Combination therapy comprising MYC modulation

Abstract

The present disclosure relates to combination therapy. The combination therapy may comprise the administration of an expression repressor and a kinase inhibitor. In some embodiments, the expression repressor comprises a targeting moiety that binds to an MYC promoter, an anchor sequence, or a super enhancer. In some embodiments, the expression repressor comprises an effector moiety that inhibits transcription or methylates DNA. These compositions and methods are useful, for example, in the treatment of cancer, such as HCC.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 354,646, filed on June 22, 2022, and U.S. Provisional Application No. 63 / 489,629, filed on March 10, 2023, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on June 19, 2023, is named O2057-7034WO_SL.xml, and is 282,044 bytes in size. Background Art

[0005] Misregulation of gene expression is the root cause of many diseases (e.g., mammals, such as humans), such as tumors, neurological diseases, metabolic disorders and obesity. Misregulation of the transcription factor MYC plays a central role in a variety of human tumors and chronic liver diseases. Due to various factors, such as the lack of a clear ligand binding site, the lack of physiological functions necessary to maintain normal tissues, etc., the MYC protein is considered to be "undruggable". The technology of regulating MYC gene expression provides a feasible alternative for treating these diseases. New tools, systems and methods are needed to stably change (e.g., reduce) the expression of disease-associated genes (e.g., MYC). Summary of the Invention

[0006] The present disclosure provides, for example, combination therapies comprising an expression repressor and a kinase inhibitor.

[0007] The present disclosure provides, inter alia, expression repressors and expression repressor systems that can be used to modulate (eg, reduce) the expression of a target gene (eg, MYC).

[0008] In some aspects, the disclosure provides a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcriptional regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene (e.g., MYC) or a sequence proximal to the transcriptional regulatory element; and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence comprising anchor sequence-mediated conjugation (ASMC) of the target gene (e.g., MYC), or a sequence proximal to the anchor sequence.

[0009] In some aspects, the disclosure relates to nucleic acids encoding a first expression repressor, a second expression repressor, both, or components thereof (e.g., gRNA, mRNA). In some embodiments, the nucleic acid encoding the expression repressor system is a polycistronic sequence. In some embodiments, the polycistronic sequence is a bicistronic sequence.

[0010] In some aspects, the disclosure relates to a method of treating a disorder associated with overexpression of a target gene (e.g., MYC) in a subject, comprising administering to the subject an expression repressor or system, nucleic acid, or vector described herein, thereby treating the disorder.

[0011] Additional features of any of the foregoing methods or compositions include one or more of the embodiments listed below.

[0012] 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.

[0013] All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences mentioned herein (e.g., in any of the tables herein) are incorporated by reference. Unless otherwise indicated, sequence accession numbers specified herein (including in any of the tables herein) refer to database entries as of December 15, 2020. When a gene or protein references multiple sequence accession numbers, all sequence variants are encompassed.

[0014] Examples

[0015] 1. An expression repressor comprising:

[0016] a targeting moiety that binds to the MYC promoter, and

[0017] Optionally, an effector moiety,

[0018] The expression repressor can reduce the expression of MYC.

[0019] 2. The expression repressor of embodiment 1, wherein the targeting portion binds to a genomic locus within 1400, 1200, 1000, 800, 600, 400, or 200 nt upstream or downstream of SEQ ID NO: 4, 199, or 201.

[0020] 3. The expression repressor of embodiment 1, wherein the targeting portion binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19 or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, 83, 85, 199 or 201.

[0021] 4. An expression repressor comprising:

[0022] Incorporates a targeting portion of a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 75, 76, 78, 79, 80, 81, 84, 85, 86, 190, 191, 192, 200, or 202 and

[0023] Optionally, an effector moiety,

[0024] The expression repressor can reduce the expression of MYC.

[0025] 5. An expression repressor comprising:

[0026] a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of a sequence of SEQ ID NO: 2, 77, 82, 83, 199, or 201, and

[0027] Optionally, an effector moiety,

[0028] The expression repressor can reduce the expression of MYC.

[0029] 6. An expression repressor comprising:

[0030] a targeting moiety that binds to the MYC locus, and

[0031] comprising an effector portion of MQ1 or a fragment or variant thereof,

[0032] The expression repressor can reduce the expression of MYC.

[0033] 7. An expression repressor comprising:

[0034] Binds to the targeted portion of the locus in the MYC super enhancer region,

[0035] Optionally an effector moiety, e.g., an effector moiety comprising a DNA methyltransferase, wherein optionally the effector moiety comprises MQ1 or a fragment or variant thereof,

[0036] The expression repressor can reduce the expression of MYC.

[0037] 8. An expression repressor comprising:

[0038] Binds to the targeted portion of the locus in the MYC super enhancer region,

[0039] comprising an effector portion of a transcriptional repressor, wherein optionally the effector portion comprises KRAB or a fragment or variant thereof,

[0040] The expression repressor can reduce the expression of MYC.

[0041] 9. The expression repressor of any one of embodiments 7 or 8, wherein the targeting portion binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of a sequence of any one of SEQ ID NOs: 96-110, 83, 199, or 201.

[0042] 10. The expression repressor of any one of embodiments 7-9, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of GRCh37:chr8:129162465-129212140, using the hg19 reference genome.

[0043] 11. The expression repressor of any one of embodiments 7-10, wherein the targeting portion binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 96 or 108.

[0044] 12. The expression repressor of any one of embodiments 7-11, wherein the targeting portion comprises a zinc finger domain or a TAL effector domain.

[0045] 13. An expression repressor comprising:

[0046] a targeting moiety that binds to a locus (e.g., the MYC locus),

[0047] a first effector portion comprising EZH2 or a fragment or variant thereof, and

[0048] a second effector moiety comprising KRAB or a fragment or variant thereof,

[0049] The expression repressor is capable of reducing the expression at the locus, for example, reducing the expression of MYC.

[0050] 14. The expression repressor of embodiment 13, wherein the targeting moiety binds to the MYC promoter, super enhancer region, or anchor sequence.

[0051] 15. The expression repressor of embodiment 13 or 14, wherein the targeting portion comprises a TAL effector domain, a CRISPR / Cas domain, or a zinc finger domain.

[0052] 16. The expression repressor of any one of embodiments 13-15, wherein the first effector moiety is N-terminal to the second effector moiety, or wherein the first effector is C-terminal to the second effector moiety.

[0053] 17. An expression repressor comprising:

[0054] a targeting moiety that binds to the MYC locus, wherein the targeting moiety comprises a zinc finger domain, and

[0055] Optionally, an effector moiety,

[0056] The expression repressor can reduce the expression of MYC.

[0057] 18. An expression repressor comprising:

[0058] A targeting moiety comprising a CRISPR / Cas domain (e.g., comprising a catalytically inactive CRISPR / Cas protein) that binds to a transcriptional regulatory element (e.g., a promoter, enhancer, super enhancer, or transcription start site (TSS)) operably linked to the MYC gene, or a sequence proximal to the transcriptional regulatory element; and

[0059] An effector portion comprising MQ1 or a functional variant or fragment thereof.

[0060] 19. An expression repressor comprising:

[0061] A targeting moiety comprising a CRISPR / Cas domain (e.g., comprising a catalytically inactive CRISPR / Cas protein) that binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene, or a sequence proximal to the transcriptional regulatory element; and

[0062] An effector portion comprising MQ1 or a functional variant or fragment thereof.

[0063] 20. An expression repressor comprising:

[0064] A targeting moiety comprising a CRISPR / Cas domain (e.g., comprising a catalytically inactive CRISPR / Cas protein) that binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene, or a sequence proximal to the transcriptional regulatory element; and

[0065] An effector portion comprising KRAB or a functional variant or fragment thereof.

[0066] 21. An expression repressor comprising:

[0067] a targeting moiety comprising a CRISPR / Cas domain (e.g., comprising a catalytically inactive CRISPR / Cas protein) that binds to an anchor sequence comprising anchor sequence-mediated conjugation (ASMC) of the MYC gene, or to a sequence proximal to the anchor sequence; and

[0068] An effector portion comprising KRAB or a functional variant or fragment thereof.

[0069] 22. An expression repressor comprising:

[0070] a targeting moiety comprising a zinc finger domain that binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to a MYC gene, or a sequence proximal to the transcriptional regulatory element; and

[0071] An effector portion comprising MQ1 or a functional variant or fragment thereof.

[0072] 23. An expression repressor comprising:

[0073] a targeting moiety comprising a zinc finger domain that binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to a MYC gene, or a sequence proximal to the transcriptional regulatory element; and

[0074] An effector portion comprising KRAB or a functional variant or fragment thereof.

[0075] 24. An expression repressor comprising:

[0076] a targeting portion that binds to a mouse genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any one of SEQ ID NOs: 190-192, and

[0077] Optionally, an effector moiety,

[0078] The expression repressor can reduce the expression of MYC.

[0079] 25. The expression repressor of claim 24, wherein the effector portion comprises a DNA methyltransferase, such as MQ1 or a fragment or variant thereof.

[0080] 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 large nuclease domain, or an oligonucleotide.

[0081] 27. The expression repressor of any one of embodiments 24-26, wherein the targeting portion comprises a zinc finger domain or a TAL effector domain.

[0082] 28. The expression suppressor of any one of embodiments 24-27, wherein the expression suppressor comprises an amino acid sequence selected from any one of SEQ ID NOs: 160-165, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0083] 29. The expression repressor of any one of embodiments 24-28, wherein the expression repressor is encoded by a nucleotide sequence selected from any one of SEQ ID NOs: 166-168, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that 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.

[0084] 30. The expression repressor of any one of embodiments 24-29, wherein the targeting portion comprises an amino acid sequence according to any one of SEQ ID NOs: 154-156, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that 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.

[0085] 31. The expression repressor of any of embodiments 24-30, wherein the targeting portion comprises a nucleic acid sequence according to any of SEQ ID NOs: 157-159, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that 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.

[0086] 32. The expression repressor of any one of embodiments 24-31, wherein the effector portion is a persistent effector portion.

[0087] 33. The expression repressor of any one of embodiments 24-32, wherein the effector portion is a transient effector portion.

[0088] 34. The expression repressor of any one of embodiments 24-33, wherein the expression repressor is a fusion molecule.

[0089] 35. The expression repressor of any one of embodiments 24-34, wherein the targeting portion comprises a zinc finger domain and the effector portion comprises an epigenetic modification portion, such as a DNA methyltransferase, such as MQ1 or a fragment or variant thereof.

[0090] 36. The expression repressor of any one of embodiments 18-20, 22, or 23, wherein the regulatory element is part of a regulatory element cluster.

[0091] 37. The expression repressor of any one of embodiments 18-20, 22, or 23, wherein the regulatory element is located in a non-coding region.

[0092] 38. The expression repressor of any one of embodiments 18-20, 22, or 23, wherein the regulatory element is a distal enhancer, eg, located at least 1,000 nt from the target gene (eg, MYC) promoter.

[0093] 39. The expression repressor of any one of embodiments 18-20, 22, 23, or 36-38, wherein the regulatory element increases expression of a target gene (eg, MYC).

[0094] 40. The expression repressor of any one of embodiments 18-20, 22, 23, or 36-39, wherein the regulatory element comprises one or more mutations.

[0095] 41. The expression repressor of any one of embodiments 18-20, 22, 23, or 36-40, wherein the regulatory element comprises at least one disease-associated single nucleotide polymorphism (SNP).

[0096] 42. The expression repressor of any one of embodiments 18-20, 22, 23, or 36-41, wherein the transcriptional regulatory element interacts with the promoter of the target gene (e.g., MYC) through an enhancer docking site.

[0097] 43. The expression repressor of embodiment 42, wherein the enhancer docking site comprises a nucleotide sequence according to SEQ ID NO: 71 or 72 herein, or any one of SEQ ID NO: 73 or 74 described in international application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0098] 44. An expression repressor comprising:

[0099] a targeting moiety comprising a zinc finger domain that binds to an anchor sequence comprising anchor sequence-mediated conjugation (ASMC) of the MYC gene, or to a sequence proximal to the anchor sequence; and

[0100] An effector portion comprising KRAB or a functional variant or fragment thereof.

[0101] 45. The expression suppressor of any one of embodiments 1-23 or 36-43, wherein the expression suppressor comprises an amino acid sequence selected from any one of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, or 177-186 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference), 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.

