repressor binds to the anchor sequence or the sequence adjacent to it, the expression of MYC decreases after transfection. , 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours. The system described in any of the above.

[0377] 337. When the first expression repressor binds to the transcriptional 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 is reduced. , the system according to any of embodiments 329-336.

[0378] 338. When the first expression repressor binds to the transcriptional regulatory element or a sequence adjacent to it, and the second expression repressor binds to the anchor sequence or a sequence adjacent to it, expression of MYC decreases after transfection. 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, 338. The system of any of embodiments 329-337, wherein the system exhibits an appreciable decrease in 76, 80, or 96 hours.

[0379] 339. For example, when measured by QPCR or ELISA, expression is 10, 20, 30, 40, 50, 60, 70, 80, compared to expression in the absence of the first and second expression repressors. 339. The system of embodiment 337 or 338, wherein the system is reduced by 90, or 100%.

[0380] 340. Binding of said first expression repressor to said transcriptional regulatory element or sequence adjacent thereto, and binding of said second expression repressor to said anchor sequence or sequence adjacent thereto, as determined by, e.g., QPCR or ELISA. when MYC expression is 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.

[0381] 341. The decrease in expression caused by the binding of the first expression repressor to the transcriptional regulatory element or a sequence adjacent thereto, and the binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto, may occur individually. is greater than the decrease in expression caused by binding of the first expression repressor to the transcriptional regulatory element or a sequence adjacent thereto or by binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto. The system according to any of embodiments 329-340.

[0382] 342. Binding of said first expressed repressor to said transcriptional regulatory element or a sequence adjacent thereto, and binding of said second expressed repressor to said anchor sequence or a sequence adjacent thereto, is determined by, for example, QPCR or ELISA. When compared individually 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. , expression is 1.05× (i.e. 1.05×), 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×.

[0383] 343. The decrease in expression caused by the binding of the first expression repressor to the transcriptional regulatory element or a sequence adjacent thereto, and the binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto, may occur individually. is longer than the decrease in expression caused by binding of the first expression repressor to the transcriptional regulatory element or a sequence adjacent thereto, or binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto. 343. The system of any of embodiments 329-342, which lasts for hours (eg, more hours, days, or cell divisions).

[0384] 344. Binding of said first expressed repressor to said transcriptional regulatory element or a sequence adjacent thereto, and binding of said second expressed repressor to said anchor sequence or a sequence adjacent thereto, is determined by, for example, QPCR or ELISA. When compared individually 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. , expression is 1.05× (i.e. 1.05×), 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., number of hours, days, or number of cell divisions) 344. The system of embodiment 343, wherein the system decreases (as measured by ).

[0385] 345. When said first expression repressor binds to said promoter or a sequence adjacent thereto, and said second expression repressor binds to said super enhancer or a sequence adjacent thereto, as determined by, for example, QPCR or ELISA, MYC expression for 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.

[0386] 346. 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, individually Embodiments 329 to 329, wherein the decrease in expression is greater than the decrease in expression caused by binding of the first expression repressor to the promoter or sequences adjacent thereto or by binding of the second expression repressor to the super enhancer or sequences adjacent thereto. Any system described in 345.

[0387] 347. When said first expression repressor binds to said promoter or a sequence adjacent thereto, and said second expression repressor binds to said super enhancer or a sequence adjacent thereto, as determined by, for example, QPCR or ELISA, Individually, when compared to either binding of the first expression repressor to the promoter or a sequence adjacent to it or binding of the second expression repressor to the super enhancer or a sequence adjacent to it, the expression is 1.05. × (i.e., 1.05 times), 1.1 The system of embodiment 346, wherein the system is reduced by 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100×.

[0388] 348. 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, individually The reduction in expression caused by the binding of the first expression repressor to the promoter or sequences adjacent to it or the binding of the second expression repressor to the super enhancer or sequences thereof over a longer period of time (e.g. , for more hours, days, or cell divisions).

[0389] 349. When said first expression repressor binds to said promoter or a sequence adjacent thereto, and said second expression repressor binds to said super enhancer or a sequence adjacent thereto, as determined by, for example, QPCR or ELISA, Individually, when compared to either binding of the first expression repressor to the promoter or a sequence adjacent to it or binding of the second expression repressor to the super enhancer or a sequence adjacent to it, the expression is 1.05. × (i.e., 1.05 times), 1.1 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) ) The system of embodiment 348, wherein the system decreases.

[0390] 350. The system of any of embodiments 329-349, wherein expression is appreciably reduced indefinitely (eg, over a longer period of time than can be measured experimentally).

[0391] 351. Any of embodiments 329-350, wherein when the first expression repressor binds to the transcriptional regulatory element or a sequence adjacent thereto, the viability of a cell containing the transcriptional regulatory element or a sequence adjacent thereto is reduced. System described.

[0392] 352. Contacting a plurality of cells with said first expressed repressor or a nucleic acid encoding said first expressed repressor reduces the viability of said plurality of cells, and optionally causes said plurality of cells to become cancerous. 352. The system of any of embodiments 329-351, comprising infected and non-cancerous cells and / or infected and uninfected cells.

[0393] 353. For example, if the viability as measured by CellTiter Glo is 10, 20, 30, 40, 50, 60, 70, 80, 90 compared to the viability in the absence of the first expressed repressor. , or 100%.

[0394] 354. By administering the first expression repressor, at least 5%, 6%, 7%, 8%, 9% 10%, 12%, 15%, 17% of target cells (e.g., cancer cells) 20 %, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of the systems according to any of embodiments 329-353, wherein apoptosis occurs in 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 75%.

[0395] 355. The method according to any of embodiments 329 to 354, wherein binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto reduces the viability of a cell containing the anchor sequence or a sequence adjacent thereto. system.

[0396] 356. The method according to any of embodiments 329-355, wherein contacting a plurality of cells with the second expression repressor or a nucleic acid encoding the second expression repressor reduces the viability of the plurality of cells. system.

[0397] 357. According to any of embodiments 329 to 356, when the second expression repressor binds to the super enhancer or a sequence adjacent thereto, the viability of a cell containing the transcriptional regulatory element or a sequence adjacent thereto is reduced. system.

[0398] 358. Contacting a plurality of cells with a nucleic acid encoding said second expressed repressor or said first expressed repressor reduces the viability of said plurality of cells, and optionally causes said plurality of cells to become cancerous. 358. The system of any of embodiments 329-357, comprising infected and non-cancerous cells and / or infected and uninfected cells.

[0399] 359. For example, if the viability as measured by CellTiter Glo is 10, 20, 30, 40, 50, 60, 70, 80, 90 compared to the viability in the absence of the second expressed repressor. , or 100%.

[0400] 360. By administering the second expression repressor, at least 5%, 6%, 7%, 8%, 9% 10%, 12%, 15%, 17% of the target cells (e.g., cancer cells) 20 %, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of the systems according to any of embodiments 329-359.

[0401] 361. When said first expression repressor binds to said transcriptional regulatory element or a sequence adjacent thereto, and said second expression repressor binds to said anchor sequence or a sequence adjacent thereto, said anchor sequence or a sequence adjacent thereto 361. The system of any of embodiments 329-360, wherein the viability of cells comprising:

[0402] 362. Embodiments wherein cell viability is reduced when said first expression repressor binds to said promoter or a sequence proximate thereto and said second expression repressor binds to said super enhancer or a sequence proximate thereto. A system described in any of 329 to 361.

[0403] 363. The system of any of embodiments 329-362, wherein contacting a plurality of cells with the system or a nucleic acid encoding the system reduces the viability of the plurality of cells.

[0404] 364. For example, the viability as measured by CellTiter Glo is 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the viability in the absence of the system. 364. The system of embodiments 329-363, wherein the system decreases.

[0405] 365. The decrease in viability caused by binding of the first expression repressor to the transcriptional regulatory element or a sequence adjacent thereto, and binding of the second expression repressor to the anchor sequence or a sequence adjacent thereto, Individually, the first expressed repressor binds to the transcriptional regulatory element or a sequence adjacent thereto, or the second expressed repressor binds to the anchor sequence or a sequence adjacent thereto. 365. The system of any of embodiments 329-364, wherein the system is large.

[0406] 366. The decrease in viability 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 may be individually Embodiments wherein the reduction in viability is greater than the reduction in viability caused by binding of the first expression repressor to the promoter or sequences adjacent thereto or binding of the second expression repressor to the super enhancer or sequences adjacent thereto. A system described in any of 329 to 365.

[0407] 367. When said first expressed repressor binds to said transcriptional regulatory element or a sequence adjacent thereto, and said second expressed repressor binds to said anchor sequence or a sequence adjacent thereto, as determined, for example, by CellTiter Glo. , individually compared to either the binding of the first expressed repressor to the transcriptional regulatory element or a sequence adjacent thereto, or the binding of the second expressed repressor to the anchor sequence or a sequence adjacent thereto, 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×.

[0408] 368. When said first expression repressor binds to said promoter or a sequence adjacent thereto, and said second expression repressor binds to said super enhancer or a sequence adjacent thereto, the individual The survival ability is 1.05 compared to either binding of the first expression repressor to the promoter or a sequence adjacent to it or binding of the second expression repressor to the super enhancer or a sequence adjacent to it. × (i.e., 1.05 times), 1.1 368. The system of embodiment 366 or 367, wherein the system is reduced by 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100×.

[0409] 369. By administering said first expressed repressor and said second expressed repressor, at least 5%, 6%, 7%, 8%, 9% 10%, 12% of target cells (e.g., cancer cells) , 15%, 17% 20%, 25% 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, wherein apoptosis occurs in any of embodiments 329-368. system.

[0410] 370. The system according to embodiments 329-369, wherein the plurality of cells includes a plurality of cancer cells and a plurality of non-cancer cells.

[0411] 371. Contacting said plurality of cells with said system or a nucleic acid encoding said system reduces the viability of said plurality of cancer cells to a greater extent than the resulting reduction in the viability of said plurality of non-cancerous cells; 370. The system according to embodiment 370.

[0412] 372. Contacting said plurality of cells with said system or a nucleic acid encoding said system reduces the viability of said plurality of cancer cells by 1.05 times the resulting reduction in the viability of said plurality of non-cancerous cells (i.e. 1.05x), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4 372. The system of embodiment 370 or 371, wherein the system decreases by a factor of x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 50x, or 100x.

[0413] 373. For example, as described in any preceding embodiment, the viability of non-cancerous cells (e.g., primary hepatocytes) is not reduced by more than 5, 10, 15, or 20% when assayed according to Example 29. Expression repressor or system.

[0414] 374. The expressed repressor or system of embodiment 320, wherein viability is assayed 72 hours after contacting the cell with the expressed repressor or system.

[0415] 375. The expressed repressor or system of embodiment 374, wherein said assay comprises contacting said non-cancer cell with 2.5, 2, 1.25, 1, 0.6, or 0.5 ug / ml of said expressed repressor or system. system.

[0416] 376. When contacted with infected cells and uninfected cells, it reduces the viability of the infected cells to a greater extent than it reduces the viability of the uninfected cells, and / or 376. The system of any of embodiments 352-375, wherein it reduces the viability of the plurality of cancerous cells to a greater extent than it reduces the viability of the plurality of non-cancerous cells.

[0417] 377.The above cancers include hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, and small cell lung cancer (SCLC). , large cell (undifferentiated) cancer, triple negative breast cancer, gastric adenocarcinoma, endometrial cancer, or pancreatic cancer.

[0418] 378. The system according to any one of embodiments 352 to 377, wherein the cancer cell is a lung cancer cell, a gastric cancer cell, a gastrointestinal cancer cell, a colorectal cancer cell, a pancreatic cancer cell, or a liver cancer cell.

[0419] 379. The system according to any of embodiments 352-378, wherein the cells are human lung epithelial cells or human lung fibroblasts.

[0420] 380. The system according to any of embodiments 352-379, wherein the infection is a viral infection.

[0421] 381. The expression repressor of embodiment 380, wherein the viral infection is hepatitis, such as hepatitis B.

[0422] 382. The system according to any of embodiments 378-381, wherein the infected cells are human hepatocytes.

[0423] 383. The system according to any of embodiments 352-382, wherein the viral infection is a chronic infection.

[0424] 384. A first amino acid region comprising a sequence encoding the first expression repressor of the system according to any of embodiments 118 to 383; a second amino acid region comprising a sequence encoding the second expression repressor described in the system according to any one of embodiments 118 to 383; fusion protein containing.

[0425] 385. The fusion protein of embodiment 384, comprising a third amino acid region, the third amino acid region being located between said first amino acid region and said second amino acid region.

[0426] 386. The fusion according to embodiment 385, wherein said third amino acid region comprises a protease cleavable peptide sequence, e.g. a self-cleaving peptide sequence, e.g. a T2A self-cleaving peptide sequence, e.g. a sequence according to SEQ ID NO: 120. protein.

[0427] 387. In embodiment 386, wherein said third amino acid region comprises a protease cleavable peptide sequence, e.g. a self-cleavable peptide sequence, e.g. a tandem 2A peptide sequence, e.g. a tPT2A sequence, e.g. a sequence according to SEQ ID NO: 124. Fusion proteins as described.

[0428] 388. The fusion protein of embodiment 385, wherein the peptide sequence comprises a T2A peptide sequence and a P2A peptide sequence.

[0429] 389. The first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; and the second expression repressor comprises the amino acid sequence according to SEQ ID NO: 24 or 142, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; The fusion protein according to any of embodiments 384-388.

[0430] 390. The first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; and the second expression repressor comprises the amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; The fusion protein according to any of embodiments 384-388.

[0431] 391. The first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; and the second expression repressor comprises the amino acid sequence according to SEQ ID NO: 179 or 185, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; The fusion protein according to any of embodiments 384-388.

[0432] 392. The amino acid sequence of SEQ ID NO: 91, 92, 121, or 122, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17 positions differing therefrom. , 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or in any of embodiments 384-391, Fusion proteins as described.

[0433] 393. The amino acid sequence of SEQ ID NO: 181, 182, 187, or 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17 positions differing therefrom. , 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or in any of embodiments 384-392, Fusion proteins as described.

[0434] 394. A nucleic acid comprising a sequence encoding the system according to any of embodiments 118-393.

[0435] 395. A nucleic acid comprising a sequence encoding a system according to embodiment 394.

[0436] 396. A first region comprising a sequence encoding the first expression repressor of the system according to any of embodiments 118 to 393; and A second region comprising a sequence encoding the second expression repressor of the system according to any one of embodiments 118 to 393. 396. The nucleic acid of embodiment 394 or 395, comprising:

[0437] 397. The nucleic acid according to any one of embodiments 394 to 396, comprising a third region located between the first region and the second region.