[0102] 46. ​​The expression repressor of any one of embodiments 1-23 or 36-45, wherein the expression repressor is encoded by a nucleotide sequence selected from any of SEQ ID NOs: 55-66, 130, 189, or 193-197 herein, or SEQ ID NOs: 67-70 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference), or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0103] 47. The expression repressor of any one of embodiments 1-23 or 36-46, wherein the targeting portion comprises an amino acid sequence according to any one of SEQ ID NOs: 5-16 or 169-172, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0104] 48. The expression repressor of any preceding embodiment, wherein the effector portion comprises an amino acid sequence according to SEQ ID NO: 18, 19, or 87, or a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical thereto, or that 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.

[0105] 49. The expression repressor of any one of embodiments 1-12, 17-19, 22, 36-42, or 44-47, wherein the effector moiety is a persistent effector moiety.

[0106] 50. The expression repressor of any one of embodiments 1-23 or 36-48, wherein the effector moiety is a transient effector moiety.

[0107] 51. The expression repressor of any one of embodiments 1-12, 17-19, 22, 36-42, or 44-48, wherein the effector portion comprises a DNA methyltransferase, eg, MQ1, or a fragment or variant thereof.

[0108] 52. The expression repressor of any one of embodiments 1-23, 36-47, or 49, wherein the effector portion comprises a transcriptional repressor, eg, comprises KRAB or a fragment or variant thereof.

[0109] 53. The expression repressor of any preceding embodiment, wherein the targeting moiety comprises a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a large nuclease domain, or an oligonucleotide.

[0110] 54. The expression repressor of embodiment 53, wherein the CRISPR / Cas domain binds a gRNA, e.g., a gRNA that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any one of SEQ ID NOs: 1-4, e.g., wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any one of SEQ ID NOs: 1-4.

[0111] 55. The expression repressor of embodiment 53, wherein the CRISPR / Cas domain binds a gRNA, e.g., a gRNA that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of a sequence of any one of SEQ ID NOs: 96-110, e.g., wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of a sequence of any one of SEQ ID NOs: 96-110.

[0112] 56. An expression repressor as described in any one of embodiments 53-55, wherein the CRISPR / Cas domain comprises a Cas protein or Cpf1 protein or any variant (e.g., mutant) thereof selected from Table 1 described in international application WO / 2022 / 132195.

[0113] 57. The expression repressor of any one of embodiments 53-56, wherein the CRISPR / Cas domain comprises a catalytically inactive CRISPR / Cas protein, e.g., dCas9.

[0114] 58. The expression repressor of embodiment 53, wherein the zinc finger domain binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any one of SEQ ID NOs: 96-110, e.g., wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any one of SEQ ID NOs: 96-110.

[0115] 59. The expression repressor of any one of embodiments 17, 22, 26-53, or 57, wherein the zinc finger domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (and optionally no more than 11, 10, 9, 8, 7, 6, or 5 zinc fingers).

[0116] 60. The expression repressor of any one of embodiments 17, 22, 26-53, 57, or 58, wherein the zinc finger domain comprises 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, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers.

[0117] 61. The expression repressor of any one of embodiments 17, 22, 26-53, or 57-59, wherein the zinc finger domain comprises 3 or 9 zinc fingers.

[0118] 62. The expression repressor of any preceding embodiment, comprising a fusion molecule.

[0119] 63. The expression repressor of any preceding embodiment, comprising a linker located between the targeting domain and the effector domain, optionally wherein the linker comprises the amino acid sequence according to SEQ ID NO: 137 or SEQ ID NO: 138.

[0120] 64. The expression repressor of any one of embodiments 1-17, 20, 21, 23, 44-48, 50, or 52-57, wherein the targeting moiety comprises a catalytically inactive CRISPR / Cas domain (e.g., dCas9) and the effector moiety comprises a transcriptional repressor, e.g., KRAB or a fragment or variant thereof.

[0121] 65. The expression repressor of any one of embodiments 1-17, 20, 21, 23, 44-48, 50, 52, or 53-64, wherein the targeting moiety comprises a zinc finger domain and the effector moiety comprises a transcriptional repressor, e.g., KRAB or a fragment or variant thereof.

[0122] 66. The expression repressor of any one 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.

[0123] 67. The expression repressor of any one of embodiments 1-12, 18-19, 22, 36-43, 45-49, 51, or 53-57, wherein the targeting portion comprises a catalytically inactive CRISPR / Cas domain (e.g., dCas9) and the effector portion comprises an epigenetic modification portion, e.g., a DNA methyltransferase, e.g., MQ1, or a fragment or variant thereof.

[0124] 68. The expression repressor of any one of embodiments 1-12, 17-19, 22, 36-43, 45-49, 51, 53, or 58-63, wherein the targeting portion comprises a zinc finger domain and the effector portion comprises an epigenetic modification portion, e.g., a DNA methyltransferase, e.g., MQ1, or a fragment or variant thereof.

[0125] 69. An expression repressor as described in any of the preceding embodiments, comprising the amino acid sequence of any one of SEQ ID NOs: 22-34, 129, 133, 134, 139-149 or 177-186 herein or any one of SEQ ID NOs: 34-37 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0126] 70. The expression repressor of any preceding embodiment, which: (i) comprises one or more nuclear localization signal sequences (NLS), or (ii) does not comprise an NLS.

[0127] 71. The expression repressor of any preceding embodiment, comprising a first NLS at the N-terminus, eg, wherein the first NLS has the sequence of SEQ ID NO: 88.

[0128] 72. The expression repressor of any preceding embodiment, comprising an NLS at the C-terminus, eg, a second NLS, eg, having a sequence of SEQ ID NO: 89.

[0129] 73. The expression repressor of any preceding embodiment, wherein the first and the second NLS have the same sequence.

[0130] 74. The expression repressor of any one of embodiments 71-73, wherein the first and the second NLS have different sequences.

[0131] 75. The expression repressor of any preceding embodiment, comprising an epitope tag.

[0132] 76. The expression repressor of embodiment 75, wherein the epitope tag is an HA tag.

[0133] 77. The expression repressor of any preceding embodiment, wherein the anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations thereto.

[0134] 78. The expression repressor of any one of embodiments 1-77, wherein the anchor sequence comprises the sequence of SEQ ID NO: 73 or 74 as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference), or a sequence having no more than 8, 7, 6, 5, 4, 3, 2 or 1 changes thereto.

[0135] 79. The expression repressor of any preceding embodiment, wherein the anchor sequence and the MYC gene are located on the same chromosome.

[0136] 80. The expression repressor of any preceding embodiment, wherein the anchor sequence is upstream of the MYC gene (eg, upstream of the TSS or upstream of the promoter).

[0137] 81. The expression repressor of any preceding embodiment, wherein the anchor sequence is at least 1, 5, 10, 50, 100, or 1000 kilobases from the MYC gene (eg, from the TSS or promoter of the MYC gene).

[0138] 82. The expression repressor of any preceding embodiment, wherein the anchor sequence is 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, 0.5-5 or 500-1000 kilobases.

[0139] 83. The expression repressor of any one of embodiments 1-79 or 81-82, wherein the target sequence is located downstream of the MYC gene (e.g., downstream of the TSS or downstream of the promoter).

[0140] 84. The expression repressor of any preceding embodiment, wherein the targeting moiety binds to chromosomal coordinates 128746342-128746364, 128746321-128746343, 128746525-128746547, 128748014-128748036, 129188878-129188900, 129188958-129188980, 129188960-129188982, 129189067-129189089, 129189457-129189479, 129189554-129189576, 129189679-12918970 1. 129209511-129209533, 129209643-129209665, 129209658-129209680, 129209856-129209878, 129189452-129189474, 129189190-129189212, 12918 9274-129189296, 129189421-129189443, 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842, or sequences proximal thereto.

[0141] 85. The expression repressor of any preceding embodiment, wherein binding of the expression repressor to the target gene locus (e.g., MYC) increases methylation at a site in the target gene locus (e.g., MYC) by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to methylation in the absence of the expression repressor, e.g., as measured by ELISA or as described in any of Examples 7 or 28 of International Application WO / 2022 / 132195 (incorporated herein by reference in its entirety), wherein optionally the site at which methylation is determined is chr8:129188693-129189048 according to the hg19 reference genome, e.g., comprising a sequence according to SEQ ID NO: 123.

[0142] 86. An expression repressor as described in any of the preceding embodiments, wherein the binding of the expression repressor to the target gene locus (e.g., MYC) causes increased methylation at a site in the target gene locus (e.g., 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 a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, for example, as described in Example 28 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0143] 87. An expression repressor as described in any of the preceding embodiments, wherein binding of the expression repressor to the MYC locus reduces expression of MYC in the cell by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 or 100% compared to expression in the absence of the expression repressor, for example as measured by ELISA or as described in any of Examples 2-7 or 9 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0144] 88. The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the MYC locus results in a significant reduction in expression of MYC for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, as measured by ELISA or as described in Examples 2-7 or 9 described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference.

[0145] 89. The expression repressor of any preceding embodiment, wherein binding of the expression repressor to the MYC locus results in a significant decrease in MYC expression 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 post-transfection.

[0146] 90. The expression repressor of any one of embodiments 1-23 or 36-89, wherein the targeting portion binds to a human genomic locus.

[0147] 91. The expression repressor of any one of embodiments 24-43, 49, 51, 53, 56-57, 59-62, 66-68, 70-89, wherein the targeting portion binds to a mouse genomic locus.

[0148] 92. The expression repressor of any preceding embodiment, wherein binding of the expression repressor to the MYC locus reduces the viability of cells (e.g., cancer cells) comprising the MYC locus.

[0149] 93. The expression repressor of any one of the preceding embodiments, wherein contacting a plurality of cells with the expression repressor or a nucleic acid encoding the expression repressor reduces the viability of the plurality of cells.

[0150] 94. The expression repressor of any of the preceding embodiments, wherein the viability is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the viability in the absence of the first expression repressor, for example as measured by CellTiter Glo or as described in any of Examples 2-7 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0151] 95. The expression repressor of any of the preceding embodiments, wherein administration of the expression repressor results in apoptosis of at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

[0152] 96. The expression repressor of any preceding embodiment, wherein the plurality of cells comprises a plurality of cancer cells and a plurality of non-cancerous cells and / or a plurality of infected cells and a plurality of uninfected cells.

[0153] 97. The expression repressor of any preceding embodiment, wherein contacting the plurality of cells with the expression repressor or a nucleic acid encoding the expression repressor reduces the viability of the plurality of cancer cells more than it reduces the viability of the plurality of non-cancerous cells.

[0154] 98. The expression suppressor of any one of the preceding embodiments, wherein contacting the plurality of cells with the expression suppressor, or a nucleic acid encoding the expression suppressor, reduces the viability of the plurality of cancer cells by 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 more than the reduction in viability of the plurality of non-cancerous cells.

[0155] 99. The expression repressor of any one of embodiments 92-97, wherein the cancer cells are lung cancer cells, gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or liver cancer cells.

[0156] 100. The expression suppressor of any one of embodiments 92-99, wherein the cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, small cell lung cancer (SCLC), large cell (undifferentiated) carcinoma, triple-negative breast cancer, gastric adenocarcinoma, endometrial cancer, or pancreatic cancer.

[0157] 101. The expression repressor of any preceding embodiment, which, when contacted with a plurality of infected cells and a plurality of uninfected cells, reduces the viability of the plurality of infected cells more than it reduces the viability of the plurality of uninfected cells.

[0158] 102. The expression repressor of any preceding embodiment, wherein the infection is a viral infection.

[0159] 103. The expression repressor of embodiment 102, wherein the viral infection is hepatitis, such as hepatitis B.

[0160] 104. The expression repressor of any one of embodiments 92-103, wherein the infected cell is a human hepatocyte.

[0161] 105. The expression suppressor of any preceding embodiment, which has an EC50 of 0.04-0.4, 0.04-0.1, 0.1-0.2, 0.2-0.3, or 0.3-0.4 μg / mL when tested in a cancer cell (e.g., HCC cell) viability assay using LNPs delivering mRNA encoding the expression suppressor (e.g., in an assay according to Example 12 described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference).