[0438] 398. The nucleic acid according to any of embodiments 394 to 397, wherein the third region encodes a ribosome skipping sequence.

[0439] 399. The nucleic acid according to embodiment 397 or 398, wherein said third region encodes a tPT2A peptide sequence, eg, a sequence according to SEQ ID NO: 124.

[0440] 400. Any of embodiments 397 to 399, wherein said third region encodes a protease cleavable peptide sequence, e.g. a self-cleaving peptide sequence, e.g. a T2A self-cleaving peptide sequence, e.g. a sequence according to SEQ ID NO: 95. Nucleic acid described in crab.

[0441] 401. Embodiment 397, wherein said third region encodes a protease cleavable peptide sequence, e.g. a self-cleavable peptide sequence, e.g. a tandem 2A peptide sequence, e.g. a tPT2A peptide sequence, e.g. a sequence according to SEQ ID NO: 124. A nucleic acid according to any of ~400.

[0442] 402. The first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, 129, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; and the second expression repressor comprises the amino acid sequence according to SEQ ID NO: 24, 142, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; The nucleic acid according to any of embodiments 394-401.

[0443] 403. The first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, 129, or a sequence at least 80, 85, 90, 95, or 99% identical thereto; and the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 177, 179, 183, or 185 or a sequence at least 80, 85, 90, 95, or 99% identical thereto; The nucleic acid according to any of embodiments 394-401.

[0444] 404. Amino acid sequence of SEQ ID NO: 91, 92, 121, 122, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17, 16 positions differing therefrom. , 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one sequence according to any of embodiments 394-403. of nucleic acids.

[0445] 405. The amino acid sequence of SEQ ID NO: 181, 182, 187, 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or any of embodiments 394-404 encoding a sequence at no more than one location. Nucleic acid as described.

[0446] 406. The nucleotide sequence of SEQ ID NO: 93, 94, 112, or 113 or a sequence at least 80, 85, 90, 95, or 99% identical thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than one sequence according to any of embodiments 394-405. of nucleic acids.

[0447] 407. The nucleotide sequence of SEQ ID NO: 196, 197, or a sequence at least 80, 85, 90, 95, or 99% identical thereto, or 20, 19, 18, 17, 16, 15, 407. The nucleic acid of any of embodiments 394-406, comprising the sequence 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than one.

[0448] 408. A nucleic acid comprising a sequence encoding the expression repressor or expression repressor system according to any of embodiments 1 to 407.

[0449] 409. The nucleic acid according to any of embodiments 394-408, which is RNA, eg, mRNA.

[0450] 410. The nucleic acid according to any of embodiments 394 to 409, for example, comprising an N7-methylated guanosine linked to the 5' end of the RNA, for example, by a reverse 5'→5' triphosphate bond. .

[0451] 411.5' UTR, the nucleic acid according to any of embodiments 394-410.

[0452] 412. The nucleic acid according to any one of embodiments 394 to 411, for example, comprising a Kozak sequence between the 5'UTR and the sequence encoding the expression repressor.

[0453] 413. A first nucleic acid comprising a sequence encoding the first expression repressor of the system according to any of embodiments 118-393; a second expression repressor, for example, a second nucleic acid comprising a sequence encoding the second expression repressor of the system according to any of embodiments 118 to 393; system containing.

[0454] 414. The first nucleic acid has the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than one nucleotide sequence, and the nucleotide sequence of SEQ ID NO: 57 , or at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 414. The system of embodiment 413, wherein the second nucleic acid has a sequence of no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence.

[0455] 415. The first nucleic acid has the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, SEQ ID NO: 189, or 194 nucleotide sequence, or a sequence at least 80, 85, 90, 95, or 99% identical thereto, or with 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 positions differing therefrom. , 10, 9, 8, 7, 6, 5, 4, 3, 2, or the second nucleic acid having the sequence at no more than one position.

[0456] 416. The first nucleic acid has the nucleotide sequence of SEQ ID NO: 189, 194, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or not more than 1 sequence, and SEQ ID NO: 63, 130. a nucleotide sequence, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 416. The system of embodiment 415, wherein the second nucleic acid has a sequence of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence.

[0457] 417. The nucleic acid or system according to any of embodiments 394-416, wherein the nucleic acid comprises mRNA.

[0458] 418. A system according to any of the preceding embodiments, or a vector comprising the above nucleic acid encoding an expression repressor.

[0459] 419. A lipid nanoparticle comprising a system, nucleic acid, mRNA, or vector according to any of the preceding embodiments.

[0460] 420. The lipid nanoparticle of embodiment 419 comprising an ionizable lipid, e.g., a cationic lipid, e.g., MC3, SSOP.

[0461] 421. Neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids, polyunsaturated lipids, structured lipids (e.g. sterols), PEG, cholesterol, or polymer conjugates. The lipid nanoparticle of embodiment 419 or 420 further comprising one or more gated lipids.

[0462] 422. A reaction mixture comprising an expression repressor, system, nucleic acid, vector, or lipid nanoparticle according to any of the preceding embodiments.

[0463] 423. The reaction mixture of embodiment 422 further comprising cells.

[0464] 424. A pharmaceutical composition comprising an expression repressor, system, nucleic acid, vector, lipid nanoparticle or reaction mixture as described in any preceding embodiment.

[0465] 425. A method of reducing MYC gene expression in a cell, the method comprising: The above cells (e.g., cancer cells) are treated with an expressed repressor, system, one or more nucleic acids, vectors, lipid nanoparticles, or pharmaceuticals encoding said system or expressed repressor according to any of embodiments 1-424. contacting the composition including; A method of reducing the expression of the MYC gene in the above cells.

[0466] 426. A method of treating cancer in a subject in need thereof, the method comprising: Administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 424 to the above subject. including; A method of treating the cancer in the subject thereby.

[0467] 427. A method of reducing tumor growth in a subject in need thereof, comprising: Administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 424 to the above subject. including; A method of reducing tumor size in a subject as described above.

[0468] 428. The method of embodiment 427, wherein the reduction in tumor growth comprises a reduction in tumor volume compared to the tumor volume at the start of treatment.

[0469] 429. The method of embodiment 428, wherein the reduction in tumor growth in the subject is greater as compared to untreated subjects.

[0470] 430. A method of increasing or ameliorating the susceptibility of a cancer to a kinase inhibitor, e.g., sorafenib, comprising administering to a subject having said cancer an expression repressor or system described herein. .

[0471] 431. Administration of the expression repressor or system may reduce the IC of the kinase inhibitor, e.g., in a cancer cell viability assay, e.g., the assay according to Example 38. 50 The method of embodiment 430, wherein is reduced by 10%, 20%, 30%, or 40%.

[0472] 432. The method of embodiment 430 or 431, wherein the kinase inhibitor inhibits one or more (eg, all) of VEGFR, PDGFR, or RAF kinases.

[0473] 433. A method of increasing or ameliorating the susceptibility of a cancer to a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1, comprising administering the susceptibility of a cancer to a bromodomain inhibitor, e.g. 1 to 423), optionally administering the expression repressor or system described above may be used in a cancer cell viability assay, e.g., Example 39. In the assay related to the above-mentioned bromodomain inhibitor, the IC 50 The method by which the amount decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0474] 434. The method of embodiment 433, wherein the bromodomain inhibitor is or comprises JQ1, BET672, or birabresib.

[0475] 435. A method of increasing or ameliorating the susceptibility of a cancer to a MEK inhibitor, e.g., trametinib, comprising administering to a subject with said cancer a drug as described herein (e.g., any of embodiments 1-423). ) administering an expression repressor, system, or nucleic acid, optionally administering said expression repressor or system, for example in a cancer cell viability assay, e.g. Inhibitor IC 50 The method by which the amount decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0476] 436. Embodiments 427-435, wherein the reduction in tumor growth in the subject is greater than or equal to the reduction in tumor size when the subject is treated with a chemotherapeutic agent or a small molecule MYC inhibitor. The method described in any of the above.

[0477] 437. The method of embodiment 436, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0478] 438. The method of embodiment 437, wherein the small molecule MYC inhibitor is MYCi975.

[0479] 439. A method of reducing tumor size in a subject in need thereof, comprising: 1 to 424 expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition to said subject, said reduction in tumor size occurs when said subject is treated with a chemotherapeutic agent. the tumor size reduction is greater than or equal to.

[0480] 440. The method of 439, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0481] 441. The method of any of the preceding embodiments, wherein the subject does not experience any significant side effects compared to when treated with a chemotherapeutic agent or a small molecule MYC inhibitor.

[0482] 442. The method according to any of embodiments 436-441, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0483] 443. The method of embodiment 442, wherein the small molecule MYC inhibitor is MYCi975.

[0484] 444. The method of any of embodiments 426-443, wherein the cancer is a Stage I, Stage II, Stage III, or Stage IV cancer.

[0485] 445. The method of any of the preceding embodiments, wherein the subject's weight remains approximately the same before and after treatment.

[0486] 446. Any of the preceding embodiments, wherein said subject experiences no weight loss, or said subject experiences less than 3%, 2%, or 1% weight loss compared to the start of treatment. The method described in.

[0487] 447. The method of any of the preceding embodiments, wherein said subject does not experience a decrease or increase in body weight after treatment compared to said subject's body weight before treatment.

[0488] 448. A method of treating liver disease in a subject in need thereof, comprising: In the above-mentioned subjects, an expression repressor, the MYC locus (e.g., the transcribed region of MYC, the MYC promoter, or the anchor sequence of the anchor sequence mediated junction (ASMC) containing the MYC gene or a sequence adjacent to the anchor sequence) administering an expression repressor comprising a targeting moiety that binds a including; A method of treating the liver disease in the subject thereby.

[0489] 449. The subject comprises a second expression repressor, a targeting moiety that binds to an anchor sequence of an anchor sequence mediated junction (ASMC) comprising a target gene, e.g. MYC, and optionally a second effector moiety; further comprising administering a second expression repressor comprising, e.g., an effector moiety described herein; e.g., KRAB; 448. The method of embodiment 447, thereby treating said liver disease in said subject.

[0490] 450. A method of treating liver disease in a subject in need thereof, the method comprising: Administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 424 to the above subject. including; A method of treating the liver disease in the subject thereby.

[0491] 451. The method of embodiment 450, wherein the liver disease is chronic liver disease.

[0492] 452. The method of embodiment 450 or 451, wherein the liver disease is virus-related or alcohol-related.

[0493] 453. The method according to any of embodiments 450 to 452, wherein the liver disease is hepatitis or hepatocellular carcinoma.

[0494] 454. The method of embodiment 453, wherein the hepatocellular carcinoma is selected from HCC subtype S1, HCC subtype S2, or HCC subtype S3.

[0495] 455. The method of embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S1.

[0496] 456. The method of embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S2.

[0497] 457. The method of any of embodiments 450-456, wherein the liver disease is caused by hepatitis B virus or hepatitis C virus.

[0498] 458. A method of treating a pulmonary disease in a subject in need thereof, the method comprising: In the above-mentioned subjects, an expression repressor, the MYC locus (e.g., the transcribed region of MYC, the MYC promoter, or the anchor sequence of the anchor sequence mediated junction (ASMC) containing the MYC gene or a sequence adjacent to the anchor sequence) administering an expression repressor comprising a targeting moiety that binds a including; A method of treating the lung disease in the subject thereby.

[0499] 459. The subject is provided with a second expression repressor, a targeting moiety that binds a genomic locus located in the super-enhancer region of a target gene, e.g. MYC, and optionally a second effector moiety, e.g. administering a second expression repressor comprising an effector moiety as described herein; e.g., KRAB; further including; 459. The method of embodiment 458, thereby treating said pulmonary disease in said subject.

[0500] 460. A method of treating a pulmonary disease in a subject in need thereof, the method comprising: Administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 424 to the above subject. including; A method of treating the lung disease in the subject thereby.

[0501] 461. The method of embodiment 459 or 460, wherein the lung disease is cancer, eg, lung cancer, eg, lung carcinoma, eg, non-small cell lung carcinoma or small cell lung carcinoma.

[0502] 462. The method of any of embodiments 425-461, wherein the contacting or administering comprises intravenous administration to the subject.

[0503] 463. The method of any of embodiments 425-462, wherein the contacting or administering comprises intratumoral delivery (eg, injection).

[0504] 464. Embodiments wherein said cancer is characterized by an increase in MYC expression compared to a reference level (e.g., compared to MYC expression in a reference cell, e.g., an otherwise similar non-cancerous cell of said subject). 425-463.

[0505] 465. The method of any of embodiments 426-464, wherein the cancer is characterized by duplication of part or all of the MYC gene.

[0506] 466. The cancer is selected from colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, endometrial cancer, liver cancer, lymphoma (e.g., Burkitt's lymphoma), cervical cancer, or gastric cancer. , the method according to any of embodiments 426-465.

[0507] 467. The method according to any of embodiments 426-466, wherein the cancer is a human chorionic gonadotropin (hCG)-secreting cancer.

[0508] 468. The method according to any of embodiments 426 to 467, wherein the cancer is hepatocellular carcinoma.

[0509] 469. The method of any of embodiments 426-468, wherein the cancer is a refractory cancer, such as a refractory hepatocellular carcinoma.

[0510] 470. The method according to any of embodiments 426-469, wherein the cancer is non-small cell lung carcinoma or small cell lung carcinoma.

[0511] 471. The cancer of embodiments 426-470, wherein the cancer overexpresses alpha-fetoprotein (AFP) (e.g., compared to AFP expression in a reference cell, e.g., an otherwise similar non-cancerous cell of the subject). Any method described.

[0512] 472. The cells of said cancer are characterized by the presence of a super-enhancer, e.g. comprising a MYC gene or comprising said anchor sequence mediated junction comprising a MYC gene, and optionally said cancer is liver cancer, colorectal cancer. , breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, or endometrial cancer.

[0513] 472. An implementation in which said expression repressor (e.g., said second expression repressor) binds to an anchor sequence of an anchor sequence mediated junction (ASMC) comprising the MYC gene, or binds to a sequence proximal to said anchor sequence. The method described in Form 471.

[0514] 473. The method of any of embodiments 426-472, wherein the cancer cell is characterized by the absence of a super-enhancer comprising the MYC gene or the anchor sequence-mediated junction comprising the MYC gene. .

[0515] 474. The method of embodiment 473, wherein said expression repressor (e.g., said first expression repressor) binds a MYC promoter.

[0516] 475. The cancer contains a super enhancer containing the MYC gene or the anchor sequence-mediated junction containing the MYC gene, and a cell containing the super enhancer containing the MYC gene or the anchor sequence-mediated junction containing the MYC gene. and a cell that does not contain a super enhancer.

[0517] 476 a cell in which said cancer is characterized by increased MYC expression compared to a reference level (e.g., compared to MYC expression in a reference cell, e.g., an otherwise similar non-cancerous cell of said subject); Cells that are not characterized by an increase in MYC expression (e.g., compared to the MYC expression of a reference cell, e.g., an otherwise similar non-cancerous cell of interest), e.g. normal MYC expression. 476. The method according to any one of embodiments 426 to 475, comprising a cell having the following.

[0518] 477. The method of any of embodiments 426-476, wherein the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition is administered as a monotherapy.

[0519] 478. administering to said subject a plurality of doses, e.g., at least 2, 3, 4, 5, or 6 doses, of said expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition; The method according to any of embodiments 426-477.

[0520] 479. The method of any of embodiments 426-479, comprising administering to the subject multiple doses of the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition at 5-day intervals. Method.

[0521] 480.a) first administering to said subject a first plurality of doses of an expression repressor or system described herein (e.g., any of embodiments 1-424), , optionally, each subsequent dose in said first plurality is administered 5 days after the previous dose in said first plurality; b) secondly, discontinuing the expression repressor or system for a period of time (a “drug washout period”, e.g. about 2 weeks); c) third, administering to said subject a second plurality of doses of said expression repressor or system, optionally a subsequent dose of said second plurality in said second plurality; be administered 5 days after the previous dose of 479. The method according to any of embodiments 426-479, comprising:

[0522] 481. The method of embodiment 480, wherein said first plurality of doses comprises 4 doses.

[0523] 482. The method of embodiment 479 or 480, wherein the second plurality of doses comprises 2 doses.

[0524] 483. The method of any of embodiments 480-482, wherein the subject does not receive any therapeutic agent during the drug withdrawal period.

[0525] 484. The method of any of embodiments 480-483, wherein the subject receives administration of a second therapeutic agent during the drug withdrawal period.

[0526] 485. The method of any of embodiments 480-484, wherein the washout period is at least twice as long as the time between administration of doses in the first plurality of doses.

[0527] 486. The method of any of embodiments 480-486, wherein the washout period is at least twice as long as the time between administration of doses in the second plurality of doses.

[0528] 487. The method of any of embodiments 426-486, wherein the volume of the tumor is reduced to an undetectable level after treatment with the expression repressor or system.

[0529] 488. The method of any of embodiments 426-487, wherein the tumor volume decreases (eg, to an undetectable level) after cessation of treatment with the expression repressor or system.

[0530] 489. said cancer does not become resistant to said expression repressor or system, or becomes resistant to said expression repressor or system within a period of 10, 20, 30, 40, 50, or 60 days; 489. The method according to any one of embodiments 425-488.

[0531] 490. The method of any of embodiments 425-489, wherein the cancer cell has a functional apoptotic pathway.

[0532] 491. The method according to any of embodiments 425-490, wherein the cancer cell has functional caspase-3.

[0533] 492. The method of embodiment 491, wherein caspase 3 is upregulated in cancer cells upon administration of said expression repressor or system to said subject.

[0534] 493. The method of any of embodiments 425-492, wherein Ki67 is downregulated in cancer cells upon administration of the expression repressor or system to the subject.

[0535] 494. The method according to any of embodiments 425-493, wherein cancer cell proliferation is reduced upon administration of the expression repressor or system to the subject.

[0536] 495.a. Contacting the cells with a second therapeutic agent; or b.Administering the second therapeutic drug to the above subjects. 495. The method according to any of embodiments 425-494, further comprising.

[0537] 496. The method of embodiment 495, wherein the second therapeutic agent is not an expression repressor that binds to the MYC promoter.

[0538] 497. Embodiment 495, wherein the second therapeutic agent is not an expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle according to any of Embodiments 1-424. or the method described in 496.

[0539] 498. Embodiment 494, wherein the second therapeutic agent is an expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle according to any of Embodiments 1-424. The method described in any of ~496.

[0540] 499. The second therapeutic agent may include immunotherapy, immune checkpoint therapy, and / or anti-vascular endothelial growth factor therapy, systemic chemotherapy, tyrosine kinase inhibitors, such as sorafenib, mitogen-activated protein kinase kinase inhibitors ( 498. The method according to any of embodiments 495-497, wherein the method is a MEK inhibitor), such as trametinib, or a bromodomain inhibitor, such as a BET inhibitor, such as JQ1 or bilabresib.

[0541] 500. The method of any of embodiments 495-499, wherein the second therapeutic agent is a tyrosine kinase inhibitor, eg, sorafenib.

[0542] 501. The method of any of embodiments 495-499, wherein the second therapeutic agent is a bromodomain inhibitor, eg, a BET inhibitor, eg, JQ1, birabresib, or BET 672.

[0543] 502. The method of any of embodiments 495-499, wherein the second therapeutic agent is a mitogen-activated protein kinase kinase inhibitor (MEK inhibitor), such as trametinib.

[0544] 503. The method of any of embodiments 495-502, further comprising administering to said subject an additional treatment.

[0545] 504. The additional treatment modalities include surgical resection, orthotopic liver transplantation, radiofrequency ablation, photodynamic therapy (PDT), laser therapy, brachytherapy, radiotherapy, transcatheter arterial chemotherapy or radioembolization, or 505. The method of embodiment 504, comprising stereotactic radiation therapy.

[0546] 505. The method of any of embodiments 495-504, wherein the second therapeutic agent is selected from a checkpoint inhibitor or a small molecule.

[0547] 506. The method of any of embodiments 495-505, wherein the second therapeutic agent is a chemotherapeutic agent, eg, a kinase inhibitor or a bromodomain inhibitor, eg, a BET inhibitor.

[0548] 507. The method of embodiment 505 or 506, wherein the second therapeutic agent is selected from sorafenib, JQ1, BET672, birabresib, or trametinib.

[0549] 508. The method of any of embodiments 495-506, wherein said expression repressor, system, or nucleic acid and said second therapeutic agent are administered simultaneously.

[0550] 509. The method of any of embodiments 495-508, wherein the expression repressor, system, or nucleic acid and the second therapeutic agent are administered sequentially.

[0551] 509. The method of any of embodiments 503-509, wherein said additional therapy is administered simultaneously.

[0552] 511. The method of any of embodiments 503-510, wherein said additional therapy is administered sequentially.

[0553] 512. Embodiments 495~, wherein the second therapeutic agent is administered simultaneously with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture of any of Embodiments 1~424. 511.

[0554] 513. Embodiments in which the second therapeutic agent is administered sequentially with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture of any of embodiments 1-424. 495-512.

[0555] 514. The method of any of embodiments 495-513, wherein said expression repressor, system, or nucleic acid is administered intravenously and said second therapy is administered orally.

[0556] 515. The method of any of the preceding embodiments, wherein the cancer is a resistant or refractory cancer.

[0557] 516. Any of the preceding embodiments, wherein the cancer is resistant or refractory to a kinase inhibitor, e.g., a kinase inhibitor that inhibits one or more of VEGFR, PDGFR, or RAF kinases, e.g., sorafenib. Method described in Crab.

[0558] 517. The method of any of the preceding embodiments, wherein the subject has an amplification of the MYC super-enhancer.

[0559] 518. A composition comprising an expressed repressor, system, one or more nucleic acids, vectors, lipid nanoparticles, reaction mixtures, or pharmaceutical compositions encoding said system or expressed repressor according to any of embodiments 1-424. and a set of instructions comprising at least one method for modulating, eg, reducing, expression of the MYC gene in a cell by said composition.

[0560] definition a, an, the: As used herein, the singular forms "a," "an," and "the" refer to the singular form "a," "an," and "the." Contains multiple referents unless explicitly indicated otherwise.

[0561] Agent: As used herein, the term "agent" refers to any chemical class of compounds, including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations or complexes thereof. Or sometimes used to refer to an entity. As will be clear from the context to one skilled in the art, in some embodiments the term is utilized to refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Sometimes it is done. Alternatively, or in addition, as one of ordinary skill in the art would appreciate in light of the context, in some embodiments the term is natural in that it is found in and / or obtainable from nature. It is also sometimes used to refer to the products of In some embodiments, the term refers to designed, engineered, and / or created through human intervention, again as would be understood in context by those skilled in the art. It may also be used to refer to one or more entities that are man-made in that they are natural and / or not found in nature. In some embodiments, the agent may be utilized in isolated or pure form; in some embodiments, the agent may be utilized in crude form. In some embodiments, potential agents may be provided as a collection or library, which, for example, can be screened to identify or characterize active agents therein. In some embodiments, the term "agent" can refer to a compound or entity that is or includes a polymer; in some embodiments, the term "agent" can refer to a compound or entity that is or includes one or more polymeric moieties; It can refer to an entity. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and / or does not include substantially any polymer and / or does not include one or more specific polymeric moieties. In some embodiments, the term may refer to a compound or entity that is devoid or substantially free of any polymeric moieties.

[0562] Anchor sequence: As used herein, the term "anchor sequence" refers to a nucleic acid sequence that is recognized by a nucleating agent and that binds sufficiently to form an anchor sequence-mediated junction, e.g., a complex. Points to an array. In some embodiments, the anchor sequence includes one or more CTCF binding motifs. In some embodiments, the anchor sequence is not located within the gene coding region. In some embodiments, the anchor sequence is located within an intergenic region. In some embodiments, the anchor sequence is not located within either the enhancer or the promoter. In some embodiments, the anchor sequence is at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, from any transcription start site. located at least 950 bp, or at least 1 kb apart. In some embodiments, the anchor sequence is located within a region that is not associated with genomic imprinting, monoallelic expression, and / or monoallelic epigenetic marks. In some embodiments, the anchor sequence is capable of binding to an endogenous nucleating polypeptide (e.g., CTCF), forming an anchor sequence-mediated conjugation by interacting with a second anchor sequence, or forming an anchor sequence-mediated conjugation. It has one or more functions selected from shielding for outer enhancers. In some embodiments of the present disclosure, the specific one or more techniques are provided that can specifically target anchor sequences; such targeting anchor sequences are sometimes referred to as "target anchor sequences." In some embodiments, the sequence and / or activity of the targeting anchor sequence is modulated but within the same system as the targeting anchor sequence (e.g., within the same cell and / or in some embodiments, the same nucleic acid molecule, The sequence and / or activity of one or more other anchor sequences that may be present (eg, on the same chromosome) is not regulated. In some embodiments, the anchor sequence includes or is a nucleating polypeptide binding motif. In some embodiments, the anchor sequence is adjacent to the nucleating polypeptide binding motif.

[0563] Anchor sequence-mediated junction: As used herein, the term "anchor sequence-mediated junction" refers to one or more polypeptides, such as a nucleating polypeptide, or one or more protein and / or nucleic acid entities. Refers to a DNA structure, in some cases a complex, that occurs and / or is maintained by the physical interaction or binding of at least two anchor sequences within DNA (such as RNA or DNA); anchor sequences to allow for spatial proximity and functional linkage between the anchor sequences (see, eg, Figure 1).

[0564] Related: As this term is used herein, two events or entities are "related" to each other when the presence, level, form and / or function of one is correlated with that of the other. . For example, in some embodiments, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) whose presence, levels, form, and / or function is associated with a particular disease, disorder, or condition. A term is considered relevant to a disease, disorder, or condition when it correlates with the incidence and / or susceptibility thereto. In some embodiments, two or more entities are in physical proximity to each other if they interact directly or indirectly and are and / or remain in physical proximity to each other. "Related". In some embodiments, two or more entities that are physically related to each other are covalently bound to each other; In some embodiments, two or more entities that are physically related to each other are covalently bonded to each other; However, they are non-covalently bonded, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, the nucleic acid is present at least partially within a target genome or transcription complex, and the expression of genes in the DNA sequence is affected by the formation or disintegration of the target genome or transcription complex. is "associated with" a target genome or transcriptional complex.

[0565] Domain: As used herein, the term "domain" refers to a section or portion of an entity. In some embodiments, a "domain" is associated with a particular structural and / or functional characteristic of the entity, and when a domain is physically separated from the rest of its parent entity, it Retains substantially or completely certain structural and / or functional characteristics. Alternatively, or in addition, in some embodiments, when a domain is separated from a (parent) entity and joined to another (recipient) entity, the domain provides the recipient entity with the ability to It may be part of, or include, an entity that substantially retains and / or imparts one or more characterized structural and / or functional characteristics. In some embodiments, a domain is or includes a section or portion of a molecule (eg, a small molecule, carbohydrate, lipid, nucleic acid, polypeptide, etc.). In some embodiments, a domain is or includes a section of a polypeptide. In some such embodiments, the domains include specific structural elements (e.g., specific amino acid sequences or sequence motifs, alpha-helical features, beta-sheet features, coiled-coil features, random-coil features, etc.), and / or specific Characterized by functional characteristics (eg, binding activity, enzymatic activity, folding activity, signal transduction activity, etc.).

[0566] Effector moiety: As used herein, the term "effector moiety" refers to a domain that is capable of modifying the expression of a target gene when located at the appropriate site within the nucleus of a cell. In some embodiments, the effector moiety recruits components of the transcriptional machinery. In some embodiments, the effector moiety inhibits recruitment of transcription factor or repressor components. In some embodiments, the effector moiety includes an epigenetic modification moiety (eg, epigenetically modifies the target DNA sequence).

[0567] Epigenetic modification moiety: As used herein, an "epigenetic modification moiety" means that when the epigenetic modification moiety is placed in the appropriate position relative to a nucleic acid (e.g., by a targeting moiety), i) a chromatin and / or ii) epigenetic markers (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone SUMOylation, histone phosphorylation, and RNA-associated silencing); Refers to the domain that modifies the domain. In some embodiments, the epigenetic modification moiety comprises an enzyme that affects (eg, increases or decreases the level of) one or more epigenetic markers, or a functional fragment or variant thereof. In some embodiments, the epigenetic modification moiety comprises a DNA methyltransferase, a histone methyltransferase, CREB binding protein (CBP), or a functional fragment of any of these.

[0568] Expression control sequence: As used herein, the term "expression control sequence" refers to a nucleic acid sequence that increases or decreases transcription of a gene, including (but not limited to) promoters and enhancers. "Enhancing sequence" refers to a subtype of expression control sequence that increases the likelihood of gene transcription. "Silencing or repressor sequence" refers to a subtype of expression control sequence that reduces the likelihood of gene transcription.