[0162] 106. The expression suppressor of any one of embodiments 1-104, which has an EC50 of 0.1-2.5, 0.5-2.2, 1.0-1.5, 1.2-2 μg / mL when tested in a cancer cell (e.g., lung cancer cell) viability assay using LNPs delivering mRNA encoding the expression suppressor (e.g., in an assay according to Example 18 described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference).

[0163] 107. The expression suppressor of any preceding embodiment, having an EC50 of 0.004-0.08, 0.004-0.01, 0.01-0.02, 0.02-0.04, or 0.04-0.08 μg / mL when tested in an assay utilizing LNP to deliver mRNA encoding the expression suppressor to reduce MYC mRNA levels in cancer cells (e.g., HCC cells) (e.g., in an assay according to Example 12 described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference).

[0164] 108. The expression suppressor of any of the preceding embodiments, having an EC50 of 0.04-0.1, 0.04-0.09, 0.05-0.09 or 0.06-0.8 μg / mL when tested in an assay that reduces MYC mRNA levels in cancer cells (e.g., lung cancer cells) using LNP to deliver mRNA encoding the expression suppressor (e.g., in an assay according to Example 18 described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference).

[0165] 109. The expression repressor of any of the preceding embodiments, which reduces the level of 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%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the level of protein in untreated cells.

[0166] 110. The expression suppressor of any preceding embodiment, which is capable of reducing tumor volume, eg, in a human subject or a mammalian model.

[0167] 111. The expression suppressor of any of the preceding embodiments, wherein the expression suppressor is capable of reducing tumor volume to a similar or greater extent than a chemotherapeutic agent, e.g., in a mammalian model, e.g., when measured on day 20 after initiation of treatment, e.g., wherein the expression suppressor is administered at a dose of 3 mg / kg every 5 days.

[0168] 112. The expression suppressor of any preceding embodiment, wherein the expression suppressor is capable of reducing tumor volume compared to a PBS control, e.g., in a mammalian model, e.g., when measured at day 20 after initiation of treatment, e.g., wherein the expression suppressor is administered at 1 mg / kg, 1.5 mg / kg, or 3 mg / kg every 5 days for 4 doses, then every 3 days for 3 doses.

[0169] 113. The expression suppressor of any one of the preceding embodiments, wherein tumor volume is reduced by at least about 10%, 20%, 30% or 40% compared to a control treated with PBS, eg, at day 20 after initiation of treatment.

[0170] 114. The expression repressor of any one of embodiments 111-113, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0171] 115. The expression suppressor of any preceding embodiment, wherein the system is capable of reducing tumor volume to a similar or greater extent than a small molecule MYC inhibitor.

[0172] 116. The expression repressor of embodiment 115, wherein the small molecule MYC inhibitor is MYCi975, wherein optionally, tumor volume is reduced by at least about 10%, 20%, 30% or 40% compared to a control treated with MYCi975, e.g., at day 20 after initiation of treatment.

[0173] 117. The expression suppressor of any preceding embodiment, which causes no weight loss, or causes less than 3%, 2% or 1% weight loss compared to the start of treatment.

[0174] 118. A system comprising:

[0175] The first expression repressor as described in any one of the preceding embodiments, and

[0176] A second expression repressor, eg, a second expression repressor described herein, eg, a second expression repressor described in any preceding embodiment.

[0177] 119. A system comprising:

[0178] a first expression repressor comprising a first targeting moiety and, optionally, a first effector moiety, wherein the first expression repressor binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to the transcriptional regulatory element, and

[0179] A second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence comprising anchor sequence mediated conjugation (ASMC) of the MYC gene, or to a sequence proximal to the anchor sequence.

[0180] 120. The system of embodiment 118 or 119,

[0181] wherein the transcriptional regulatory element comprises a promoter, and

[0182] The anchor sequence comprises a CTCF binding motif.

[0183] 121. The system of any one of embodiments 118-120, wherein the second expression repressor binds to a downstream region adjacent to the CTCF binding motif.

[0184] 122. The system of any one of embodiments 118-120, wherein the second expression repressor binds to an upstream region adjacent to the CTCF binding motif.

[0185] 123. A system as described in any of embodiments 118-122, wherein

[0186] The first expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 3, 4, 71, 72, 75-86, or 200-206 herein, or SEQ ID NO: 73 or 74 set forth in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference); and

[0187] The second expression repressor comprises a targeting portion that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 3, 4, 71, 72, 75-86, or 200-206 herein, or SEQ ID NO: 73 or 74 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0188] 124. A system as described in any of embodiments 118-123, wherein

[0189] The first expression repressor binds to a targeting portion of a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of any one of SEQ ID NOs: 96-110.

[0190] 125. The system of any one of embodiments 118-124, wherein:

[0191] The first expression repressor binds to a targeting portion of a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 83; and

[0192] The second expression repressor binds to a targeting portion of a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO:77.

[0193] 126. A system comprising:

[0194] a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a sequence operably linked to a promoter of a MYC gene or proximal to the promoter, and

[0195] A second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an enhancer (eg, a super enhancer) of the MYC gene.

[0196] 127. The system of embodiment 126, wherein:

[0197] The first expression repressor binds to a targeting portion of a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 204, and

[0198] The second expression repressor binds to a targeting portion of a genomic locus comprising at least 16, 17, 18, 19 or 20 nucleotides of the sequence of any one of SEQ ID NO: 199 or 201.

[0199] 128. A system for reducing MYC expression, the system comprising:

[0200] a) a first expression repressor comprising:

[0201] 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 that differs therefrom in 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

[0202] 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 that differs therefrom in 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

[0203] b) a second expression repressor comprising:

[0204] i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7 169 or 171, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs therefrom in 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

[0205] 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 that 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.

[0206] 129. The system of embodiment 128, wherein the first expression repressor further comprises a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the N-terminus of the first targeting moiety.

[0207] 130. The system of embodiment 128 or 129, wherein the first expression repressor further comprises a second nuclear localization signal, e.g., a nucleoprotein NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the C-terminus of the first effector portion.

[0208] 131. The system of any one of embodiments 128-130, wherein the second expression repressor further comprises a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located N-terminus of the second targeting moiety.

[0209] 132. The system of any one of embodiments 128-131, wherein the second expression repressor further comprises a second nuclear localization signal, e.g., a nucleoplasmic protein NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the C-terminus of the second targeting moiety.

[0210] 133. The system of any one of embodiments 128-132, wherein the first expression repressor further comprises a first linker between the first targeting moiety and the first effector moiety, wherein optionally the first linker has an amino acid sequence according to SEQ ID NO: 137 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0211] 134. The system of any one of embodiments 128-133, wherein the second expression repressor further comprises a second linker positioned between the second targeting moiety and the second effector moiety, wherein optionally the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0212] 135. The system of any one of embodiments 128-134, wherein the first expression repressor further comprises an amino acid sequence at the C-terminus of 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 a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity thereto.

[0213] 136. The system of any one of embodiments 128-132, wherein the second expression repressor further comprises an amino acid sequence at the N-terminus of the second targeting moiety, such as a sequence of up to 30, 25, 20, or 18 amino acids, or amino acid P.

[0214] 137. The system of any one of embodiments 128-136, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0215] 138. The system of any one of embodiments 128-137, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0216] 139. The system of any one of embodiments 128-137, 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.

[0217] 140. The system of any one of embodiments 128-137, 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.

[0218] 141. The system of any one of embodiments 128-140, 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.

[0219] 142. The system of any one of embodiments 128-140, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 179, 185, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0220] 143. A nucleic acid encoding a first expression repressor and a second repressor of the system of any one of embodiments 128-142.

[0221] 144. A nucleic acid encoding a system for reducing MYC expression, the nucleic acid comprising:

[0222] a) a first region encoding a first expression repressor, the first expression repressor comprising:

[0223] 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 that differs therefrom in 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

[0224] 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 that differs therefrom in 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

[0225] b) a second region encoding a second expression repressor, the second expression repressor comprising:

[0226] i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs therefrom in 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

[0227] 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 that 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.

[0228] 145. The nucleic acid of embodiment 144, wherein the first region is 5' of the second region.

[0229] 146. The nucleic acid of embodiment 144, wherein the first region is 3' to the second region.

[0230] 147. The nucleic acid of embodiment 145 or 146, wherein the first region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the N-terminus of the first targeting moiety.

[0231] 148. The nucleic acid of any one of embodiments 145-147, wherein the first region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmic protein NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located C-terminus of the first effector portion.

[0232] 149. The nucleic acid of any one of embodiments 145-148, wherein the second region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the N-terminus of the second targeting portion.

[0233] 150. The nucleic acid of any one of embodiments 145-149, wherein the second region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmic protein NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the C-terminus of the second effector portion.

[0234] 151. The nucleic acid of any one of embodiments 145-150, wherein the first region further comprises a nucleotide sequence encoding a first linker positioned between the first targeting portion and the first effector portion, wherein optionally the first linker has an amino acid sequence according to SEQ ID NO: 137 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0235] 152. The nucleic acid of any one of embodiments 145-151, wherein the second region further comprises a nucleotide sequence encoding a second linker positioned between the second targeting portion and the second effector portion, wherein optionally the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0236] 153. The nucleic acid of any one of embodiments 145-152, wherein the first region further comprises a nucleotide sequence encoding an amino acid sequence at the C-terminus of the first effector portion, which amino acid sequence is, for example, a sequence of up to 30, 25, 20 or 18 amino acids, such as a sequence according to SEQ ID NO: 126, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0237] 154. The nucleic acid of any one of embodiments 145-153, wherein the second region further comprises a nucleotide sequence encoding an amino acid sequence at the N-terminus of the second targeting portion, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, or amino acid P.

[0238] 155. The nucleic acid of any one of embodiments 145-154, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0239] 156. The nucleic acid of any one of embodiments 145-155, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0240] 157. The nucleic acid of any of embodiments 145-156, wherein the first region comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 46 or 131, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs from it in 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.

[0241] 158. The nucleic acid of any one of embodiments 145-157, wherein the first region comprises a nucleotide sequence encoding the first effector portion, wherein the nucleotide sequence encoding the first effector portion 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.

[0242] 159. The nucleic acid of any one of embodiments 145-158, wherein the second region comprises a nucleotide sequence encoding the second targeting portion, wherein the nucleotide sequence encoding the second targeting portion comprises a sequence according to SEQ ID NO:40, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs from it in 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.

[0243] 160. The nucleic acid of any one of embodiments 145-159, wherein the first region comprises a nucleotide sequence encoding the first effector portion, wherein the nucleotide sequence encoding the first effector portion comprises a sequence according to SEQ ID NO: 51, 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.

[0244] 161. The nucleic acid of any one of embodiments 145-160, wherein the first region comprises a nucleotide sequence according to SEQ ID NO: 63 or 130, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that differs therefrom in 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 poly A sequence is optional.

[0245] 162. The nucleic acid of any one of embodiments 145-161, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 57, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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 poly A sequence is optional.

[0246] 163. A nucleic acid encoding a system for reducing MYC expression, the nucleic acid comprising:

[0247] a) a first region encoding a first expression repressor, the first expression repressor comprising:

[0248] 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 that differs therefrom in 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

[0249] 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 that differs therefrom in 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

[0250] b) a second region encoding a second expression repressor, the second expression repressor comprising:

[0251] i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 169, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs therefrom in 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

[0252] 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 that 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.

[0253] 164. A nucleic acid encoding a system for reducing MYC expression, the nucleic acid comprising:

[0254] a) a first region encoding a first expression repressor, the first expression repressor comprising:

[0255] 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 that differs therefrom in 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

[0256] 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 that differs therefrom in 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

[0257] b) a second region encoding a second expression repressor, the second expression repressor comprising:

[0258] i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 171, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs therefrom in 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

[0259] 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 that 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.

[0260] 165. The nucleic acid of embodiment 163 or 164, wherein the first region is 5' of the second region.

[0261] 166. The nucleic acid of embodiment 163 or 164, wherein the first region is 3' to the second region.

[0262] 167. The nucleic acid of any one of embodiments 163-166, wherein the first region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the N-terminus of the first targeting portion.

[0263] 168. The nucleic acid of any one of embodiments 163-167, wherein the first region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmic protein NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the C-terminus of the first effector portion.

[0264] 169. The nucleic acid of any one of embodiments 163-168, wherein the second region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the N-terminus of the second targeting portion.

[0265] 170. The nucleic acid of any one of embodiments 163-169, wherein the second region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmic protein NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, e.g., located at the C-terminus of the second effector portion.

[0266] 171. The nucleic acid of any one of embodiments 163-170, wherein the first region further comprises a nucleotide sequence encoding a first linker positioned between the first targeting portion and the first effector portion, wherein optionally the first linker has an amino acid sequence according to SEQ ID NO: 137 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0267] 172. The nucleic acid of any one of embodiments 163-171, wherein the second region further comprises a nucleotide sequence encoding a second linker positioned between the second targeting portion and the second effector portion, wherein optionally the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0268] 173. The nucleic acid of any one of embodiments 163-171, wherein the first region further comprises a nucleotide sequence encoding an amino acid sequence at the C-terminus of the first effector portion, which amino acid sequence is, for example, a sequence of up to 30, 25, 20 or 18 amino acids, such as a sequence according to SEQ ID NO: 126, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0269] 174. The nucleic acid of any one of embodiments 163-173, wherein the second region further comprises a nucleotide sequence encoding an amino acid sequence at the N-terminus of the second targeting portion, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, or amino acid P.

[0270] 175. The nucleic acid of any one of embodiments 163-174, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto.

[0271] 176. The nucleic acid of any one of embodiments 144-175, 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.

[0272] 177. The nucleic acid of any one of embodiments 144-176, 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.

[0273] 178. The nucleic acid of any one of embodiments 144-177, wherein 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%, 99% or 100% identical thereto, and the second expression repressor 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.

[0274] 179. The nucleic acid of any one of embodiments 144-178, wherein the first region comprises a nucleotide sequence encoding the first targeting portion, wherein the nucleotide sequence encoding the first targeting portion comprises a sequence according to SEQ ID NO: 46 or 131, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs from it in 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.

[0275] 180. The nucleic acid of any one of embodiments 144-179, wherein the first region comprises a nucleotide sequence encoding the first effector portion, wherein the nucleotide sequence encoding the first effector portion 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.

[0276] 181. The nucleic acid of any one of embodiments 144-180, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 173, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that differs therefrom in 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 poly A sequence is optional.

[0277] 182. The nucleic acid of any one of embodiments 144-181, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 175, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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 poly A sequence is optional.

[0278] 183. The nucleic acid of any one of embodiments 144-182, wherein the second region comprises a nucleotide sequence encoding the second effector portion, wherein the nucleotide sequence encoding the second effector portion comprises a sequence according to SEQ ID NO: 51, 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.

[0279] 184. The nucleic acid of any one of embodiments 144-183, wherein the first region comprises a nucleotide sequence according to SEQ ID NO: 63 or 130, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that differs therefrom in 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 poly A sequence is optional.

[0280] 185. The nucleic acid of any one of embodiments 144-184, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 189, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that differs therefrom in 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 poly A sequence is optional.

[0281] 186. The nucleic acid of any one of embodiments 144-185, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 194, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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 the poly A sequence is optional

[0282] 187. The nucleic acid of any one of embodiments 144-186, wherein the first region comprises a nucleotide sequence encoding the first effector portion, wherein the nucleotide sequence encoding the first effector portion 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.

[0283] 188. The nucleic acid of any of embodiments 144-187, wherein the first region comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 46 or 131, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that differs from it in 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.

[0284] 189. The nucleic acid of any one of embodiments 144-188, wherein the second region comprises a nucleotide sequence encoding the second effector portion, wherein the nucleotide sequence encoding the second effector portion comprises a sequence according to SEQ ID NO: 51, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that differs from it 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.

[0285] 190. The nucleic acid of any one of embodiments 144-189, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 189, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0286] 191. The nucleic acid of any one of embodiments 144-190, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 194, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0287] 192. The nucleic acid of any one of embodiments 144-191, having a nucleotide sequence according to SEQ ID NO: 93, 112, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0288] 193. The nucleic acid of any one of embodiments 144-192, having a nucleotide sequence according to SEQ ID NO: 196 or 197, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0289] 194. The system or nucleic acid of any one of embodiments 118-193, wherein the first expression repressor comprises the first effector portion.

[0290] 195. The system or nucleic acid of any one of embodiments 118-194, wherein the second expression repressor comprises the second effector portion.

[0291] 196. The system or nucleic acid of any of embodiments 118-195, wherein the first effector moiety has a different amino acid sequence than the second effector moiety.

[0292] 197. The system or nucleic acid of any one of embodiments 118-196, wherein the first effector moiety is a persistent effector moiety.

[0293] 198. The system or nucleic acid of any one of embodiments 118-125 or 144-197, wherein the first effector moiety is a transient effector moiety.

[0294] 199. The system or nucleic acid of any one of embodiments 118-198, wherein the first effector moiety is an epigenetic modification moiety.

[0295] 200. The system or nucleic acid of any one of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone methyltransferase.

[0296] 201. The system or nucleic acid of embodiment 200, wherein the first effector portion comprises a protein selected from the group consisting of SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any one thereof, e.g., a SET domain of any one thereof.

[0297] 202. The system or nucleic acid of any one of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone demethylase (eg, a lysine demethylase).

[0298] 203. The system or nucleic acid of embodiment 202, wherein the first effector portion comprises a protein selected from the group consisting of KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or fragment of any one thereof).

[0299] 204. The system or nucleic acid of any one of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone deacetylase.

[0300] 205. The system or nucleic acid of embodiment 204, wherein the first effector portion comprises a protein selected from the group consisting of: 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 one thereof.

[0301] 206. The system or nucleic acid of any one of embodiments 118-197 or 200, wherein the first effector moiety comprises a DNA methyltransferase.

[0302] 207. The system or nucleic acid of embodiment 206, wherein the first effector portion comprises a protein selected from the group consisting of: MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any one thereof.

[0303] 208. The system or nucleic acid of any one of embodiments 118-143, 160-196, or 198, wherein the first effector moiety is a transcriptional repressor moiety, eg, comprises a transcriptional repressor.

[0304] 209. The system or nucleic acid of embodiment 198 or 199, wherein the first effector moiety comprises a protein selected from the group consisting of KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any one thereof.

[0305] 210. The system or nucleic acid of any one of embodiments 118-209, wherein the first effector moiety promotes epigenetic modification of a transcriptional regulatory element or a proximal sequence thereof.

[0306] 211. The system or nucleic acid of any one of embodiments 118-210, wherein the first effector portion catalyzes epigenetic modification of a transcriptional regulatory element or a proximal sequence thereof.

[0307] 212. The system or nucleic acid of any one of embodiments 118-125, 194, or 197-211, wherein the second expression repressor does not comprise an effector portion.

[0308] 213. The system or nucleic acid of any one of embodiments 118-212, wherein the second effector moiety is a transient effector moiety.

[0309] 214. The system or nucleic acid of any one of embodiments 118-125 or 194-211, wherein the second effector moiety is a persistent effector moiety.

[0310] 215. The system or nucleic acid of any one of embodiments 118-211 or 214, wherein the second effector moiety is an epigenetic modification moiety.

[0311] 216. The system or nucleic acid of any one of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone methyltransferase.

[0312] 217. The system or nucleic acid of embodiment 216, wherein the second effector portion comprises a protein selected from the group consisting of SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any one thereof, e.g., a SET domain of any one thereof.

[0313] 218. The system or nucleic acid of any one of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone demethylase (eg, a lysine demethylase).

[0314] 219. The system or nucleic acid of embodiment 218, wherein the second effector portion comprises a protein selected from the group consisting of KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or fragment of any one thereof).

[0315] 220. The system or nucleic acid of any one of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone deacetylase.

[0316] 221. The system or nucleic acid of embodiment 220, wherein the second effector portion comprises a protein selected from the group consisting of: 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 one thereof.

[0317] 222. The system or nucleic acid of any one of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a DNA methyltransferase.

[0318] 223. The system or nucleic acid of embodiment 222, wherein the second effector portion comprises a protein selected from the group consisting of: MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any one thereof.

[0319] 224. The system or nucleic acid of any one of embodiments 118-211 or 213, wherein the second effector moiety is a transcriptional repressor moiety.

[0320] 225. The system or nucleic acid of embodiment 224, wherein the second effector moiety promotes epigenetic modification of the anchor sequence or a sequence proximal thereto.

[0321] 226. The system or nucleic acid of embodiment 223 or 224, wherein the second effector moiety binds one or more endogenous epigenetic modifying proteins or one or more endogenous transcriptional modifying proteins.

[0322] 227. The system or nucleic acid of any one 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 one thereof.

[0323] 228. The system or nucleic acid of any one of embodiments 118-197, 199-207, 210-211, 213, or 224-227, wherein:

[0324] The first effector moiety is a persistent effector moiety, and

[0325] The second effector moiety is a transient effector moiety.

[0326] 229. The system or nucleic acid of embodiment 228, wherein the first effector moiety is an epigenetic modification moiety.

[0327] 230. The system or nucleic acid of embodiment 227 or 228, wherein the second effector moiety is a transcriptional repressor moiety.

[0328] 231. The system or nucleic acid of any one of embodiments 227-230, wherein:

[0329] The first effector moiety comprises a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any one thereof, or any combination thereof, and

[0330] The second effector moiety comprises a transcriptional repressor or a functional variant or fragment of any thereof.

[0331] 232. The system or nucleic acid of any one of embodiments 118-125, 194, 197, 199-207, 210-212, or 190, wherein:

[0332] The first effector moiety comprises a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any one thereof, or any combination thereof, and

[0333] The second expression repressor does not comprise a second effector moiety.

[0334] 233. The system or nucleic acid of any one of embodiments 118-125, 199-207, 210-211, 213, 214, or 224-231, wherein:

[0335] The first effector portion comprises 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, HDA C5, 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 one thereof, or any combination thereof, and

[0336] The second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, a functional variant or fragment of any one thereof, or any combination thereof.

[0337] 234. The system or nucleic acid of any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, or 233, wherein:

[0338] The first effector moiety comprises a DNA methyltransferase, and

[0339] The second effector moiety comprises a transcriptional repressor.

[0340] 235. The system or nucleic acid of any one of embodiments 118-125, 194, 197, 200, 206-207, 210-212, or 232, wherein:

[0341] The first effector moiety comprises a DNA methyltransferase, and

[0342] The second expression repressor does not comprise a second effector moiety.

[0343] 236. The system or nucleic acid of any one of embodiments 118-125, 200, 206-207, 210-235, wherein the first effector portion comprises MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any one thereof.

[0344] 237. The system or nucleic acid of any one of embodiments 118-211, 214, 224-234, or 236, wherein the second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any one thereof.

[0345] 238. The system or nucleic acid of any one of embodiments 118-211, 199, 206-207, 210-211, 213, 224-234, or 236-237, wherein:

[0346] The first effector portion comprises MQ1 or a functional variant or fragment thereof, and

[0347] The second effector comprises KRAB or a functional variant or fragment of any one thereof.

[0348] 239. The system or nucleic acid of any one of embodiments 118-125, 194, 197, 199-207, or 210-212, 229, 232, 235, or 236, wherein:

[0349] The first effector portion comprises MQ1 or a functional variant or fragment thereof, and

[0350] The second expression repressor does not comprise a second effector moiety.

[0351] 240. The system or nucleic acid of any of embodiments 118-200, wherein the first expression repressor comprises an amino acid sequence selected from any of SEQ ID NOs: 22-33, 129, 133, 134, 139-149, 177-180, or 183-186 herein, or SEQ ID NOs: 34-37 described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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.

[0352] 241. The system or nucleic acid of any one of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240, wherein the second expression repressor comprises an amino acid sequence selected from any of SEQ ID NOs: 22-33, 129, 133, 134, 139-149, 177-180, or 183-186 herein, or SEQ ID NOs: 34-37 as described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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.

[0353] 242. The system or nucleic acid of any one of embodiments 118-198, 200, 206-211, 213-216, 222, -223, 236-237, or 240-241, wherein: wherein the first expression repressor comprises the amino acid sequence of SEQ ID NO: 30, 129, 133, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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 the second expression repressor comprises SEQ ID NO: or an amino acid sequence of 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 a sequence that 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.

[0354] 243. The system or nucleic acid of any one of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240-242, wherein the first expression repressor is encoded by the first nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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 the second expression repressor is encoded by the first nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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, or a second nucleotide sequence of NO:57, 189 or 194, or a sequence at least 80%, 85%, 90%, 95% or 99% identical thereto, or encoding a sequence that 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.