[0569] Expression repressor: As used herein, the term "expression repressor" reduces the expression of a target gene within a cell and Refers to an agent or entity that has one or more functionalities that specifically bind to a transcription control element (to which it is operably linked). Expression repressors include at least one targeting moiety and, optionally, one effector moiety.

[0570] Expression silencing system: As used herein, the term "expression silencing system" refers to multiple expression repressors that reduce the expression of target genes within a cell. In some embodiments, the expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor and the second expression repressor (or the first expression repressor and the second expression repressor) (nucleic acids encoding expression repressors) are present together in a single composition, a mixture, or a pharmaceutical composition. In some embodiments, the expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor and the second expression repressor (or the first expression repressor and the second expression repressor) The nucleic acid encoding the expression repressor) is present in a separate composition or a pharmaceutical composition. In some embodiments, the first expressed repressor and the second expressed repressor are present within the same cell at the same time. In some embodiments, the first expressed repressor and the second expressed repressor are not present in the same cell at the same time, eg, they are present sequentially. For example, after the first expressed repressor is present within the cell for a first period, the second expressed repressor may be present within the cell for a second period, where the first and second expressed repressor are present within the cell for a second period. The periods may be overlapping or non-overlapping.

[0571] Fusion molecule: As used herein, the term "fusion molecule" refers to a compound that includes two or more moieties that are covalently linked, eg, a targeting moiety and an effector moiety. Fusion molecules and portions thereof may include any combination of polypeptides, nucleic acids, glycans, small molecules, or other components described herein (e.g., the targeting moiety may include a nucleic acid, the effector moiety may include a polypeptide, may contain peptides). In some embodiments, the fusion molecule is a fusion protein that includes one or more polypeptide domains covalently linked, eg, by peptide bonds. In some embodiments, the fusion molecule includes a targeting moiety and an effector moiety that are linked by a covalent bond other than a peptide bond or a phosphodiester bond (e.g., a targeting moiety that is linked by a covalent bond other than a peptide bond or a phosphodiester bond) , a targeting moiety comprising a nucleic acid, and an effector moiety comprising a polypeptide). In some embodiments, the expression repressor is or comprises a fusion molecule.

[0572] Genomic complex: As used herein, the term "genomic complex" refers to the formation of one or more chromosomes by interactions between multiple proteins and / or other components (possibly including genomic sequence elements). It is a complex that brings together two genomic sequence elements spaced apart from each other. In some embodiments, the genomic sequence element is an anchor sequence to which one or more protein components of the complex bind. In some embodiments, the genomic complex may include anchor sequence-mediated junctions. In some embodiments, the genomic sequence element may be or include a CTCF binding motif, promoter and / or enhancer. In some embodiments, the genomic sequence element includes at least one or both of a promoter and / or a regulatory site (eg, an enhancer). In some embodiments, complex formation is nucleated at the genomic sequence element and / or by association of the genomic sequence element and one or more protein components. As will be appreciated by those skilled in the art, in some embodiments, co-localization (e.g., conjugation) of genomic sites through the formation of complexes allows for the co-localization of genomic sites at or near (e.g., some In embodiments of the invention, the DNA topology between sequences) is altered. In some embodiments, the genomic complex includes anchor sequence-mediated conjugation, which includes one or more loops. In some embodiments, the genomic complexes described herein are nucleated with a nucleating polypeptide, such as, for example, CTCF and / or cohesin. In some embodiments, the genomic complexes described herein include, e.g., CTCF, cohesin, non-coding RNAs (e.g., eRNAs, transcriptional machinery proteins (e.g., RNA polymerases, e.g., TFIIA, TFIIB, TFIID) , one or more transcription factors selected from the group consisting of , TFIIE, TFIIF, TFIIH, etc.), transcription regulatory factors (e.g., Mediator, P300, enhancer binding protein, repressor binding protein, histone modification factor, etc.) In some embodiments, the genomic complexes described herein can include one or more polypeptide components and / or one or more nucleic acid components (e.g., one (e.g., an anchor sequence, a promoter sequence, a regulatory sequence (e.g., an enhancer sequence) The interaction can trap a section of genomic DNA into a topological configuration (eg, a loop) that is not adopted unless a complex is formed.

[0573] Moiety: As used herein, the term "moiety" refers to a defined chemical group or entity with a specific structure and / or activity, as described herein.

[0574] Modulator: As used herein, the term "modulator" refers to an agent capable of modifying (e.g., increasing or decreasing) the expression of a target gene, e.g., MYC, with one or more targeting moieties. Refers to a drug that contains one or more effector moieties.

[0575] MYC: As used herein, the term "MYC locus" refers to the MYC polypeptide (e.g., the polypeptide disclosed in NCBI Accession No. NP002458.2, or a mutant thereof) within the human genome; Refers to a portion encoding a promoter operably linked to MYC (“MYC promoter”) and an anchor sequence that forms an ASMC containing the MYC gene. In some embodiments, the MYC locus encodes a nucleic acid having NCBI Accession Number NM-002467. In some embodiments, the MYC gene is a proto-oncogene, and in some embodiments the MYC gene is an oncogene. In a particular example, the MYC gene is found on chromosome 8, at 8q24.21. In a particular example, the MYC gene starts 128,816,862 bp from pter and ends 128,822,856 bp from pter. In a particular example, the MYC gene is approximately 6kb. In a particular example, the MYC gene encodes at least eight distinct mRNA sequences - five alternatively spliced ​​variants and three non-spliced ​​variants.

[0576] Nucleic acid: As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to individual Refers to an oligonucleotide chain containing nucleic acid residues. In some embodiments, a "nucleic acid" is or includes RNA; in some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid comprises or consists of one or more "peptide nucleic acids," which are known in the art and include peptide bonds instead of phosphodiester bonds in the backbone. and are considered to be within the scope of this disclosure. Alternatively, or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than a phosphodiester linkage. In some embodiments, the nucleic acid is or includes one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). , or consisting of it. In some embodiments, the nucleic acid contains one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5propynyl- Cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine , 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intervening bases, and combinations thereof) , including or consisting of. In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to that in naturally occurring nucleic acids. . In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or protein. In some embodiments, the nucleic acid includes one or more introns. In some embodiments, the nucleic acid is isolated from a natural source, enzymatically synthesized (in vivo or in vitro) by complementary template-based polymerization, replicated in recombinant cells or systems, and chemically synthesized. prepared by more than one. In some embodiments, the nucleic acid has a length of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, the nucleic acid is partially or completely single-stranded; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a polypeptide or includes at least one element that is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0577] Nucleating polypeptide: As used herein, the term "nucleating polypeptide" or "junction nucleating polypeptide," as used herein, refers to a polypeptide that is associated directly or indirectly with an anchor sequence. one such protein that forms a dimer (or higher order structure) comprising two or more such junction nucleating polypeptides, which may or may not be identical to each other. Refers to a protein capable of interacting with a junction nucleating polypeptide (which may interact with an anchor sequence or other nucleic acid). When junction nucleating polypeptides associated with different anchor sequences associate with each other in a manner that keeps their different anchor sequences in physical proximity to each other, the resulting structure is an anchor sequence-mediated junction. . That is, the interaction and close physical proximity of a nucleating polypeptide-anchor sequence with another nucleating polypeptide-anchor sequence results in anchor sequence-mediated junctions that begin and end at the anchor sequence. (e.g., DNA loops in some cases). As those skilled in the art will readily understand upon reading this specification, terms such as "nucleating polypeptide," "nucleating molecule," "nucleating protein," and "junction nucleating protein" are sometimes used interchangeably. Sometimes used to refer to junction nucleating polypeptides. As will also be readily understood by those skilled in the art upon reading this specification, a collection of two or more junction nucleating polypeptides (which in some embodiments may include multiples of the same agent) and / or in some embodiments one or more of each of a plurality of different agents) is a "conjugate," "dimer," or "multimer." etc. may be called.

[0578] operably linked: As used herein, the phrase "operably linked" refers to a juxtaposition in which the components described are in a relationship permitting them to function in their intended manner. . A transcriptional control element "operably linked" to a functional element, e.g., a gene, is such that expression and / or activity of the functional element, e.g., a gene, is achieved under conditions compatible with the transcriptional control element. combined. In some embodiments, a transcriptional control element that is "operably linked" is contiguous with (e.g., shares) a coding element of interest, e.g., a gene; A transcriptional control element linked to acts in trans to, or otherwise at a distance from, the functional element of interest, eg, a gene. In some embodiments, operably linked means that two nucleic acid sequences are contained on the same nucleic acid molecule. In another embodiment, operably linked further refers to two nucleic acid sequences that are in close proximity to each other on the same nucleic acid molecule, e.g., within 1000, 500, 100, 50, or 10 base pairs of each other. or adjacent to each other.

[0579] Peptide, polypeptide, protein: As used herein, the terms "peptide," "polypeptide," and "protein" refer to amino acids covalently linked by peptide bonds or by means other than peptide bonds. Refers to a compound consisting of residues. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein that includes two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to, for example, short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as proteins (of which there are many types). Refers to both.

[0580] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a modulating agent, e.g., a subversive agent) formulated with one or more pharmaceutically acceptable carriers. refers to In some embodiments, the active agent is administered in unit doses suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving the desired therapeutic effect when administered to a relevant population. exist. In some embodiments, the pharmaceutical composition is adapted for oral administration, e.g., by drenching (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and Intended for systemic absorption, bolus, powder, granules, paste applied to the tongue; parenteral administration, e.g. by subcutaneous, intramuscular, intravenous or epidural injection, e.g. as a sterile solution or suspension. for topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, e.g., as a pessary, cream, or foam. specially formulated for administration in solid or liquid form, including sublingually; intraocularly; transdermally; or intranasally, intrapulmonarily, and / or to other mucosal surfaces. Good too.

[0581] Proximity: As used herein, "proximity" means that the binding of the expressed repressor at the first site and / or the modification of the first site by the expressed repressor is the same or substantial as the binding and / or modification of the other site. Refers to the closeness of two sites, such as nucleic acid sites, that would produce the same effect in terms of For example, a targeting moiety may bind to a first site, which first site is in close proximity to an enhancer (second site), and an effector moiety associated with said targeting moiety epigenetically modifies the first site. , the effect of the enhancer on the expression of the target gene may be modified in substantially the same way that the second site (enhancer sequence) is bound and / or modified. In some embodiments, proximal to a target gene (e.g., an exon, intron, or splice site within the target gene), proximal to a transcriptional control element operably linked to the target gene, or proximal to an anchor sequence. The site is 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, Less than 400, 300, 200, 100, 50, or 25 base pairs (and optionally at least 20 base pairs from the target gene (e.g., an exon, intron, or splice site located within the target gene), transcriptional control element, or anchor sequence) , 25, 50, 100, 200, or 300 base pairs).

[0582] Specific: As used herein, the term "specific," referring to an active agent, is understood by those skilled in the art to mean that the agent distinguishes between potential target entities or conditions. be understood. For example, in some embodiments, an agent binds "specifically" to a target if it preferentially binds to that target in the presence of one or more competing targets. It is said. In some embodiments, specific interactions depend on the presence of certain structural features (eg, epitopes, clefts, binding sites) of the target entity. It should be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to the specificity of the binding agent for one or more other potential target entities (eg, competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binding agent. In some embodiments, specificity is assessed relative to the specificity of a reference non-specific binding agent. In some embodiments, an agent or entity exhibits no detectable binding to another competing target under conditions in which it binds to its target entity. In some embodiments, the binding agent has a fast on-rate, slow off-rate, high affinity, low degree of dissociation, and / or high stability when compared to one or more competing targets. Bind to target entity.

[0583] Specific binding: As used herein, the term "specific binding" refers to the ability to discriminate between possible binding partners in the environment in which binding occurs. In some embodiments, a binding agent that interacts with one particular target when other potential targets are present is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between a binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of association between a binding agent and its partner; It is assessed by detecting or determining the degree of dissociation of the complex. In some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete with alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination at various concentrations.

[0584] Substantially: As used herein, the term "substantially" refers to a qualitative condition exhibiting the full range or nearly the full range or degree of the characteristic or characteristic in question. Those skilled in the art will appreciate that biological and chemical phenomena rarely, if ever, reach completion and / or progress to completeness or achieve or avoid absolute outcomes. be understood. Thus, the term "substantially" may be used in some embodiments herein to capture the potential for lack of integrity inherent in many biological and chemical phenomena. .

[0585] Symptoms are reduced: As used herein, the phrase "symptoms are reduced" refers to the extent (e.g., intensity, severity, etc.) of one or more symptoms of a particular disease, disorder, or condition. / or may be used if the frequency is reduced. In some embodiments, delaying the onset of a particular symptom is considered a form of reducing the frequency of that symptom.

[0586] Target: An agent or entity is considered to "target" another agent or entity according to this disclosure if it specifically binds to the targeting agent or entity under conditions in which they are in contact with each other. In some embodiments, for example, the antibody (or antigen-binding fragment thereof) targets its cognate epitope or antigen. In some embodiments, a nucleic acid having a particular sequence targets a nucleic acid of substantially complementary sequence.

[0587] Target gene: As used herein, the term "target gene" refers to a gene that is targeted for regulation, eg, regulation of expression. In some embodiments, the target gene is part of a targeted genomic complex (e.g., a gene that has at least a portion of its genomic sequence as part of the targeted genomic complex within an anchor sequence-mediated conjugation). and this genomic complex is targeted by one or more regulatory agents described herein. In some embodiments, modulation includes inhibition of target gene expression. In some embodiments, the target gene is regulated by contacting the target gene or a transcriptional control element operably linked to the target gene with an expression repression system described herein, e.g., an expression repressor. . In some embodiments, the target gene is aberrantly expressed (eg, overexpressed) in a cell, eg, a cell of a subject (eg, a patient).

[0588] Targeting moiety: As used herein, the term "targeting moiety" refers to an agent or entity that specifically targets, eg, binds to, a genomic sequence element (eg, an expression control sequence or anchor sequence). In some embodiments, the genomic sequence element is proximal to and / or operably linked to a target gene (eg, MYC).

[0589] Therapeutic agent: As used herein, the phrase "therapeutic agent" has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. Refers to drugs. In some embodiments, a therapeutic agent alleviates, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or one or more symptoms or characteristics of a disease, disorder, and / or condition. Any substance that can be used to reduce incidence. In some embodiments, the therapeutic agent comprises an expression suppression system described herein, eg, an expression repressor. In some embodiments, the therapeutic agent comprises a nucleic acid encoding an expression repression system, eg, an expression repressor, as described herein. In some embodiments, the therapeutic agent comprises a pharmaceutical composition described herein.