[0355] 244. The system or nucleic acid of any one of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240-243, wherein the first repressor and the second repressor are encoded by a nucleic acid sequence of SEQ ID NO: 93, 94, 112, 113, 196, or 197, or a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical thereto, or that 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.

[0356] 245. The system or nucleic acid of embodiment 244, comprising 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 sequence that 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.

[0357] 246. The system or nucleic acid of any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, wherein:

[0358] The first expression repressor comprises, from N-terminus to C-terminus:

[0359] (i) a first nuclear localization signal, such as the SV40 NLS; for example, a sequence according to SEQ ID NO: 135;

[0360] (ii) a first targeting moiety, such as a zinc finger binding domain, such as ZF9; for example, a sequence according to SEQ ID NO: 13;

[0361] (iii) a first effector moiety, such as a DNA methyltransferase, such as MQ1; for example, a sequence according to SEQ ID NO: 19 or 87;

[0362] (iv) a second nuclear localization signal, such as a nucleoplasmin NLS; for example, a sequence according to SEQ ID NO: 136;

[0363] And the second expression repressor comprises from N-terminus to C-terminus:

[0364] (v) a third nuclear localization signal, such as SV40 NLS; for example, a sequence according to SEQ ID NO: 135;

[0365] (vi) a second targeting moiety, such as a zinc finger binding domain, such as ZF3; for example, a sequence according to SEQ ID NO: 7;

[0366] (vii) a second effector moiety, such as KRAB, such as a sequence according to SEQ ID NO: 18; and

[0367] (viii) a fourth nuclear localization signal, such as a nucleoplasmin NLS, such as a sequence according to SEQ ID NO: 136.

[0368] 247. The system or nucleic acid of any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, wherein:

[0369] The first expression repressor comprises, from N-terminus to C-terminus:

[0370] (i) a first nuclear localization signal, such as the SV40 NLS; for example, a sequence according to SEQ ID NO: 135;

[0371] (ii) a first targeting moiety, such as a zinc finger binding domain, such as ZF9; for example, a sequence according to SEQ ID NO: 13;

[0372] (iii) a first effector moiety, such as a DNA methyltransferase, such as MQ1; for example, a sequence according to SEQ ID NO: 19 or 87;

[0373] (iv) a second nuclear localization signal, such as a nucleoplasmin NLS; for example, a sequence according to SEQ ID NO: 136;

[0374] And the second expression repressor comprises from N-terminus to C-terminus:

[0375] (v) a third nuclear localization signal, such as SV40 NLS; for example, a sequence according to SEQ ID NO: 135;

[0376] (vi) a second targeting moiety, such as a zinc finger binding domain, such as ZF54; for example, a sequence according to SEQ ID NO: 169;

[0377] (vii) a second effector moiety, such as KRAB, such as a sequence according to SEQ ID NO: 18; and

[0378] (viii) a fourth nuclear localization signal, such as a nucleoplasmin NLS, such as a sequence according to SEQ ID NO: 136.

[0379] 248. The system or nucleic acid of any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, wherein:

[0380] The first expression repressor comprises, from N-terminus to C-terminus:

[0381] (i) a first nuclear localization signal, such as the SV40 NLS; for example, a sequence according to SEQ ID NO: 135;

[0382] (ii) a first targeting moiety, such as a zinc finger binding domain, such as ZF9; for example, a sequence according to SEQ ID NO: 13;

[0383] (iii) a first effector moiety, such as a DNA methyltransferase, such as MQ1; for example, a sequence according to SEQ ID NO: 19 or 87;

[0384] (iv) a second nuclear localization signal, such as a nucleoplasmin NLS; for example, a sequence according to SEQ ID NO: 136;

[0385] And the second expression repressor comprises from N-terminus to C-terminus:

[0386] (v) a third nuclear localization signal, such as SV40 NLS; for example, a sequence according to SEQ ID NO: 135;

[0387] (vi) a second targeting moiety, such as a zinc finger binding domain, such as ZF67; for example, a sequence according to SEQ ID NO: 171;

[0388] (vii) a second effector moiety, such as KRAB, such as a sequence according to SEQ ID NO: 18; and

[0389] (viii) a fourth nuclear localization signal, such as a nucleoplasmin NLS, such as a sequence according to SEQ ID NO: 136.

[0390] 249. The system of any one of embodiments 118-248, wherein the system is capable of reducing MYC expression to a greater extent than the first expression repressor alone or the second expression repressor alone.

[0391] 250. The system of any one of embodiments 128-194 or 242-249, wherein the system, alone or in combination, is capable of reducing MYC expression to a greater extent than any of the expression repressors of SEQ ID NOs: 22, 23, 25-29, 31-33 herein or SEQ ID NOs: 34-37 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0392] 251. The system of any one of embodiments 118-250, which is capable of reducing tumor volume, e.g., in a human subject or a mammalian model.

[0393] 252. The system of any of embodiments 128-193 or 242-209, wherein the system is capable of reducing tumor volume to a similar or greater extent than a chemotherapeutic agent, e.g., in a mammalian model, e.g., when measured 20 days after initiation of treatment, e.g., wherein the expression repressor is administered at a dose of 3 mg / kg every 5 days, e.g., in a model system as described in Example 15 of international application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0394] 253. The system of any of embodiments 128-193 or 242-252, wherein the system is capable of reducing tumor volume to a greater extent than a chemotherapeutic agent, e.g., in a mammalian model, e.g., when measured 15 days after initiation of treatment, e.g., wherein the expression repressor is administered at a dose of 6 mg / kg every 5 days, e.g., in a model system as described in Example 14 of international application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0395] 254. The system of any one of embodiments 128-193 or 242-253, wherein tumor volume is reduced by at least about 10%, 20%, 30%, 40%, 50% or 60% compared to a control treated with PBS, e.g., at day 20 after initiation of treatment.

[0396] 255. The system of embodiment 254, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0397] 256. The system of any one of embodiments 128-193 or 242-253, wherein the system is capable of reducing tumor volume to a similar or greater extent than a small molecule MYC inhibitor.

[0398] 257. The system of embodiment 256, wherein the small molecule MYC inhibitor is MYCi975, wherein optionally, tumor volume is reduced by at least about 10%, 20%, 30% or 40% compared to a control treated with MYCi975, e.g., at day 20 after initiation of treatment.

[0399] 258. The system of any one of embodiments 118-257, which causes no weight loss, or causes less than 3%, 2% or 1% weight loss compared to the start of treatment.

[0400] 259. The system or nucleic acid of any one of embodiments 118-258, wherein the first targeting moiety is selected from a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0401] 260. The system or nucleic acid of any one of embodiments 118-260, wherein the second targeting moiety is selected from a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0402] 261. The system or nucleic acid of any one of embodiments 118-260, wherein the first targeting moiety comprises a CRISPR / Cas domain (e.g., a first CRISPR / Cas domain).

[0403] 262. The system or nucleic acid of any one of embodiments 118-261, wherein the second targeting moiety comprises a second CRISPR / Cas domain (e.g., a second CRISPR / Cas domain).

[0404] 263. The system or nucleic acid of embodiment 262, wherein: i) the first CRISPR / Cas domain binds a first guide RNA, ii) the second CRISPR / Cas domain binds a second guide RNA, or iii) both (i) and (ii).

[0405] 264. The system or nucleic acid of embodiment 262 or 263, wherein the first CRISPR / Cas domain does not bind the second guide RNA or binds to the second guide RNA with a K of at least 10, 20, 50, 100, 1000, or 10,000 nM. D The second CRISPR / Cas domain binds to the first guide RNA, and the second CRISPR / Cas domain does not bind to the first guide RNA or binds to the first guide RNA with a K of at least 10, 20, 50, 100, 1000, or 10,000 nM. D combination.

[0406] 265. The system or nucleic acid of any one of embodiments 260-264, wherein the first CRISPR / Cas domain comprises an amino acid sequence that is different from the second CRISPR / Cas domain.

[0407] 266. The system or nucleic acid of any one of embodiments 260-265, wherein the first or second CRISPR / Cas domain comprises an amino acid sequence selected from the Cas protein or Cpf1 protein of Table 1 described in International Application WO / 2022 / 132195, or a variant (e.g., mutant) of either thereof.

[0408] 267. The system or nucleic acid of any of embodiments 260-266, wherein the first CRISPR / Cas domain comprises an amino acid sequence of a Cas protein or Cpf1 protein selected from Table 1 as described in international application WO / 2022 / 132195, or any variant (e.g., mutant) thereof, and the second CRISPR / Cas domain comprises an amino acid sequence of a different Cas protein or Cpf1 protein selected from Table 1 as described in international application WO / 2022 / 132195, or any variant (e.g., mutant) thereof.

[0409] 268. The system or nucleic acid of any one of embodiments 118-260, wherein the first targeting moiety comprises a zinc finger domain (e.g., a first zinc finger domain).

[0410] 269. The system or nucleic acid of any one of embodiments 118-260 or 268, wherein the second targeting moiety comprises a zinc finger domain (e.g., a second zinc finger domain).

[0411] 270. The system or nucleic acid of any one 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.

[0412] 271. The system or nucleic acid of any one of embodiments 268-270, wherein the first zinc finger domain and the second zinc finger domain bind to the same genomic locus, eg, have the same amino acid sequence.

[0413] 272. The system or nucleic acid of any one of embodiments 268-271, wherein the first zinc finger domain and the second zinc finger domain have different amino acid sequences or bind different genomic loci.

[0414] 273. The system or nucleic acid of any one of embodiments 118-261 or 267-272, wherein the first zinc finger molecule comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally no more than 11, 10, 9, 8, 7, 6, or 5 zinc fingers).

[0415] 274. The system or nucleic acid of any one of embodiments 267-273, wherein the first zinc finger molecule comprises 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, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers.

[0416] 275. The system or nucleic acid of any one of embodiments 268-274, wherein the first zinc finger domain comprises 3 or 9 zinc fingers.

[0417] 276. The system or nucleic acid of any one of embodiments 268-275, wherein the second zinc finger domain comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally no more than 11, 10, 9, 8, 7, 6, or 5 zinc fingers).

[0418] 277. The system or nucleic acid of any one of embodiments 268-276, wherein the second zinc finger domain comprises 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, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers.

[0419] 278. The system or nucleic acid of any one of embodiments 268-277, wherein the second zinc finger domain comprises 3 or 9 zinc fingers.

[0420] 279. The system or nucleic acid of any one of embodiments 118-278, wherein the first targeting moiety comprises a TAL effector domain (e.g., a first TAL effector domain).

[0421] 280. The system or nucleic acid of any one of embodiments 118-260 or 279, wherein the second targeting moiety comprises a TAL effector domain (eg, a second TAL effector domain).

[0422] 281. The system or nucleic acid of any of embodiments 279 or 280, wherein the first TAL effector domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, no more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).

[0423] 282. The system or nucleic acid of any of embodiments 279-281, wherein the first TAL effector domain comprises 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 2-5 central repeats.

[0424] 283. The system or nucleic acid of any of embodiments 279-282, wherein the second TAL effector domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, no more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).

[0425] 284. The system or nucleic acid of any of embodiments 279-283, wherein the second TAL effector domain comprises 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 2-5 central repeats.

[0426] 285. The system or nucleic acid of any one of embodiments 118-284, wherein the first targeting moiety comprises a nucleic acid (e.g., a first nucleic acid).

[0427] 286. The system of any one of embodiments 129-285, wherein the second targeting moiety comprises a nucleic acid (e.g., a second nucleic acid).

[0428] 287. The system or nucleic acid of any one of embodiments 129-286, wherein the first targeting moiety comprises a polypeptide (e.g., a first polypeptide).

[0429] 288. The system or nucleic acid of any one of embodiments 129-287, wherein the second targeting moiety comprises a polypeptide (e.g., a second polypeptide).

[0430] 289. The system of embodiment 287 or 288, wherein the nucleic acid is covalently linked to the polypeptide.

[0431] 290. The system of embodiment 288 or 289, wherein the nucleic acid is non-covalently bound to the polypeptide.

[0432] 291. The system or nucleic acid of any of embodiments 275-290, wherein the nucleic acid comprises a sequence that is complementary to the transcriptional regulatory element or a sequence proximal thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches relative to the transcriptional regulatory element or a sequence proximal thereto.