[0590] Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" refers to a substance (e.g., a therapeutic agent, composition, and / or preparation). In some embodiments, a therapeutically effective amount of a substance causes the disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. An amount sufficient to treat, diagnose, prevent and / or delay the onset thereof. As will be appreciated by those skilled in the art, an effective amount of a given substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, in some embodiments, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition ameliorates one or more symptoms or characteristics of the disease, disorder, and / or condition. is an amount that alleviates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of. In some embodiments, the therapeutically effective amount is administered in a single dose; in some embodiments, multiple doses are required to deliver the therapeutically effective amount.

[0591] The following detailed description of embodiments of the disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of explaining the disclosure, embodiments illustrated herein are shown in the drawings. It is to be understood, however, that this disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of drawings]

[0592] [Figure 1A] two based on persistent blocking of the MYC promoter using a DBD fused to a DNA methyltransferase and a transient (48 / 72 h) block of the CTCF / TF site using a DBD or a DBD fused to a short-term effector. Draw a schematic diagram of the heavy-target approach. [Figure 1B-1] Depict the chromatin context of the guide RNA localization and target sites (CTCF and promoter) for the MYC gene. From top to bottom, these graphs show H3K4me3 (histone H3 Ky trimethylation) levels; H3K9me3 (histone H3 K9 trimethylation) levels (replicate 1); H3K9me3 (histone H3 K9 trimethylation) levels for the MYC locus in HepG2 cells. level (replicate 2); H3K27me3 (histone H3 K27 trimethylation) level; H3K27ac (histone H3 K27 acetylation) level; GROseq_fwdStrand level (binding to forward strand by transcriptionally active RNA pol II); GROseq_revStrand level (transcriptionally active RNAseq_rep2 levels (MYC transcript levels measured using RNAseq, replicate 2); DNA methylation levels as measured by WGBS (Whole Genome Bisulfite Sequencing), and Represents CTCF binding level. The positions of the four gRNAs are indicated by arrows. gRNA GD-28859, GD-28616, and GD-28862 target the upstream anchor site of MYC or its vicinity, and gRNA GD-28617 targets the MYC promoter. In this disclosure, GD-28859 is also referred to as GD-59; GD-28616 is also referred to as GD-16; GD-28862 is also referred to as GD-62; and GD-28617 is also referred to as GD-17. [Figure 1B-2] Same as above. [Figure 1B-3] Same as above. [Figure 1C] Figure 2 shows a schematic diagram of an exemplary bicistronic construct. The 5' end of this construct has a cap structure defined by an N7-methylated guanosine linked to the first nucleotide of the mRNA by a reverse 5'→5' triphosphate bond. In some embodiments, the cap structure promotes protein translation and stability. Downstream of the cap structure is an untranslated region (5'UTR) designed to promote high-level protein translation, followed by a canonical "Kozak" sequence that initiates protein translation upon recognition by the ribosome. A first expression repressor comprising a first targeting moiety and a first effector moiety, followed by a "Kozak" sequence, and a second targeting moiety and a second effector moiety, separated by a tPT2A "ribosome skipping" sequence (linker). There is a CDS that is a single contiguous sequence that includes a second expression repressor. Without wishing to be bound by theory, when the ribosome reaches the tPT2A linker, it begins translating the linker into amino acids. The first 18 amino acids created from the P2A linker remain at the C-terminal end of the first expressed repressor (including, for example, the ZF DBD and MQ1), which is then released by the ribosome. The ribosome then continues onward until it finally reaches the T2A linker, where the first 17 residues of the T2A linker are translated and released. A second polypeptide is then translated, containing a single amino acid, and then a second expression repressor (eg, containing a second ZF DBD and KRAB) is initiated. After the CDS is the 3'UTR, which is designed to aid in high-level translation and also to stabilize the mRNA. Finally, at the very end of the 3' end of the mRNA, there is a polyA tail. In some embodiments, the polyA tail promotes protein translation and mRNA stability.

Figure 2

[0593] The present disclosure provides techniques for modulating, eg, reducing, expression of a target gene, eg, MYC, in cells of a subject or patient using the expression repressors or systems described herein.

[0594] Many different diseases and syndromes can be caused by misregulation of gene expression, including cancer, autoimmunity, cardiovascular disease, and obesity. In particular, it has long been known that overexpression of transcription factors contributes to tumorigenesis, and recent studies have pointed out that overexpression of oncogenic transcription factors can alter central cellular autoregulatory circuits. There is.

[0595] MYC, a transcription factor and cellular master regulator, is frequently dysregulated in over 50% of human cancers and plays a central role in almost all aspects of the tumorigenic process. With the exception of early response genes, MYC typically upregulates gene expression. MYC is the most frequently amplified oncogene, and elevated expression of its gene product is associated with tumor attack and poor clinical outcome. Elevated levels of c-MYC can promote tumor development in a wide range of tissues. Most tumor cells depend on the transcription factor c-MYC for their growth and proliferation. Overexpression of MYC is also associated with chronic liver diseases, such as virus-related and alcohol-related liver diseases. MYC overexpression levels vary with specific cancer subtypes. Without wishing to be bound by theory, modulating the levels of MYC (e.g., globally or in a specific target tissue or tissues) in a subject suffering from a MYC misregulation disorder, e.g. , or may alleviate or eliminate the symptoms of MYC misregulation disorder.

[0596] The present disclosure relates, in part, to a targeting moiety that binds to a target gene promoter, e.g., MYC promoter, or is operably linked to a target gene, e.g., the MYC gene, and when localized by the targeting moiety, the target gene, e.g. and an effector moiety capable of modulating (eg, reducing) expression of MYC. In some embodiments, the expression repressors disclosed herein include a targeting moiety to an expression control element (e.g., a promoter or enhancer, repressor or silencer) operably linked to a target gene, e.g., MYC. and the effector moiety modulates the expression of the target gene, eg, MYC. In some embodiments, the expression repressors disclosed herein include a targeting moiety at or in a sequence proximal to an anchor sequence of an anchor sequence mediated junction (ASMC) that includes a target gene, e.g., MYC. and the effector moiety modulates the expression of the target gene, eg, MYC. In some embodiments, the expression repressor disclosed herein specifically binds to a genomic locus located in the super-enhancer region of a target gene, e.g. , regulates the expression of MYC.

[0597] The present disclosure further provides, in part, expression silencing systems that include two or more expression repressors, each of which includes a targeting moiety and, optionally, an effector moiety. In some embodiments, these targeting moieties target two or more different sequences (eg, each expressed repressor may target a different sequence). In some embodiments, the first expression repressor binds to a transcriptional regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to the target gene, e.g., MYC, and the second expression repressor binds to the anchor sequence of the anchor sequence mediated junction (ASMC) containing the target gene, eg MYC. In some embodiments, the first expression repressor binds to a transcriptional regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to the target gene, e.g., MYC, and the second expression repressor binds to an expression control element (eg, an enhancer, super-enhancer, repressor, or silencer) operably linked to a target gene, eg, MYC. In some embodiments, the first expressed repressor binds to a target gene, e.g., an anchor sequence of an anchor sequence mediated junction (ASMC) that includes MYC, and the second expressed repressor is operable to affect the target gene. Binds to linked expression control elements (eg, enhancers, super-enhancers, repressors, or silencers). Generally, regulation of expression of a target gene, eg, MYC, by an expression silencing system involves binding of a first expression repressor and a second expression repressor to first and second DNA sequences, respectively. Upon binding of the first and second DNA sequences, the functions of the first and second effector moieties are localized to those sites. While not wishing to be bound by theory, in some embodiments the functions of both the first and second effector moieties are utilized, such that the first and / or second expressor moieties are expression of a target gene containing or containing the target gene is stably suppressed, e.g., where the first and / or second DNA sequences are sequences of the target gene or one or more operably linked transcriptional control elements. is or contains it. In some embodiments, the expression repressor system is encoded by a bicistronic nucleic acid sequence.

[0598] The present disclosure further provides nucleic acids encoding said expressed repressors and / or expressed repressor systems, compositions comprising said expressed repressors and / or expressed repressor systems, and methods of delivery of said nucleic acids. Further provided are methods of increasing target gene expression in a cell, such as MYC gene expression, using the expression repressors or expression repressor systems described herein.

[0599] expression repressor As described herein, the present disclosure provides, in part, expression repressors for modulating, eg, reducing, expression of a target gene, eg, MYC. In some embodiments, the expression repressor may include a targeting moiety that binds to a target gene promoter, eg, the MYC promoter, and optionally an effector moiety. In some embodiments, the targeting moiety specifically binds to a target DNA sequence, eg, a MYC DNA sequence, thereby localizing the function of the expression repressor to the DNA sequence. In some embodiments, the expression repressor includes a targeting moiety and one effector moiety. In some embodiments, the expression repressor comprises a targeting moiety and multiple effector moieties (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (and optionally 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 , 7, 6, 5, 4, 3, or less than 2 effector domains)).

[0600] Expression repressors may include multiple effector moieties, where each effector moiety includes a different function from other effector moieties. For example, an expression repressor may include two effector moieties, where a first effector moiety includes a DNA methylase function and a second effector moiety includes a transcriptional repressor function. In some embodiments, the expression repressor comprises effector moieties whose functions are complementary to each other with respect to reducing expression of a target gene, e.g., MYC, where their functions together result in inhibition of expression. and optionally, when individually present, do not or only negligibly inhibit expression. In some embodiments, the expression repressor comprises multiple effector moieties, where each effector moiety is complementary to each other and each effector moiety reduces expression of a target gene, e.g., MYC.

[0601] In some embodiments, the expression repressor comprises a combination of effector moieties whose functions are synergistic with each other in reducing expression of a target gene, e.g., MYC. Without wishing to be bound by theory, in some embodiments, epigenetic modifications to genomic loci include multiple transcriptional activating epigenetic markers (e.g., multiple different types of epigenetic markers and or more extensive markings of a given type) individually together inhibit expression with greater efficacy than the individual modifications alone (e.g., a greater reduction in expression and / or a longer lasting effect on expression). cumulative in that it causes a reduction). In some embodiments, the expression repressor comprises multiple effector moieties, where each effector moiety acts synergistically with each other, e.g., each effector moiety reduces expression of a target gene, e.g., MYC. . In some embodiments, an expressed repressor (comprising multiple effector moieties that synergize with each other) is more effective at inhibiting expression of a target gene, e.g., MYC, than an expressed repressor containing individual effector moieties. expensive. In some embodiments, the expressed repressor comprising a plurality of effector moieties has an efficacy in reducing expression of a target gene, e.g., MYC, that is at least 1.05 times that of the expressed repressor comprising individual effector moieties (i.e. 1.05x), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85 ×, 1.9×, 1.95×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, or 100×.

[0602] In some embodiments, the expression repressor includes one or more targeting moieties, such as a Cas domain, a TAL effector domain, or a Zn finger domain. In certain embodiments, when the expression repressor system comprises two or more targeting moieties of the same type, e.g., two or more Cas domains, those targeting moieties specifically bind two or more different sequences. . For example, in an expression repressor system containing two or more Cas domains, the two or more Cas domains are such that they appreciably bind only to the gRNA corresponding to their target sequence (e.g., and to another Cas domain). may be selected or modified such that they do not appreciably bind to the gRNA corresponding to the domain's target.

[0603] In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety covalently linked, for example, by a peptide bond. In some embodiments, the targeting moiety and the effector moiety are located on the same polypeptide chain, eg, connected by one or more peptide bonds and / or linkers. In some embodiments, the expression repressor is or includes a fusion molecule that includes a targeting moiety and an effector moiety, eg, connected by a peptide bond and / or a linker. In some embodiments, the expression repressor includes a targeting moiety located N-terminal to the effector moiety on the same polypeptide chain. In some embodiments, the expression repressor includes a targeting moiety located C-terminal to the effector moiety on the same polypeptide chain. In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety covalently linked by a non-peptide bond. In some embodiments, the targeting moiety is conjugated to the effector moiety through a non-peptide bond. In some embodiments, the expression repressor comprises a targeting moiety and a plurality of effector moieties, wherein the targeting moiety and the plurality of effector moieties are covalently linked, e.g., by a peptide bond. (For example, a targeting moiety and multiple effector moieties are all connected by a series of covalent bonds, but each individual moiety does not share covalent bonds with every other effector moiety. Sometimes).

[0604] In other embodiments, the expression repressor comprises a targeting moiety and an effector moiety that are not covalently linked, eg, non-covalently associated with each other. In some embodiments, the expression repressor comprises a targeting moiety non-covalently linked to an effector moiety, or vice versa. In some embodiments, the expression repressor comprises a targeting moiety and a plurality of effector moieties, wherein the targeting moiety and the at least one effector moiety are not covalently linked, e.g., non-covalently linked to each other. bindingly associated and wherein the targeting moiety and at least one other effector moiety are covalently linked, eg, by a peptide bond.

[0605] Generally, an expression repressor as described herein binds (eg, via a targeting moiety) to a genomic sequence element proximal to and / or operably linked to a target gene, eg, MYC. In some embodiments, binding of an expression repressor to a genomic sequence element modulates (eg, reduces) expression of a target gene, eg, MYC. For example, binding of an expression repressor containing an effector moiety that recruits or inhibits the recruitment of components of the transcriptional machinery to a genomic sequence element may modulate (e.g., reduce) the expression of a target gene, e.g., MYC. . As a further example, binding of an expression repressor that includes an effector moiety with enzymatic activity (e.g., an epigenetic modification moiety) can modulate the expression of a target gene (e.g., MYC) through localization of the enzymatic activity of the effector moiety. (e.g. may be decreased). As a further example, both binding to genomic sequence elements by an expression repressor and localization of the expression repressor's enzymatic activity contribute to the regulation (e.g., reduction) of the resulting expression of a target gene, e.g., MYC. It is possible.

[0606] In some embodiments, the expression repressor comprises an effector moiety, wherein the effector moiety is MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e. LSD1), KDM1B (i.e. LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e. G9A), EHMT1 (i.e. GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB( KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof.

[0607] In some embodiments, the expression repressor comprises a first effector moiety and a second effector moiety, wherein the first effector moiety is MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e. LSD1), KDM1B (i.e. LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e. G9A), EHMT1 (i.e. GLP), SUV39H1, EZH2, EZH1, a protein selected from SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB (e.g., KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof; , MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3 , SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e. LSD1), KDM1B (i.e. LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e. , G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB (e.g., KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant thereof different proteins selected from the body or fragments.