[0433] 292. The system or nucleic acid of any one of embodiments 275-291, wherein the nucleic acid comprises a sequence that is complementary to an anchor sequence or a sequence proximal thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches relative to the anchor sequence or a sequence proximal thereto.

[0434] 293. The system of any one of embodiments 275-292, wherein the nucleic acid comprises DNA, peptide nucleic acid (PNA), peptide-oligonucleotide conjugate, locked nucleic acid (LNA), bridged nucleic acid (BNA), polyamide, triplex-forming oligonucleotide, antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecule.

[0435] 294. The system of any one of embodiments 275-293, wherein the nucleic acid comprises gRNA.

[0436] 295. The system of any of embodiments 275-294, wherein the nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 1-4, or that differs therefrom at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions.

[0437] 296. The system of any of embodiments 275-295, wherein the first nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 1-4, or differs from it in no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions, and the second nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 1-4, or differs from it in no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions.

[0438] 297. The system of any of embodiments 275-295, wherein the first nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 96-110, or differs from it at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions, and the second nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical to any one of SEQ ID NOs: 96-110, or differs from it at no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions.

[0439] 298. The system of any one of embodiments 118-297, wherein the transcriptional regulatory element comprises a promoter.

[0440] 299. The system of any one of embodiments 118-298, wherein the transcriptional regulatory element comprises an enhancer; e.g., a super enhancer.

[0441] 300. The system of any one of embodiments 118-299, wherein the anchor sequence comprises a CTCF binding motif.

[0442] 301. The system of any one of embodiments 118-300, wherein the anchor sequence comprises a YY1 binding motif, such as described in international application WO / 2022 / 132195 (incorporated herein by reference in its entirety).

[0443] 302. The system of any one of embodiments 118-301, wherein the anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations thereto.

[0444] 303. The system of any one of embodiments 118-302, wherein the anchor sequence comprises the sequence of SEQ ID NO: 73 or 74 as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference), or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 changes thereto.

[0445] 304. The system of any one of embodiments 118-303, wherein the anchor sequence is located on the same chromosome as the MYC gene.

[0446] 305. The system of any one of embodiments 118-304, wherein the anchor sequence is upstream of the MYC gene (eg, upstream of the TSS or upstream of the promoter).

[0447] 306. The system of any one of embodiments 118-305, wherein the anchor sequence is at least 1, 5, 10, 50, 100, or 1000 kilobases from the MYC gene (eg, from the TSS or promoter of the MYC gene).

[0448] 307. The system of any one of embodiments 118-306, wherein the anchor sequence is 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 or 500-1000 kilobases.

[0449] 308. The system of any one of embodiments 118-303 or 305-307, wherein the anchor sequence and the MYC gene are on different chromosomes.

[0450] 309. The system of any of embodiments 118-308, wherein the second targeting moiety binds to the anchor sequence or a sequence proximal to the anchor sequence with an affinity sufficient to compete with an endogenous polypeptide (e.g., CTCF or YY1) for binding.

[0451] 310. The system of any one of embodiments 118-309, wherein the first targeting moiety binds to a sequence at or proximal to chromosome coordinates 128746342-128746364, 128746321-128746343, or 128746525-128746547.

[0452] 311. The system of any one of embodiments 118-309, wherein the first targeting moiety binds to a sequence at or proximal to chromosome coordinates 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842.

[0453] 312. The system of any one of embodiments 118-311, wherein the second targeting moiety binds to a sequence at chromosome coordinates 128748014-128748036 or a sequence proximal thereto.

[0454] 313. The system of any one of embodiments 118-311, wherein the second targeting moiety binds to a sequence at or proximal to chromosome coordinates 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842.

[0455] 314. The system of any one of embodiments 118-314, wherein the first expression repressor is a fusion molecule.

[0456] 315. The system of any one of embodiments 118-314, wherein the second expression repressor is a fusion molecule.

[0457] 316. The system of any one of embodiments 118-315, wherein the first expression repressor comprises a linker.

[0458] 317. The system of any one of embodiments 118-316, wherein the second expression repressor comprises a linker.

[0459] 318. The system of any one of embodiments 118-267 or 285-317, wherein:

[0460] The first expression repressor comprises a targeting moiety and an effector moiety, the targeting moiety comprising a first CRISPR / Cas molecule, for example comprising a first catalytically inactive CRISPR / Cas protein, and the effector moiety comprising an epigenetic modification moiety; and

[0461] The second expression repressor comprises a targeting moiety comprising a second CRISPR / Cas molecule, e.g., comprising a second catalytically inactive CRISPR / Cas protein, and an effector moiety optionally comprising a transcriptional repressor.

[0462] 319. The system of any one of embodiments 118-260, 268-278, or 285-317, wherein:

[0463] The first expression repressor comprises a targeting portion comprising a first zinc finger domain and an effector portion comprising an epigenetic modification portion; and

[0464] The second expression repressor comprises a targeting portion comprising a second zinc finger domain and an optional effector portion comprising a transcriptional repressor.

[0465] 320. The system of any one of embodiments 118-120, 262, 268, or 275-318, wherein:

[0466] The first expression repressor comprises a targeting moiety and an effector moiety, wherein the targeting moiety comprises a CRISPR / Cas molecule, for example, a catalytically inactive CRISPR / Cas protein, and the effector moiety comprises an epigenetic modification moiety; and

[0467] The second expression repressor comprises a targeting portion comprising a zinc finger domain and an optional effector portion comprising a transcriptional repressor.

[0468] 321. The system of any one of embodiments 118-260, 268, or 275-318, wherein:

[0469] The first expression repressor comprises a targeting portion comprising a zinc finger domain and an effector portion comprising an epigenetic modification portion; and

[0470] 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.

[0471] 322. The system of any one of embodiments 260, 268-278, or 275-318, wherein the zinc finger domain (e.g., a first or second zinc finger domain) comprises 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, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers, such as 3 or 9 zinc fingers.

[0472] 323. The system of any one of embodiments 322, wherein the epigenetic modification moiety comprises a DNA methyltransferase.

[0473] 324. The system of any one of embodiments 118-323, wherein the epigenetic modification portion comprises MQ1 or a functional variant or fragment thereof.

[0474] 325. The system of any one of embodiments 118-324, wherein the second expression repressor comprises an effector portion comprising a transcriptional repressor.

[0475] 326. The system of any one of embodiments 118-323, wherein the transcriptional repressor comprises KRAB or a functional variant or fragment thereof.

[0476] 327. The system of any one of embodiments 118-326, wherein the first expression repressor comprises the amino acid sequence of any one of SEQ ID NOs: 28-33, 145-149, 151, or 152 herein or SEQ ID NOs: 35-37 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference), 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.

[0477] 328. The system of any one of embodiments 118-327, wherein the second expression repressor comprises the amino acid sequence of any one of SEQ ID NOs: 22-27, 139-144, 150, 177-180, 183-186 herein or SEQ ID NO: 34 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference), 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.

[0478] 329. The system of any one of embodiments 118-328, wherein binding of the first expression repressor to the transcriptional regulatory element or a sequence proximal thereto reduces expression of MYC in the cell.

[0479] 330. The system of embodiment 327, wherein expression is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to expression in the absence of the first expression repressor, e.g., as measured by QPCR or ELISA.

[0480] 331. The system of embodiment 326 or 327, wherein binding of the first expression repressor to the transcriptional regulatory element results in a significant reduction in expression of MYC for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA.

[0481] 332. The system of any of embodiments 329-331, wherein binding of the first expression repressor to the transcriptional regulatory element results in a significant decrease in 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 post-transfection.

[0482] 333. The system of any one of embodiments 328-332, wherein binding of the second expression repressor to the anchor sequence or a sequence proximal thereto reduces expression of MYC in the cell.

[0483] 334. The system of embodiment 333, wherein expression is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to expression in the absence of the second expression repressor, e.g., as measured by QPCR or ELISA.

[0484] 335. The system of embodiment 333 or 334, wherein binding of the second expression repressor to the anchor sequence or its proximal sequence significantly reduces expression of MYC for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA.

[0485] 336. The system of any of embodiments 334-335, wherein binding of the second expression repressor to the anchor sequence or a sequence proximal thereto significantly 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 post-transfection.

[0486] 337. The system of any one of embodiments 329-336, wherein binding of the first expression repressor to the transcriptional regulatory element or a sequence proximal thereto and binding of the second expression repressor to the anchor sequence or a sequence proximal thereto reduces expression of MYC in the cell.

[0487] 338. The system of any of embodiments 329-337, wherein the binding of the first expression repressor to the transcriptional regulatory element or a sequence proximal thereto and the binding of the second expression repressor to the anchor sequence or a sequence proximal thereto significantly reduces the 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 post-transfection.

[0488] 339. The system of any of embodiments 337 or 338, wherein expression is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to expression in the absence of the first and second expression repressors, e.g., as measured by QPCR or ELISA.

[0489] 340. The system of any one of embodiments 329-339, wherein binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence and binding of the second expression repressor to the anchor sequence or its proximal sequence significantly reduces expression of MYC 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 for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA.

[0490] 341. The system of any of embodiments 329-340, wherein the reduction in expression caused by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence and binding of the second expression repressor to the anchor sequence or its proximal sequence is greater than the reduction in expression caused by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence or binding of the second expression repressor to the anchor sequence or its proximal sequence alone.

[0491] 342. The system of embodiment 341, wherein the reduction in expression by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence and binding of the second expression repressor to the anchor sequence or its proximal sequence is 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 the reduction in expression by either binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence or binding of the second expression repressor to the anchor sequence or its proximal sequence, e.g., as measured by QPCR or ELISA.

[0492] 343. The system of any of embodiments 329-342, wherein the reduction in expression caused by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence and binding of the second expression repressor to the anchor sequence or its proximal sequence persists for a longer period of time (e.g., more hours, days, or cell divisions) than the reduction in expression caused by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence or binding of the second expression repressor to the anchor sequence or its proximal sequence alone.

[0493] 344. The system of embodiment 343, wherein the reduction in expression by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence and binding of the second expression repressor to the anchor sequence or its proximal sequence is longer (e.g., measured in hours, days, or cell divisions) than the reduction in expression by either binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence or binding of the second expression repressor to the anchor sequence or its proximal sequence, e.g., as measured by QPCR or ELISA.

[0494] 345. The system of any one of embodiments 329-344, wherein binding of the first expression repressor to the promoter or its proximal sequence and binding of the second expression repressor to the super enhancer or its proximal sequence significantly reduces expression of MYC for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or 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 a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA.

[0495] 346. The system of any of embodiments 329-345, wherein the reduction in expression caused by binding of the first expression repressor to the promoter or its proximal sequence and binding of the second expression repressor to the super enhancer or its proximal sequence is greater than the reduction in expression caused by binding of the first expression repressor to the promoter or its proximal sequence or binding of the second expression repressor to the super enhancer or its proximal sequence alone.

[0496] 347. The system of embodiment 346, wherein the reduction in expression by binding of the first expression repressor to the promoter or its proximal sequence and binding of the second expression repressor to the super enhancer or its proximal sequence is 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 the reduction in expression by either binding of the first expression repressor to the promoter or its proximal sequence or binding of the second expression repressor to the super enhancer or its proximal sequence, e.g., as measured by QPCR or ELISA.

[0497] 348. The system of any of embodiments 329-347, wherein the reduction in expression caused by binding of the first expression repressor to the promoter or its proximal sequence and binding of the second expression repressor to the super enhancer or its proximal sequence persists for a longer period of time (e.g., more hours, days, or cell divisions) than the reduction in expression caused by binding of the first expression repressor to the promoter or its proximal sequence or binding of the second expression repressor to the super enhancer or its proximal sequence alone.

[0498] 349. The system of embodiment 348, wherein the reduction in expression by binding of the first expression repressor to the promoter or its proximal sequence and the binding of the second expression repressor to the super enhancer or its proximal sequence is longer (e.g., measured in hours, days, or cell divisions) than the reduction in expression by either binding of the first expression repressor to the promoter or its proximal sequence or binding of the second expression repressor to the super enhancer or its proximal sequence, e.g., as measured by QPCR or ELISA.

[0499] 350. The system of any one of embodiments 329-349, wherein expression is significantly reduced indefinitely (eg, for a period longer than can be measured experimentally).

[0500] 351. The system of any of embodiments 329-350, wherein binding of the first expression repressor to the transcriptional regulatory element or a proximal sequence thereof reduces the viability of a cell comprising the transcriptional regulatory element or a proximal sequence thereof.

[0501] 352. The system of any one of embodiments 329-351, wherein contacting a plurality of cells with the first expression repressor or the nucleic acid encoding the first expression repressor reduces the viability of the plurality of cells, optionally wherein the plurality of cells comprises cancerous and non-cancerous cells and / or infected and uninfected cells.

[0502] 353. The system of embodiment 352, wherein the viability is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the viability in the absence of the first expression repressor, e.g., as measured by CellTiter Glo.

[0503] 354. The system of any of embodiments 329-353, wherein administration of the first expression repressor results in apoptosis of at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

[0504] 355. The system of any one of embodiments 329-354, wherein binding of the second expression repressor to the anchor sequence or a sequence proximal thereto reduces the viability of a cell comprising the anchor sequence or a sequence proximal thereto.

[0505] 356. The system of any one of embodiments 329-355, wherein contacting a plurality of cells with the second expression repressor or the nucleic acid encoding the second expression repressor reduces viability of the plurality of cells.

[0506] 357. The system of any one of embodiments 329-356, wherein binding of the second expression repressor to the super enhancer or a sequence proximal thereto reduces the viability of a cell comprising the transcriptional regulatory element or a sequence proximal thereto.

[0507] 358. The system of any one of embodiments 329-357, wherein contacting a plurality of cells with the second expression repressor or the nucleic acid encoding the first expression repressor reduces the viability of the plurality of cells, optionally wherein the plurality of cells comprises cancerous and non-cancerous cells and / or infected and uninfected cells.

[0508] 359. The system of embodiment 358, wherein the viability is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the viability in the absence of the second expression repressor, e.g., as measured by CellTiter Glo.

[0509] 360. The system of any of embodiments 329-359, wherein administration of the second expression repressor results in apoptosis of at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

[0510] 361. The system of any of embodiments 329-360, wherein binding of the first expression repressor to the transcriptional regulatory element or a sequence proximal thereto and binding of the second expression repressor to the anchor sequence or a sequence proximal thereto reduces the viability of a cell comprising the anchor sequence or a sequence proximal thereto.

[0511] 362. The system of any one of embodiments 329-361, wherein binding of the first expression repressor to the promoter or a proximal sequence thereof and binding of the second expression repressor to the super enhancer or a proximal sequence thereof reduces cell viability

[0512] 363. The system of any one of embodiments 329-362, wherein contacting a plurality of cells with the system or a nucleic acid encoding the system reduces viability of the plurality of cells.

[0513] 364. The system of embodiments 329-363, wherein the viability is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the viability in the absence of the system, e.g., as measured by CellTiter Glo.

[0514] 365. The system of any of embodiments 329-364, wherein the reduction in motility caused by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence and binding of the second expression repressor to the anchor sequence or its proximal sequence is greater than the reduction in motility caused by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence or binding of the second expression repressor to the anchor sequence or its proximal sequence alone.

[0515] 366. The system of any of embodiments 329-365, wherein the reduction in motility caused by binding of the first expression repressor to the promoter or its proximal sequence and binding of the second expression repressor to the super enhancer or its proximal sequence is greater than the reduction in motility caused by binding of the first expression repressor to the promoter or its proximal sequence or binding of the second expression repressor to the super enhancer or its proximal sequence alone.

[0516] 367. The system of embodiment 366, wherein the reduction in viability by binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence and binding of the second expression repressor to the anchor sequence or its proximal sequence is 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 the reduction in viability by either binding of the first expression repressor to the transcriptional regulatory element or its proximal sequence or binding of the second expression repressor to the anchor sequence or its proximal sequence, e.g., as measured by CellTiter Glo.

[0517] 368. The system of embodiment 366 or 367, wherein the reduction in viability by binding of the first expression repressor to the promoter or its proximal sequence and binding of the second expression repressor to the super enhancer or its proximal sequence is 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 the reduction in viability by either binding of the first expression repressor to the promoter or its proximal sequence or binding of the second expression repressor to the super enhancer or its proximal sequence, e.g., as measured by CellTiter Glo.

[0518] 369. The system of any of embodiments 329-368, wherein administration of the first expression repressor and the second expression repressor results in apoptosis of at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

[0519] 370. The system of embodiments 329-369, wherein the plurality of cells comprises a plurality of cancer cells and a plurality of non-cancerous cells.

[0520] 371. The system of embodiment 370, wherein contacting the plurality of cells with the system or nucleic acid encoding the system reduces the viability of the plurality of cancer cells more than it reduces the viability of the plurality of non-cancerous cells.

[0521] 372. The system of embodiment 370 or 371, wherein contacting the plurality of cells with the system, or a nucleic acid encoding the system, reduces the viability of the plurality of cancer cells by 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 more than the reduction in viability of the plurality of non-cancerous cells.

[0522] 373. An expression repressor or system as described in any preceding embodiment, which does not reduce the viability of non-cancerous cells (e.g., primary hepatocytes) by more than 5%, 10%, 15% or 20%, for example, when determined according to Example 29 as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0523] 374. The expression repressor or system of embodiment 320, wherein viability is determined 72 hours after contacting the cells with the expression repressor or system.

[0524] 375. The expression repressor or system of embodiment 374, wherein the assay comprises contacting the non-cancerous cells with 2.5, 2, 1.25, 1, 0.6 or 0.5 μg / ml of the expression repressor or system.

[0525] 376. The system of any of embodiments 352-375, which, when contacted with a plurality of infected cells and a plurality of uninfected cells, reduces the viability of the plurality of infected cells more than the viability of the plurality of uninfected cells, and / or reduces the viability of the plurality of cancerous cells more than the viability of the plurality of non-cancerous cells.

[0526] 377. The system of any one of embodiments 352-376, wherein the cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, small cell lung cancer (SCLC), large cell (undifferentiated) carcinoma, triple-negative breast cancer, gastric adenocarcinoma, endometrial cancer, or pancreatic cancer.

[0527] 378. The system of any of embodiments 352-377, wherein the cancer cells are lung cancer cells, gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or liver cancer cells.

[0528] 379. The system of any one of embodiments 352-378, wherein the cells are human lung epithelial cells or human lung fibroblasts

[0529] 380. The system of any one of embodiments 352-379, wherein the infection is a viral infection.

[0530] 381. The expression repressor of embodiment 380, wherein the viral infection is hepatitis, eg, hepatitis B.

[0531] 382. The system of any one of embodiments 378-381, wherein the infected cells are human hepatocytes.

[0532] 383. The system of any one of embodiments 352-382, wherein the viral infection is a chronic infection.

[0533] 384. A fusion protein comprising:

[0534] a first amino acid region comprising a sequence encoding a first expression repressor of the system of any one of embodiments 118-383; and

[0535] A second amino acid region comprising a sequence encoding a second expression repressor of the system of any one of embodiments 118-383.

[0536] 385. The fusion protein of embodiment 384, comprising a third amino acid region, wherein the third amino acid region is located between the first amino acid region and the second amino acid region.

[0537] 386. The fusion protein of embodiment 385, wherein the third amino acid region comprises a protease cleavage peptide sequence, such as a self-cleavage peptide sequence, such as a T2A self-cleavage peptide sequence, such as a sequence according to SEQ ID NO: 120.

[0538] 387. The fusion protein of embodiment 386, wherein the third amino acid region comprises a protease cleavage peptide sequence, such as a self-cleavage peptide sequence, such as a tandem 2A peptide sequence, such as a tPT2A sequence, such as a sequence according to SEQ ID NO: 124.

[0539] 388. The fusion protein of embodiment 385, wherein the peptide sequence comprises a T2A peptide sequence and a P2A peptide sequence.

[0540] 389. The fusion protein of any one of embodiments 384-388, wherein:

[0541] The first expression suppressor 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

[0542] 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.

[0543] 390. The fusion protein of any one of embodiments 384-388, wherein:

[0544] The first expression suppressor 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

[0545] 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.

[0546] 391. The fusion protein of any one of embodiments 384-388, wherein:

[0547] The first expression suppressor 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

[0548] 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.

[0549] 392. The fusion protein of any one of embodiments 384-391, comprising the amino acid sequence of SEQ ID NO: 91, 92, 121 or 122, or a sequence that is at least 80%, 85%, 90%, 95% or 99% identical thereto, or a sequence that 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.

[0550] 393. The fusion protein of any one of embodiments 384-392, comprising the amino acid sequence of SEQ ID NO: 181, 182, 187, or 188, or a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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.

[0551] 394. A nucleic acid encoding the sequence of the system of any one of embodiments 118-393.

[0552] 395. A nucleic acid encoding the sequence of the system of embodiment 394.

[0553] 396. The nucleic acid of embodiment 394 or 395, comprising:

[0554] a first region comprising a sequence encoding a first expression repressor of the system of any one of embodiments 118-393; and

[0555] A second region comprising a sequence encoding a second expression repressor of the system of any one of embodiments 118-393.

[0556] 397. The nucleic acid of any one of embodiments 394-396, comprising a third region, wherein the third region is located between the first region and the second region.

[0557] 398. The nucleic acid of any one of embodiments 394-397, wherein the third region encodes a ribosomal skipping sequence.

[0558] 399. The nucleic acid of embodiment 397 or 398, wherein the third region encodes a tPT2A peptide sequence, eg, a sequence according to SEQ ID NO: 124.

[0559] 400. The nucleic acid of any one of embodiments 397-399, wherein the third region encodes a protease-cleaving 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.

[0560] 401. The nucleic acid of any one of embodiments 397-400, wherein the third region encodes a protease-cleavage peptide sequence, e.g., a self-cleavage 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.

[0561] 402. The nucleic acid of any one of embodiments 394-401, wherein

[0562] The first expression suppressor 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

[0563] The second expression suppressor 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.

[0564] 403. The nucleic acid of any one of embodiments 394-401, wherein

[0565] The first expression suppressor 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

[0566] The second expression repressor comprises the 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.

[0567] 404. The nucleic acid of any one of embodiments 394-403, which encodes the amino acid sequence of SEQ ID NO: 91, 92, 121, 122, or a sequence at least 80%, 85%, 90%, 95% or 99% identical thereto, or a sequence that 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.

[0568] 405. The nucleic acid of any one of embodiments 394-404, which encodes the amino acid sequence of SEQ ID NO: 181, 182, 187, 188, or a sequence at least 80%, 85%, 90%, 95% or 99% identical thereto, or a sequence that 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.

[0569] 406. The nucleic acid of any one of embodiments 394-405, comprising 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 a sequence that 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.

[0570] 407. The nucleic acid of any one of embodiments 394-406, comprising the nucleotide sequence of SEQ ID NO: 196, 197, or a sequence at least 80%, 85%, 90%, 95% or 99% identical thereto, or a sequence that 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.

[0571] 408. A nucleic acid comprising a sequence encoding the expression repressor or expression repressor system of any one of embodiments 1-407.

[0572] 409. The nucleic acid of any one of embodiments 394-408, which is RNA, eg, mRNA.

[0573] 410. The nucleic acid of any one of embodiments 394-409, comprising an N7-methylated guanosine, eg, an N7-methylated guanosine linked to the 5' end of the RNA via an inverted 5' to 5' triphosphate bond.

[0574] 411. The nucleic acid of any one of embodiments 394-410, comprising a 5'UTR.

[0575] 412. The nucleic acid of any one of embodiments 394-411, comprising a Kozak sequence, eg, between the 5'UTR and the sequence encoding the expression repressor.

[0576] 413. A system comprising:

[0577] a first nucleic acid comprising a sequence encoding a first expression repressor of the system of any one of embodiments 118-393; and

[0578] A second nucleic acid comprising a sequence encoding a second expression repressor, eg, a second expression repressor of the system of any one of embodiments 118-393.

[0579] 414. The system of embodiment 413, wherein the first nucleic acid has the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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 the second nucleic acid has SEQ ID NO: or a nucleotide sequence of NO:57, or a sequence at least 80%, 85%, 90%, 95% or 99% identical thereto, or a sequence that 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.

[0580] 415. The system of embodiment 414, wherein the first nucleic acid has the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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 the second nucleic acid has SEQ ID NO: or a nucleotide sequence of NO:189 or 194, or a sequence at least 80%, 85%, 90%, 95% or 99% identical thereto, or a sequence that 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.