[0608] In some embodiments, the present disclosure provides nucleic acid sequences encoding expression repressors, expression repressor systems, targeting moieties, and / or effector moieties as described herein. Those skilled in the art will recognize that the nucleic acid sequence of RNA is typically identical to the corresponding DNA sequence, except that thymine (T) is typically replaced by uracil (U). If the nucleotide sequence is represented by a DNA sequence (e.g., containing A, T, G, C), this disclosure also provides that the corresponding RNA sequence (e.g., A, (including U, G, and C). Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is the 5' direction. It is called.

[0609] Due to the degeneracy of the genetic code, numerous nucleotide sequences can be produced that encode expression repressors, including targeting and / or effector moieties, some of which are described herein. Those of ordinary skill in the art will appreciate that the nucleic acid sequences have similarity, eg, 90%, 95%, 96%, 97%, 98%, or 99% identity, to the nucleic acid sequences disclosed in . For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acid molecules of the invention where arginine is specified by a codon, the codon can be modified to any of the corresponding codons described above without modifying the encoded polypeptide.

[0610] In some embodiments, the nucleic acid aggregate encoding an expression repressor that includes a targeting moiety and / or an effector moiety is a portion of a codon-optimized coding region that has been optimized according to codon usage in a mammal, e.g., a human. It can be all. In some embodiments, the nucleic acid aggregates encoding the targeting and / or effector moieties are codons optimized to increase protein expression and / or to increase the duration of protein expression. In some embodiments, the protein produced by the codon-optimized nucleic acid sequence is at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% higher.

[0611] Expression suppression system The expression suppression system of the present disclosure can include two or more expression repressors. In some embodiments, the silencing system has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more (and optionally 15, 14, 13) , 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less) expression repressors. In some embodiments, the expression silencing system comprises two or more different sequences (e.g., first and second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth , 11th, 12th, and / or more DNA sequences, and optionally 20th, 19th, 18th, 17th, 16th, 15th, 14th, 13th, 12th, 11th, 10th, 9th, 8th, 6th, 5th, 4th, 3rd, or 2nd or less sequences). In some embodiments, the expression silencing system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors does not detectably bind to another member of the plurality of expression repressors; For example, do not combine. In some embodiments, the expression repression system comprises a first expression repressor and a second expression repressor, wherein said first expression repressor does not detectably bind to said second expression repressor. , e.g., do not combine.

[0612] In some embodiments, the expression repression systems of the present disclosure include two or more expression repressors, wherein the expression repressors are present together in a composition, pharmaceutical composition, or mixture. In some embodiments, the expression repression systems of the present disclosure include two or more expression repressors, where one or more expression repressors are not mixed with at least one other expression repressor. For example, the expression repression system may include a first expressed repressor and a second expressed repressor, where the presence of the first expressed repressor in the nucleus of a cell is the reason for the presence of the second expressed repressor in the nucleus of the same cell. Here, the expression suppression system achieves a reduction in the expression of the MYC gene by the non-overlapping presence of the first and second expression repressors. In some embodiments, the expression silencing system achieves a greater reduction in MYC gene expression compared to the reduction in MYC gene expression achieved by the first or second expression repressor alone.

[0613] In some embodiments, the expressed repressors of the expressed repressor system each include a different targeting moiety (e.g., the first, second, third, or more expressed repressors each have different targeting moieties). (including parts). For example, an expression repression system may include a first expression repressor and a second expression repressor, wherein the first expression repressor has a first targeting moiety (e.g., a Cas9 domain, a TAL effector domain, or a Zn finger The second expression repressor includes a second targeting moiety (eg, a Cas9 domain, a TAL effector domain, or a Zn finger domain) that is different from the first targeting moiety. In some embodiments, different means including distinct types of targeting moieties, e.g., a first targeting moiety includes a Cas9 domain and a second DNA targeting moiety includes a Zn finger domain. obtain. In other embodiments, different includes individual variants of the same type of targeting moiety, e.g., the first targeting moiety comprises a first Cas9 domain (e.g., from a first species). , can mean that the second targeting moiety comprises a second Cas9 domain (eg, from a second species). In certain embodiments, when the expression repressor system comprises two or more targeting moieties of the same type, e.g. two or more Cas9 or ZF domains, those targeting moieties are specific for two or more different sequences. Join. For example, in an expression repressor system containing two or more Cas9 domains, the two or more Cas9 domains are such that they appreciably bind only gRNAs corresponding to their target sequences (e.g., and may be selected or modified such that they do not appreciably bind to the gRNA corresponding to the domain's target. In a further example, in an expressed repressor system that includes two or more effector moieties, the two or more effector moieties are such that they appreciably bind only to their target sequence (e.g., and may be selected or modified such that they do not appreciably bind to the target sequence.

[0614] In some embodiments, the expressed repressor system includes three or more expressed repressors, and the two or more expressed repressors include the same targeting moiety. For example, an expressed repressor system may include three expressed repressors, where the first and second expressed repressors both contain a first targeting moiety, and the third expressed repressor contains a second different targeting moiety. Contains targeting part. For a further example, an expressed repressor system may include four expressed repressors, where the first and second expressed repressors both include a first targeting moiety, and the third and fourth expressed repressors Contains a second different targeting portion. For a further example, an expressed repressor system may include five expressed repressors, where the first and second expressed repressors both include a first targeting moiety, and the third and fourth expressed repressors , both contain a second different targeting moiety and the fifth expressed repressor contains a third different targeting moiety. As described above, different can mean including different types of targeting moieties or including individual variants of the same type of targeting moieties.

[0615] In some embodiments, the expression repressors of the expression repressor system each bind to a different DNA sequence (e.g., the first, second, third, or more expression repressors are each different from each other). (binds to DNA sequences). For example, an expression repression system may include a first expression repressor and a second expression repressor, wherein the first expression repressor binds to a first DNA sequence and the second expression repressor binds to a first expression repressor. Binds to the DNA sequence of 2. In some embodiments, different means that there is at least one position between the DNA sequence that one expressed repressor binds and the DNA sequence that the other expressed repressor binds that is not identical; It can be meant that there is at least one position present in the DNA sequence to which an expression repressor binds that is not present in the DNA sequence to which the other expression repressor binds.

[0616] In some embodiments, a first DNA sequence may be located on a first genomic DNA strand and a second DNA sequence may be located on a second genomic DNA strand. In some embodiments, the first DNA sequence may be located on the same genomic DNA strand as the second DNA sequence.

[0617] In some embodiments, the expression repressor system comprises three or more expression repressors, and the two or more expression repressors bind to the same DNA sequence. For example, an expression repressor system may include three expression repressors, where the first and second expression repressors both bind to the first DNA sequence and the third expression repressor binds to the second expression repressor. Binds to different DNA sequences. For a further example, an expression repressor system may include four expression repressors, where the first and second expression repressors both bind to the first DNA sequence and the third and fourth expression repressors bind to the first DNA sequence. Both pressers bind to the second DNA sequence. For a further example, an expression repressor system may include five expression repressors, where the first and second expression repressors both bind to the first DNA sequence and the third and fourth expression repressors bind to the first DNA sequence. Both pressers bind to the second DNA sequence and the fifth expressed repressor binds to the third DNA sequence. As described above, different means that there is at least one non-identical position between the DNA sequence bound by one expressed repressor and the DNA sequence bound by the other expressed repressor, or It may mean that there is at least one position present in the DNA sequence to which the repressor binds that is not present in the DNA sequence to which the other expressed repressor binds.

[0618] In some embodiments, the expression silencing system comprises two or more (e.g., two) expression repressors, and more than one (e.g., two) of the expression repressors bind to different DNA sequences. Contains targeting part. In such embodiments, the first targeting moiety may bind to the first DNA sequence and the second DNA targeting moiety may bind to the second DNA sequence, where the first and second DNA sequences are different and non-overlapping. In some such embodiments, the first DNA sequence has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and optionally , 500, 400, 300, 200, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 base pairs or less) apart. In some such embodiments, the first DNA sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs or less apart ( and optionally, no base pairs apart, eg, the first and second sequences are directly adjacent to each other).

[0619] In some embodiments, the expressed repressors of the expressed repressor system each include a different effector moiety (e.g., the first, second, third, or more expressed repressors are each different from each other). (including effector part). For example, an expression repression system may include a first expression repressor and a second expression repressor, wherein the first expression repressor comprises a first effector moiety (e.g., a DNA methyltransferase or a functional fragment thereof). The second expression repressor includes a second effector moiety that is different from the first effector moiety (eg, one containing a transcriptional repressor (eg, KRAB) or a functional fragment thereof). In some embodiments, different can mean including distinct types of effector moieties. In other embodiments, different includes individual variants of the same type of effector moiety, e.g., the first effector moiety differs from the first DNA methyltransferase (e.g., the first site specificity or amino acid sequence). may mean that the second effector moiety comprises a second DNA methyltransferase (eg, one having a second site specificity or amino acid sequence).

[0620] In some embodiments, the expressed repressor system includes a first expressed repressor that includes a first effector moiety and a second expressed repressor that includes a second effector moiety, wherein the first effector moiety is MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e. LSD1), KDM1B (i.e. LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e. G9A), EHMT1 (i.e. GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof The second effector part includes MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e. LSD1), KDM1B (i.e. LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e. G9A), EHMT1 (i.e. GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or Includes different proteins selected from functional variants or fragments.

[0621] In some embodiments, the first or second effector moiety has DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any of these) the other effector moiety comprises a transcriptional repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these) , the first or second effector moiety contains histone methyltransferase activity, and the other effector moiety contains histone deacetylase activity (e.g., 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. In some embodiments, the first or second effector moiety is The other effector moieties include histone methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or the functionality of any of these). In some embodiments, the first or second effector moiety includes DNA methyltransferase activity and the other effector moiety includes transcriptional repressor activity. In some embodiments, the first or second effector moiety includes a transcriptional repressor activity. or the second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a transcriptional repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or In some embodiments, the first or second effector moiety comprises a transcriptional repressor activity and the other effector moiety comprises a different transcriptional repressor activity. In some embodiments, the first or second effector moiety comprises a different transcriptional repressor activity. The second effector moiety contains DNA methyltransferase activity and the other effector moiety contains the same DNA methyltransferase activity. In some embodiments, the first or second effector moiety includes DNA methyltransferase activity and the other effector moiety includes histone deacetylase activity. In some embodiments, the first or second effector moiety includes histone demethylase activity and the other effector moiety includes DNA methyltransferase activity. In some embodiments, the first or second effector moiety includes histone methyltransferase activity and the other effector moiety includes DNA demethylase activity. In some embodiments, the first or second effector moiety includes histone demethylase activity and the other effector moiety includes transcriptional repressor activity. In some embodiments, the first or second effector moiety includes a histone demethylase activity and the other effector moiety includes a different histone demethylase activity. In some embodiments, the first or second effector moiety includes histone demethylase activity and the other effector moiety includes the same histone demethylase activity. In some embodiments, the first or second effector moiety includes histone deacetylase activity and the other effector moiety includes DNA methyltransferase activity. In some embodiments, the first or second effector moiety includes histone deacetylase activity and the other effector moiety includes DNA demethylase activity. In some embodiments, the first or second effector moiety includes histone deacetylase activity and the other effector moiety includes transcriptional repressor activity. In some embodiments, the first or second effector moiety includes a histone deacetylase activity and the other effector moiety includes a different histone deacetylase activity. In some embodiments, the first or second effector moiety includes histone deacetylase activity and the other effector moiety includes the same histone deacetylase activity. In some embodiments, the first or second effector moiety includes DNA methyltransferase activity and the other effector moiety includes DNA demethylase activity. In some embodiments, the first or second effector moiety includes DNA demethylase activity and the other effector moiety includes transcriptional repressor activity. In some embodiments, the first or second effector moiety includes a DNA methyltransferase activity and the other effector moiety includes a different DNA methyltransferase activity. In some embodiments, the first or second effector moiety includes DNA methyltransferase activity and the other effector moiety includes the same DNA methyltransferase activity. In some embodiments, the first or second effector moiety includes DNA demethylase activity and the other effector moiety includes transcriptional repressor activity. In some embodiments, the first or second effector moiety includes a DNA demethylase activity and the other effector moiety includes a different DNA demethylase activity. In some embodiments, the first or second effector moiety includes DNA demethylase activity and the other effector moiety includes the same DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a transcriptional repressor activity and the other effector moiety comprises a different transcriptional repressor activity. In some embodiments, the first or second effector moiety includes transcriptional repressor activity and the other effector moiety includes the same transcriptional repressor activity.

[0622] In some embodiments, the expressed repressor system comprises three or more expressed repressors, and the two or more expressed repressors contain the same DNA targeting moiety. For example, an expressed repressor system may include three expressed repressors, where the first and second expressed repressors both contain a first effector moiety, and the third expressed repressor contains a second different expressed repressor. Includes effector part. For a further example, an expression repressor system may include four expression repressors, where the first and second expression repressors both include a first effector moiety, and the third and fourth expression repressors including a second different effector portion. For a further example, an expression repressor system may include five expression repressors, where the first and second expression repressors both include a first effector moiety, and the third and fourth expression repressors , both contain a second different effector moiety, and the fifth expressed repressor contains a third different effector moiety. As described above, different can mean including different types of effector moieties, or including individual variants of the same type of effector moieties.

[0623] In some embodiments, the two or more (e.g., all) expressed repressors of the expressed repressor system are not covalently associated with each other, e.g., each expressed repressor is It is also not covalently associated with repressors. In another embodiment, the two or more expressed repressors of the expressed repressor system are covalently associated with each other. In certain embodiments, the expression repression system comprises a first expressed repressor and a second expressed repressor placed on the same polypeptide, e.g., as a fusion molecule, e.g., connected by a peptide bond and optionally a linker. include. In some embodiments, the peptide is a self-cleaving peptide, such as a T2A self-cleaving peptide. In certain embodiments, the expression repression system includes a first expression repressor and a second expression repressor that are connected by a non-peptide bond, eg, conjugated to each other.

[0624] linker An expression repressor or expression repressor system as disclosed herein may include one or more linkers. A linker can connect a targeting moiety to an effector moiety, an effector moiety to another effector moiety, or a targeting moiety to another targeting moiety. A linker can be a chemical bond, such as one or more covalent or non-covalent bonds. In some embodiments, the linker is covalent. In some embodiments, the linker is non-covalent. In some embodiments, the linker is a peptide linker. Such linkers include 2~30, 5~30, 10~30, 15~30, 20~30, 25~30, 2~25, 5~25, 10~25, 15~25, 20~25, 2~ 20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, 5-10, or 2-5 amino acids long, or 2, 5, 10, 15, It can be 20, 25, or 30 amino acids or more in length (and optionally 50, 40, 30, 25, 20, 15, 10, or 5 amino acids or less in length). In some embodiments, a linker can be used to separate a first domain or moiety from a second domain or moiety, eg, a DNA targeting moiety from an effector moiety. In some embodiments, for example, a linker can be positioned between the DNA targeting moiety and the effector moiety to provide, for example, molecular flexibility in secondary and tertiary structure. Linkers can include flexible, rigid, and / or cleavable linkers as described herein. In some embodiments, the linker provides flexibility by including at least one glycine, alanine, and serine amino acids. In some embodiments, the linker is a hydrophobic linker, such as containing a negatively charged sulfonic acid group, a polyethylene glycol (PEG) group, or a pyrophosphate diester group. In some embodiments, the linker is cleavable to selectively release the moiety (eg, polypeptide) from the modulator, but is sufficiently stable to prevent premature cleavage.

[0625] In some embodiments, one or more portions and / or domains of an expression repressor described herein are joined with one or more linkers. In some embodiments, silencing may include a linker located between the targeting moiety and the effector moiety. In some embodiments, the linker can have the sequence ASGSGGGSGGARD (SEQ ID NO: 137), or ASGSGGGSGG (SEQ ID NO: 138). In some embodiments, a system comprising a first and a second repressor comprises a first linker located between the first targeting moiety and the first effector moiety; and a second linker located in between. In some embodiments, the first and second linkers can be the same. In some embodiments, the first and second linkers can be different. In some embodiments, the first linker may comprise an amino acid sequence according to SEQ ID NO: 137 or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto, and the second linker comprises: It may include the amino acid sequence according to SEQ ID NO: 138 or a sequence at least 80, 85, 90, 95, 99, or 100% identical thereto.

[0626] As will be known to those skilled in the art, commonly used flexible linkers have sequences consisting primarily of a stretch of Gly and Ser residues ("GS" linkers). Flexible linkers may be useful for joining domains / moieties that require some degree of movement or interaction, and include small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. obtain. Incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the moiety / domain. I can do it. In some embodiments, the linker is a GS linker or a variant thereof, eg, G4S (SEQ ID NO: 207).

[0627] Rigid linkers are useful to maintain a constant distance between domains / parts and maintain their independent functions. Rigid linkers may also be useful when spatial separation of domains is critical to ensuring stability or biological activity of one or more components within the fusion. Rigid linkers are α-helical structures or Pro-rich sequences, (XP) n (wherein X means any amino acid, preferably Ala, Lys, or Glu).

[0628] A cleavable linker can release a free functional domain in vivo. In some embodiments, the linker may be cleaved under special conditions, such as in the presence of a reducing reagent or protease. In vivo cleavable linkers can take advantage of the reversible nature of disulfide bonds. An example is a thrombin sensitive sequence (eg, PRS) located between two Cys residues. Thrombin treatment of CPRSC residues in vitro results in cleavage of the thrombin-sensitive sequences while leaving the reversible disulfide bonds intact. Such linkers are known and are described, for example, in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369. In vivo cleavage of the linker within the fusion can also be performed under certain conditions in vivo by a protease that is expressed in specific cells or tissues or confined within certain intracellular compartments. The specificity of many proteases results in delayed cleavage of the linker in the constrained compartment. In some embodiments, the cleavable linker can be a self-cleavable linker, such as a T2A peptide linker. In some embodiments, the linker can include a "ribosome-skipping" sequence, such as a tPT2A linker.

[0629] Examples of molecules suitable for use in the linkers described herein include negatively charged sulfonic acid groups; lipids, such as poly(-CH 2 -) hydrocarbon chains, such as polyethylene glycol (PEG) groups, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants; carbon-free linkers; These include carbohydrate linkers; phosphodiester linkers, or other molecules that can covalently link two or more components of an expression repressor. Non-covalent linkers, such as leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or a series of residues rich in other hydrophobic residues, Also included are hydrophobic lipid globules to which the polypeptide is linked through a hydrophobic region of the peptide or a hydrophobic stretch of the polypeptide. Components of an expressed repressor may be linked using charge-based chemistry, such as a positively charged component of an expressed repressor linked to a negatively charged component of another component.

[0630] targeting part The present disclosure provides for targeting, e.g., binding, that specifically targets, e.g. provides an expression repressor comprising a moiety. The targeting moiety can specifically bind a DNA sequence, eg, a DNA sequence associated with a target gene, eg, MYC. Any molecule or compound that specifically binds a DNA sequence can be used as a targeting moiety.

[0631] In some embodiments, the targeting moiety targets, eg, binds to, a component of a genomic complex (eg, ASMC). In some embodiments, the targeting moiety targets, eg, binds to, an expression control sequence (eg, a promoter or enhancer) operably linked to the target gene. In some embodiments, the targeting moiety targets, eg, binds to, a target gene, or a portion of a target gene. The target of a targeting moiety may be referred to as its targeting moiety. The targeting component can be any genomic sequence element operably linked to the target gene, or the target gene itself, including but not limited to promoters, enhancers, anchor sequences, exons, introns, UTR coding sequences, splices, etc. site, or transcription initiation site. In some embodiments, the targeting moiety specifically binds to one or more target anchor sequences (eg, within a cell) and does not bind to non-target anchor sequences (eg, within the same cell).

[0632] In some embodiments, the targeting moiety can be or include a CRISPR / Cas domain, a TAL effector domain, a Zn finger domain, a peptide nucleic acid (PNA) or a nucleic acid molecule. In some embodiments, the expression repressor includes one effector moiety. In some embodiments, the expression repressor comprises multiple targeting moieties, where each targeting moiety does not detectably bind, eg, does not bind, to another targeting moiety. In some embodiments, the expression silencing system includes a plurality of expression repressors, wherein each member of the plurality of expression repressors includes a targeting moiety, wherein each targeting moiety is linked to another targeting moiety. does not detectably bind to, e.g. In some embodiments, the expression repression system includes a first expression repressor that includes a first targeting moiety and a second expression repressor that includes a second targeting moiety, wherein the first targeting moiety is a first expression repressor that includes a second targeting moiety. 2 does not detectably bind to the targeting moiety, e.g. In some embodiments, the expression repression system includes a first expression repressor that includes a first targeting moiety and a second expression repressor that includes a second targeting moiety, wherein the first targeting moiety is does not detectably bind, eg, does not bind, to a first targeting moiety of another, and the second targeting moiety does not detectably bind, eg, does not bind, to another second targeting moiety. In some embodiments, targeting moieties used in the compositions and methods described herein are functional (eg, attached to a sequence) in a monomeric, eg, non-dimeric, state.

[0633] In some embodiments, binding of the targeting moiety to the targeting moiety reduces the binding affinity of the targeting moiety for another transcription factor, genomic complex component, or genomic sequence element. In some embodiments, the targeting portion is 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001nM K below D (and optionally at least 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 , 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001nM K D ) to bind to its target sequence. In some embodiments, the targeting moiety has a K of 0.001 nM to 500 nM, such as 0.1 nM to 5 nM, such as about 0.5 nM. D binds to its target sequence. In some embodiments, the targeting moiety has a K of at least 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, or 100,000 nM D(and optionally does not appreciably bind to non-target sequences). In some embodiments, the targeting moiety does not bind to non-target sequences.

[0634] In some embodiments, the targeting moiety comprises a nucleic acid sequence complementary to the targeting moiety, eg, a regulatory element (eg, promoter or enhancer) of the target gene, eg, MYC. In some embodiments, the targeting moiety is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% with the targeting moiety. %, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementary.

[0635] In some embodiments, the targeting moiety can be or include a CRISPR / Cas domain, a TAL effector domain, a Zn finger domain, or a nucleic acid molecule.

[0636] In some embodiments, the targeting moiety of the expression repressor is 100, 90, 80, 70, 60, 50, 40, 30, or 20 or fewer nucleotides (and optionally at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides). In some embodiments, the effector portion of the expression repressor or fusion molecule is 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 or fewer amino acids (and optionally at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 , 1600, 1700, 1800, or 1900 amino acids). In some embodiments, the effector portion of the expressed repressor or fusion molecule is 100-2000, 100-1900, 100-1800, 100-1700, 100-1600, 100-1500, 100-1400, 100-1300, 100~1200, 100~1100, 100~1000, 100~900, 100~800, 100~700, 100~600, 100~500, 100~400, 100~300, 100~200, 200~2000, 200~ 1900, 200~1800, 200~1700, 200~1600, 200~1500, 200~1400, 200~1300, 200~1200, 200~1100, 200~1000, 200~900, 200~800, 200~700, 200~600, 200~500, 200~400, 200~300, 300~2000, 300~1900, 300~1800, 300~1700, 300~1600, 300~1500, 300~1400, 300~1300, 300~ 1200, 300~1100, 300~1000, 300~900, 300~800, 300~700, 300~600, 300~500, 300~400, 400~2000, 400~1900, 400~1800, 400~1700, 400~1600, 400~1500, 400~1400, 400~1300, 400~1200, 400~1100, 400~1000, 400~900, 400~800, 400~700, 400~600, 400~500, 500~ 2000, 500~1900, 500~1800, 500~1700, 500~1600, 500~1500, 500~1400, 500~1300, 500~1200, 500~1100, 500~1000, 500~900, 500~800, 500~700, 500~600, 600~2000, 600~1900, 600~1800, 600~1700, 600~1600, 600~1500, 600~1400, 600~1300, 600~1200, 600~1100, 600~ 1000, 600~900, 600~800, 600~700, 700~2000, 700~1900, 700~1800, 700~1700, 700~1600, 700~1500, 700~1400, 700~1300, 700~1200, 700~1100, 700~1000, 700~900, 700~800, 800~2000, 800~1900, 800~1800, 800~1700, 800~1600, 800~1500, 800~1400, 800~1300, 800~ 1200, 800~1100, 800~1000, 800~900, 900~2000, 900~1900, 900~1800, 900~1700, 900~1600, 900~1500, 900~1400, 900~1300, 900~1200, 900 ~ 1100, 900 ~ 1000, 1000 ~ 2000, 1000 ~ 1900, 1000 ~ 1800, 1000 ~ 1700, 1000 ~ 1600, 1000 ~ 1500, 1000 ~ 1500, 1000 ~ 1400, 1000 ~ 1300, 1000 ~ 1200, 1000 ~ 1200, 1100 ~ 1100 ~ 2000, 1100~1900, 1100~1800, 1100~1700, 1100~1600, 1100~1500, 1100~1400, 1100~1300, 1100~1200, 1200~2000, 1200~1900, 1200~1800, 1200~170 0, 1200~1600, 1200~1500, 1200~1400, 1200~1300, 1300~2000, 1300~1900, 1300~1800, 1300~1700, 1300~1600, 1300~1500, 1300~1400, 1400~2000, 140 0~ 1900, 1400~1800, 1400~1700, 1400~1600, 1400~1500, 1500~2000, 1500~1900, 1500~1800, 1500~1700, 1500~1600, 1600~2000, 1600~1900, 1600~180 0, Contains 1600-1700, 1700-2000, 1700-1900, 1700-1800, 1800-2000, 1800-1900, or 1900-2000 amino acids.

[0637] An expression repressor or system comprising an expressor as disclosed herein can include a nucleic acid, eg, one or more nucleic acids. The term "nucleic acid" refers to any compound that is or can be incorporated into an oligonucleotide chain. In some embodiments, the nucleic acid is an oligonucleotide chain or a compound and / or substance that can be incorporated into it via a phosphodiester bond. As will be clear from the context, in some embodiments "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); refers to an oligonucleotide chain containing nucleic acid residues. In some embodiments, a "nucleic acid" is or includes RNA; in some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, the nucleic acid is or comprises greater than 50% ribonucleotides and is referred to herein as ribonucleic acid (RNA). In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have phosphodiester bonds in the backbone. Alternatively, it has a peptide bond and is considered to be within the scope of this invention. Alternatively, or in addition, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is or includes one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or consisting of it. In some embodiments, the nucleic acid contains one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5propynyl- Cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine , 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intervening bases, and combinations thereof) , including or consisting of. In some embodiments, the nucleic acid contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those present in naturally occurring nucleic acids. )including. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or protein. In some embodiments, the nucleic acid includes one or more introns. In some embodiments, the nucleic acids are isolated from natural sources, enzymatically synthesized by complementary template-based polymerization (in vivo or in vitro), reproduced in recombinant cells or systems, and chemically synthesized. prepared by one or more. As used herein, "recombinant" when used to describe a nucleic acid refers to any nucleic acid that is not naturally occurring. In some embodiments, the nucleic acids are at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425 , 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues in length or more. In some embodiments, the nucleic acid is about 2 to about 5000 nt, about 10 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to 250 nt, about 200 to about 300 nt, about 250 to about 350 nt. , about 300 to about 500 nt, about 10 to about 1000 nt, about 50 to about 1000 nt, about 100 to about 1000 nt, about 1000 to about 2000 nt, about 2000 to about 3000 nt, about 3000 to about 4000 nt, about 4000 to about 5000 nt, or They can have any range of lengths therebetween. In some embodiments, the nucleic acid is partially or completely single-stranded; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a polypeptide or includes at least one element that is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0638] In some embodiments, the targeting moiety comprises or is a nucleic acid sequence, protein, protein fusion, or membrane transport polypeptide. In some embodiments, the targeting moiety is selected from an exogenous junctional nucleating molecule, a nucleic acid encoding a junctional nucleating molecule, or a fusion of a sequence targeting polypeptide and a junctional nucleating molecule. Junction nucleation molecules can be, for example, CTCF, cohesin, USF1, YY1, TATA box-associated protein-associated factor 3 (TAF3), ZNF143 binding motif. In some embodiments, the targeting moiety comprises a polymer or polymer moiety, such as a polymer of nucleotides (such as an oligonucleotide), a peptide nucleic acid, a peptide-nucleic acid mixer, a peptide or polypeptide, a polyamide, a carbohydrate, or the like. That's it.

[0639] In some embodiments, the targeting moiety comprises or is a nucleic acid. In some embodiments, the effector moiety comprises or is a nucleic acid. In some embodiments, the nucleic acids that may be included in the moieties may be or contain DNA, RNA, and / or artificial or synthetic nucleic acids or nucleic acid analogs or mimetics. For example, in some embodiments, the nucleic acid is: genomic DNA (gDNA), complementary DNA (cDNA), peptide nucleic acid (PNA), peptide-nucleic acid mixmer, peptide oligonucleotide conjugate, locked nucleic acid (LNA), Targeting cross-linked nucleic acids (BNA), polyamides, triplex-forming oligonucleotides, antisense oligonucleotides, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecules (e.g., non-coding RNAs as described herein) and / or targeting the expression products of particular genes associated with the targeted genomic complexes described herein). Nucleic acid sequences suitable for use in modulators can include modified oligonucleotides (e.g., chemical modifications, such as modifications that alter backbone linkages, sugar molecules, and / or nucleobases), and / or artificial nucleic acids. . In some embodiments, the nucleic acid sequence includes, but is not limited to, genomic DNA, cDNA, peptide nucleic acids (PNAs) or peptide oligonucleotide conjugates, locked nucleic acids (LNAs), bridged nucleic acids (BNAs), polyamides, triplex-forming oligos. Includes nucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules. In some embodiments, the nucleic acid may include one or more residues that are not naturally occurring DNA or RNA residues and one or more linkages that are not phosphodiester bonds (e.g., phosphorothioate bonds). etc.) and / or may include one or more modifications, such as a 2'O modification, such as, for example, 2'-OmeP. Various nucleic acid structures useful for preparing synthetic nucleic acids are known in the art (see, e.g., WO 2017 / 062862l and WO 2014 / 012081), and those skilled in the art will appreciate that these , may be used in accordance with this disclosure.

[0640] Some examples of nucleic acids include, but are not limited to, nucleic acids that hybridize to a target gene, such as MYC (e.g., gRNA or antisense ssDNA as described elsewhere herein), viral DNA, or Nucleic acids that hybridize to exogenous nucleic acids such as RNA; nucleic acids that hybridize to RNA; nucleic acids that interfere with gene transcription; nucleic acids that interfere with RNA translation; stabilize RNA through targeting for degradation or destabilize RNA; Nucleic acids that interfere with a DNA or RNA binding factor through interference with its expression or function, and nucleic acids that are linked to an intracellular protein or protein complex and regulate its function.

[0641] In some embodiments, the expression repressor comprises one or more nucleoside analogs. In some embodiments, the nucleic acid sequence contains one or more naturally occurring nucleosides, e.g., purines or pyrimidines, e.g., adenine, cytosine, guanine, thymine, and uracil, in addition to or in place of one or more natural nucleosides. May contain multiple nucleoside analogs. In some embodiments, the nucleic acid sequence includes one or more nucleoside analogs. Nucleoside analogs include, but are not limited to, nucleoside analogs such as 5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 4-methylbenzimidazole, 5- (Carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine, β-D-galactosylcuosine, inosine, N6-isopentenyl adenine, 1- Methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylcuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v ), waibutoxocin, pseudouracil, cuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v ), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, 3-nitropyrrole, inosine, thiouridine, cuosine , wyosin, diaminopurine, isoguanine, isocytosine, diaminopyrimidine, 2,4-difluorotoluene, isoquinoline, pyrrolo[2,3-β]pyridine, and any other that can base pair with the purine or pyrimidine side chain. Things can be mentioned.

[0642] CRISPR / Cas domain In some embodiments, the targeting moiety is or includes a CRISPR / Cas domain. A CRISPR / Cas protein can include a CRISPR / Cas effector and optionally one or more other domains. CRISPR / Cas domains typically have structural and / or functional relationships with proteins involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., Cas proteins. have similar similarities. The CRISPR / Cas domain optionally includes a guide RNA, such as a single guide RNA (sgRNA). In some embodiments, the CRISPR / Cas domain non-covalently binds to the gRNA comprised by the CRISPR / Cas domain.

[0643] The CRISPR system is an adaptive defense system first discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases called CRISPR-associated or "Cas" endonucleases (eg, Cas9 or Cpf1) to cleave foreign DNA. For example, in a typical CRISPR / Cas system, an endonuclease is activated by a sequence-specific, non-coding "guide RNA" that targets single- or double-stranded DNA sequences (e.g., the genome to which the sequence is being edited). Directed to the inner part). Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class of II CRISPR systems includes type II Cas endonucleases, such as Cas9, CRISPR RNA (“crRNA”), and trans-acting crRNA (“tracrRNA”). crRNA includes a "guide RNA", which is typically an approximately 20 nucleotide RNA sequence and corresponds to a target DNA sequence. crRNA also contains a region that binds to tracrRNA to form a partially double-stranded structure, which is cleaved by RnaseIII to yield a crRNA / tracrRNA hybrid. The crRNA / tracrRNA hybrid then directs Cas9 endonuclease to recognize and cleave the target DNA sequence. The target DNA sequence must generally be flanked by a "protospacer adjacent motif" ("PAM") specific for a given Cas endonuclease; PAM sequences occur throughout a given genome. CRISPR endonucleases identified from diverse prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA ( Examples include Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endoglucanases, e.g., Cas9 endonuclease, bind to G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleavage of target DNA at a position 3 nucleotides upstream (5') from the PAM site. Implement. Another class II CRISPR system includes the V-type endonuclease Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Can be mentioned. Cpf1-associated CRISPR arrays are processed into mature crRNAs without the need for tracrRNA; in other words, the Cpf1 system only requires Cpf1 nuclease and crRNA to cleave target DNA sequences. Cpf1 endonuclease binds T-rich PAM sites, such as 5'-TTN. Cpf1 also recognizes the 5'-CTA PAM motif. Cpf1 introduces offset or staggered double-strand breaks with 4- or 5-nucleotide 5' overhangs, e.g., 18 nucleotides downstream (3' to) the PAM on the coding strand and the PAM on the complementary strand. Cleave the target DNA by cutting the target DNA with a 5-nucleotide offset or staggered cleavage located 23 nucleotides downstream from Compared to insertion-based DNA insertion, homologous recombination-based DNA insertion enables more precise genome editing. See, for example, Zetsche et al. (2015) Cell, 163:759-771.

[0644] A wide variety of CRISPR-associated (Cas) genes or proteins can be used with the t...

Claims

1. a first targeting moiety comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical thereto; a first effector moiety comprising a DNA methyltransferase; An expression repressor comprising:

2. 2. The expression repressor of claim 1, wherein the first targeting portion comprises an amino acid sequence according to SEQ ID NO: 13 or a sequence that is at least 95, 99, or 100% identical thereto or differs therefrom at 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.

3. The first effector portion i) comprises MQ1 or a functional variant or fragment thereof; ii) comprising a sequence of SEQ ID NO: 19 or 87, or a sequence which is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; and / or iii) comprising a sequence of SEQ ID NO: 30 or 129, or a sequence which is at least 80, 85, 90, 95, 99, or 100% identical thereto, or which differs therefrom at 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; 3. An expression repressor according to claim 1 or 2.

4. i) the first targeting moiety is positioned N-terminal to the first effector moiety on the same polypeptide chain. ii) the expression repressor comprises a nuclear localization signal (NLS), optionally wherein the NLS comprises the amino acid sequence of SEQ ID NO: 135 or SEQ ID NO: 136; iii) the expression repressor comprises the amino acid sequence of SEQ ID NO: 88 at its N-terminus; and / or iv) the expression repressor comprises a linker located between the first targeting moiety and the first effector moiety, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO: 137; An expression repressor according to any one of claims 1 to 3.

5. A first expression repressor according to any one of claims 1 to 4, A second expression repressor and A system including:

6. the second expression repressor a second targeting moiety that binds to a genomic locus; and A second effector part and and optionally i) the second targeting moiety binds to a genomic locus that includes at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 77, 199, or 201; ii) the second targeting moiety comprises a zinc finger domain; iii) the second targeting moiety comprises an amino acid sequence according to any one of SEQ ID NOs: 7, 169, or 171, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; iv) the second effector moiety comprises KRAB or a functional variant or fragment thereof; v) the second effector moiety comprises an amino acid sequence according to SEQ ID NO: 18, or a sequence which is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; and / or vi) the second expression repressor comprises an amino acid sequence according to any one of SEQ ID NOs: 24, 134, 141, 177, 183, 179, or 185, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; The system of claim 5.

7. a) a first expression repressor, i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; A first expression repressor comprising: b) a second expression repressor, i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7 or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; and a second expression repressor comprising An expression suppression system comprising:

8. a) a first expression repressor, i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence which is at least 80, 85, 90, 95, 99, or 100% identical thereto or which differs therefrom at 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; ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; A first expression repressor comprising: b) a second expression repressor, i) a second targeting moiety having an amino acid sequence according to any one of SEQ ID NOs: 169 or 171, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; and a second expression repressor comprising An expression suppression system comprising:

9. The method of claim 1, wherein the expression repressor or expression suppression system appreciably reduces expression of MYC for 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 for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions. ii) binding of the expression repressor to the MYC locus appreciably reduces expression of MYC 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 with the expression repressor or expression suppression system; iii) contacting a plurality of cells (e.g., a plurality of cancer cells and a plurality of non-cancer cells) with the expression repressor, expression repressor system, or nucleic acid encoding the expression repressor or the first expression repressor and the second expression repressor reduces viability of the plurality of cells (e.g., a plurality of cancer cells and a plurality of non-cancer cells), and optionally, said contacting reduces viability of the plurality of cancer cells thereby reducing viability of the plurality of non-cancer cells. a greater decrease in viability than a decrease in viability of a number of non-cancerous cells, and optionally a decrease in viability of the plurality of cancer cells is greater than 1.05x (i.e., 1.05-fold), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 50x, or 100x greater than a decrease in viability of the plurality of non-cancerous cells. iv) administration of the expression repressor or expression suppression system induces apoptosis 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); and / or v) administration of the expression repressor or expression suppression system increases methylation at the site within the genomic locus; An expression repressor or an expression suppression system according to any one of claims 1 to 8.

10. The expression repressor, expression suppression system, e.g., a first expression repressor and a second expression repressor, one or more nucleic acids encoding a targeting moiety and / or an effector moiety according to any one of claims 1 to 9, optionally comprising: i) a nucleotide sequence encoding said first targeting moiety, said nucleotide sequence encoding said first targeting moiety comprising a sequence according to SEQ ID NO: 46 or 131 or a sequence which is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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. ii) a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto or differs therefrom at 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; iii) a nucleotide sequence according to SEQ ID NO: 63 or 130, or a sequence which is at least 80, 85, 90, 95, 99 or 100% identical thereto or which differs therefrom at 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, wherein a polyA sequence is optional, and / or iv) a nucleic acid encoding the first expression repressor and the second expression repressor, optionally comprising a nucleotide sequence according to SEQ ID NO: 113, 196, or 197, and optionally a polyA sequence; A nucleic acid comprising:

11. A vector comprising the system according to any one of claims 1 to 9, or a nucleic acid encoding an expression repressor.

12. A pharmaceutical composition comprising an expression repressor, system, nucleic acid or vector according to any one of claims 1 to 11.

13. Use of an expression repressor, system, or a nucleic acid encoding an expression repressor or system as defined in any one of claims 1 to 10 in the manufacture of a medicament, composition or product for treating cancer, treating hepatitis, treating liver disease, treating lung disease, reducing tumor growth, reducing tumor size, reducing expression of a target gene, reducing cell viability, or altering the level of an epigenetic marker associated with a target gene, in a subject in need thereof, wherein the subject in need thereof exhibits overexpression of MYC in at least some of its cells.

14. A pharmaceutical composition for treating cancer, hepatitis, liver disease, lung disease, reducing tumor growth, reducing tumor size, reducing expression of a target gene, reducing cell viability, or altering the level of an epigenetic marker associated with a target gene in a subject in need thereof, the pharmaceutical composition comprising an expression repressor, system, or a nucleic acid encoding an expression repressor or system described in any one of claims 1 to 10, wherein the subject in need thereof exhibits overexpression of MYC in at least some of its cells.

15. Use of an expression repressor, system, or a nucleic acid encoding an expression repressor or system according to any one of claims 1 to 10 in the manufacture of a medicament, composition, or product, comprising: i) for increasing or restoring the sensitivity of a cancer to a kinase inhibitor, e.g., sorafenib, and optionally the medicament, composition or product exhibits an IC ５０ by 10%, 20%, 30%, or 40%; ii) for increasing or restoring the sensitivity of a cancer to a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1, and optionally said medicament, composition or product reduces the IC50 of said bromodomain inhibitor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, e.g., in a cancer cell viability assay; and / or iii) for increasing or restoring the sensitivity of a cancer to a MEK inhibitor, e.g., trametinib, and optionally said medicament, composition or product reduces the IC50 of said MEK inhibitor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, e.g., in a cancer cell viability assay; Optionally, the subject exhibits overexpression of MYC in at least some cells.

16. i) the cancer includes cells characterized by increased MYC expression compared to a reference level (e.g., compared to the MYC expression of reference cells, e.g., otherwise similar non-cancerous cells of the subject) and cells that are not characterized by increased MYC expression compared to a reference level (e.g., compared to the MYC expression of reference cells, e.g., otherwise similar non-cancerous cells of the subject), e.g., cells having normal MYC expression; ii) said use comprising a) first administering to the subject a first plurality of doses of said medicament, composition, product or pharmaceutical composition, optionally wherein each subsequent dose in said first plurality is administered 5 days after a previous dose in said first plurality; b) secondly, discontinuing said medicament, composition, product or pharmaceutical composition for a period of time (a "drug holiday"), e.g., about two weeks; c) third, administering to the subject a second plurality of doses of the medicament, composition, product, or pharmaceutical composition, optionally wherein the second plurality of subsequent doses is administered 5 days after a previous dose in the second plurality. Including, iii) tumor volume is reduced (e.g., to undetectable levels) following cessation of treatment with the medicament, composition, product or pharmaceutical composition; and / or iv) said use comprising a) contacting the cells with a second therapeutic agent; and / or b) administering to the subject a second therapeutic agent, optionally wherein the second therapeutic agent is not an expression repressor, system, nucleic acid, vector, or pharmaceutical composition according to any one of claims 1 to 12 (e.g., the second therapeutic agent is an immunotherapy, one or both of an immune checkpoint therapy and an anti-vascular endothelial growth factor therapy, a systemic chemotherapy, a tyrosine kinase inhibitor, e.g., sorafenib, a mitogen-activated protein kinase kinase inhibitor, e.g., trametinib, or a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1, e.g., virabresib), and optionally wherein (I) the first and second therapeutic agents are administered simultaneously, or (II) the first and second therapeutic agents are administered sequentially. Further comprising:

16. The use according to claim 15.

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