[0581] 416. The system of embodiment 415, wherein the first nucleic acid has the nucleotide sequence of SEQ ID NO: 189, 194, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or a sequence that 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 the second nucleic acid has SEQ ID NO: NO:63, 130, or a sequence at least 80%, 85%, 90%, 95% or 99% identical thereto, or a sequence that 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.

[0582] 417. The nucleic acid or system of any one of embodiments 394-416, wherein the nucleic acid comprises mRNA.

[0583] 418. A vector comprising a nucleic acid encoding the system or expression repressor of any preceding embodiment.

[0584] 419. A lipid nanoparticle comprising the system, nucleic acid, mRNA or vector of any preceding embodiment.

[0585] 420. The lipid nanoparticle of embodiment 419, comprising an ionizable lipid, e.g., a cationic lipid, e.g., MC3, SSOP.

[0586] 421. The lipid nanoparticle of embodiment 419 or 420, further comprising one or more neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids, polyunsaturated lipids, structured lipids (e.g., sterols), PEG, cholesterol, or polymer-conjugated lipids.

[0587] 422. A reaction mixture comprising the system, nucleic acid, vector or lipid nanoparticle of any preceding embodiment.

[0588] 423. The reaction mixture of embodiment 422, further comprising cells.

[0589] 424. A pharmaceutical composition comprising the expression repressor, system, nucleic acid, vector, lipid nanoparticle or reaction mixture of any preceding embodiment.

[0590] 425. A method for reducing MYC gene expression in a cell, the method comprising:

[0591] contacting the cell (e.g., a cancer cell) with an expression suppressor, system, one or more nucleic acids encoding the system or expression suppressor, vector, lipid nanoparticle, or pharmaceutical composition of any one of embodiments 1-424,

[0592] This reduces the expression of the MYC gene in the cell.

[0593] 426. A method for treating cancer in a subject in need thereof, the method comprising:

[0594] administering to the subject the expression suppressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition of any one of embodiments 1-424,

[0595] The cancer is thereby treated in the subject.

[0596] 427. A method of reducing tumor growth in a subject in need thereof, the method comprising:

[0597] administering to the subject the expression suppressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition of any one of embodiments 1-424,

[0598] thereby reducing tumor size in the subject.

[0599] 428. The method of embodiment 427, wherein the reduction in tumor growth comprises a decrease in tumor volume compared to the tumor volume at the start of treatment.

[0600] 429. The method of embodiment 428, wherein the reduction in tumor growth is greater in the subject compared to untreated subjects.

[0601] 430. A method of increasing or restoring sensitivity of cancer to a kinase inhibitor, such as a VEGFR1-3 inhibitor (e.g., lenvatinib), a tyrosine kinase inhibitor (e.g., sorafenib), an ERK inhibitor (e.g., uritinib), an AKT inhibitor (e.g., MK-2206, ARQ092, or TAS-117), a MEK inhibitor (e.g., trametinib or cobimetinib), or an EGFR TKI (e.g., osimertinib, erlotinib, afatinib, or gefitinib), the method comprising administering to a subject having cancer an expression repressor or system described herein.

[0602] 431. The method of embodiment 430, wherein administration of the expression inhibitor or system reduces the IC of the kinase inhibitor. 50 A 10%, 20%, 30% or 40% decrease, for example in a cancer cell viability assay, for example, an assay according to Example 38 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0603] 432. The method of embodiment 430 or 431, wherein the kinase inhibitor inhibits one or more (e.g., all) of VEGFR, PDGFR, MEK, ERK, AKT, EGFR, or RAF kinases.

[0604] 433. A method of increasing or restoring the sensitivity of cancer to a bromodomain inhibitor, such as a BET inhibitor, such as JQ1, comprising administering to a subject having cancer an expression repressor, system, or nucleic acid described herein (e.g., any one of Examples 1-423), wherein optionally administration of the expression repressor or system results in an IC of the bromodomain inhibitor being reduced. 50 A decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, for example in a cancer cell viability assay, for example, an assay according to Example 39 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0605] 434. The method of embodiment 433, wherein the bromodomain inhibitor is or comprises JQ1, BET672, or birabresib.

[0606] 435. A method of increasing or restoring sensitivity of cancer to a MEK inhibitor, such as trametinib, comprising administering to a subject having cancer an expression suppressor, system, or nucleic acid described herein (e.g., any one of Examples 1-423), wherein optionally administration of the expression suppressor or system results in an IC of the MEK inhibitor being reduced. 50A decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, for example in a cancer cell viability assay, such as the assay according to Example 51 described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0607] 436. The method of any one of embodiments 427-435, wherein the reduction in tumor growth in the subject is greater than or similar to the reduction in tumor size when the subject is treated with a chemotherapeutic agent or a small molecule MYC inhibitor.

[0608] 437. The method of embodiment 436, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0609] 438. The method of embodiment 437, wherein the small molecule MYC inhibitor is MYCi975.

[0610] 439. A method of reducing tumor size in a subject in need thereof, the method comprising:

[0611] The expression suppressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition of 1-424 is administered to the subject, wherein the reduction in tumor size is greater than or similar to the reduction in tumor size when the subject is treated with a chemotherapeutic agent.

[0612] 440. The method of 439, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0613] 441. The method of any preceding embodiment, wherein the subject does not experience any significant side effects compared to treatment with a chemotherapeutic agent or a small molecule MYC inhibitor.

[0614] 442. The method of any one of embodiments 436-441, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0615] 443. The method of embodiment 442, wherein the small molecule MYC inhibitor is MYCi975.

[0616] 444. The method of any one of embodiments 426-443, wherein the cancer is stage I, stage II, stage III, or stage IV cancer.

[0617] 445. The method of any preceding embodiment, wherein the subject's body weight remains approximately the same before and after treatment.

[0618] 446. The method of any one of the preceding embodiments, wherein the subject does not experience weight loss, or wherein the subject experiences less than 3%, 2%, or 1% weight loss compared to the start of treatment.

[0619] 447. The method of any one of the preceding embodiments, wherein the subject does not lose or gain weight after treatment compared to the subject's weight before treatment.

[0620] 448. A method for treating liver disease in a subject in need thereof, the method comprising:

[0621] administering to the subject an expression suppressor, wherein the expression suppressor comprises a targeting moiety that binds to a MYC locus (e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence comprising anchor sequence-mediated conjugation (ASMC) of a MYC gene or a sequence proximal to the anchor sequence), and optionally an effector moiety, e.g., an effector moiety described herein;

[0622] The liver disease in the subject is thereby treated.

[0623] 449. The method of embodiment 447, further comprising administering to the subject a second expression suppressor comprising a targeting moiety that binds an anchor sequence comprising anchor sequence-mediated conjugation (ASMC) of a target gene (e.g., MYC), and optionally a second effector moiety, e.g., an effector moiety described herein; e.g., KRAB;

[0624] The liver disease in the subject is thereby treated.

[0625] 450. A method for treating liver disease in a subject in need thereof, the method comprising:

[0626] administering to the subject the expression suppressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition of any one of embodiments 1-424,

[0627] The liver disease in the subject is thereby treated.

[0628] 451. The method of embodiment 450, wherein the liver disease is a chronic liver disease.

[0629] 452. The method of embodiment 450 or 451, wherein the liver disease is viral or alcohol related.

[0630] 453. The method of any one of embodiments 450-452, wherein the liver disease is hepatitis or hepatocellular carcinoma.

[0631] 454. The method of embodiment 453, wherein the hepatocellular carcinoma is selected from HCC subtype S1, HCC subtype S2, or HCC subtype S3.

[0632] 455. The method of embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S1.

[0633] 456. The method of embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S2.

[0634] 457. The method of any one of embodiments 450-456, wherein the liver disease is caused by hepatitis B virus or hepatitis C virus.

[0635] 458. A method for treating a lung disease in a subject in need thereof, the method comprising:

[0636] administering to the subject an expression suppressor, wherein the expression suppressor comprises a targeting moiety that binds to a MYC locus (e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence comprising anchor sequence-mediated conjugation (ASMC) of a MYC gene or a sequence proximal to the anchor sequence), and optionally an effector moiety, e.g., an effector moiety described herein;

[0637] The lung disease in the subject is thereby treated.

[0638] 459. The method of embodiment 458, further comprising administering to the subject a second expression repressor comprising a targeting portion that binds to a genomic locus located in a super enhancer region of the target gene (e.g., MYC), and optionally a second effector portion, e.g., an effector portion described herein; e.g., KRAB;

[0639] thereby treating the subject's lung disease

[0640] 460. A method for treating a lung disease in a subject in need thereof, the method comprising:

[0641] administering to the subject the expression suppressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition of any one of embodiments 1-424,

[0642] The lung disease in the subject is thereby treated.

[0643] 461. The method of embodiment 459 or 460, wherein the lung disease is cancer, e.g., lung cancer, e.g., lung tumor, e.g., non-small cell lung cancer or small cell lung cancer.

[0644] 462. The method of any one of embodiments 425-461, wherein contacting or administering comprises intravenous administration to the subject.

[0645] 463. The method of any one of embodiments 425-462, wherein contacting or administering comprises intratumoral delivery (e.g., injection).

[0646] 464. The method of any one of embodiments 425-463, wherein the cancer is characterized by increased MYC expression relative to a reference level (e.g., relative to MYC expression in a reference cell, such as an otherwise similar non-cancerous cell of the subject).

[0647] 465. The method of any one of embodiments 426-464, wherein the cancer is characterized by duplication of part or all of the MYC gene.

[0648] 466. The method of any one of embodiments 426-465, wherein 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.

[0649] 467. The method of any one of embodiments 426-466, wherein the cancer is a human chorionic gonadotropin (hCG) secreting cancer.

[0650] 468. The method of any one of embodiments 426-467, wherein the cancer is liver cancer.

[0651] 469. The method of any one of embodiments 426-468, wherein the cancer is a non-responsive cancer, e.g., a non-responsive liver cancer.

[0652] 470. The method of any one of embodiments 426-469, wherein the cancer is non-small cell lung cancer or small cell lung cancer.

[0653] 471. The method of any one of embodiments 426-470, wherein the cancer overexpresses alpha-fetoprotein (AFP) (e.g., relative to AFP expression of a reference cell, e.g., an otherwise similar non-cancerous cell of the subject).

[0654] 472. The method of any one of embodiments 431-471, wherein the cells of the cancer are characterized by the presence of a super enhancer, e.g., comprising the MYC gene or comprising an anchor sequence-mediated junction comprising the MYC gene, wherein optionally, the cancer is selected from liver cancer, colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, or endometrial cancer.

[0655] 473. The method of embodiment 472, wherein the expression repressor (eg, a second expression repressor) binds to an anchor sequence of anchor sequence-mediated conjugation (ASMC) comprising the MYC gene, or binds to a sequence proximal to the anchor sequence.

[0656] 474. The method of any one of embodiments 426-473, wherein the cells of the cancer are characterized by the absence of a super enhancer comprising the MYC gene or comprising an anchor sequence-mediated junction comprising the MYC gene.

[0657] 475. The method of embodiment 474, wherein the expression repressor (e.g., a first expression repressor) binds to the MYC promoter.

[0658] 476. The method of any one of embodiments 426-475, wherein the cancer comprises cells containing a super enhancer comprising the MYC gene or comprising an anchor sequence-mediated junction comprising the MYC gene, and cells not containing a super enhancer comprising the MYC gene or comprising an anchor sequence-mediated junction comprising the MYC gene.

[0659] 477. The method of any one of embodiments 426-476, wherein the cancer comprises cells characterized by increased expression of MYC relative to a reference level (e.g., relative to MYC expression of a reference cell, e.g., an otherwise similar non-cancerous cell of the subject), and cells not characterized by increased expression of MYC relative to a reference level (e.g., relative to MYC expression of a reference cell, e.g., an otherwise similar non-cancerous cell of the subject), e.g., cells with normal MYC expression.

[0660] 478. The method of any one of embodiments 426-477, wherein the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition is administered as a monotherapy.

[0661] 479. The method of any one of embodiments 426-478, comprising administering to the subject multiple doses, e.g., at least 2, 3, 4, 5, or 6 doses of the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition.

[0662] 480. The method of any one 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.

[0663] 481. The method of any one of embodiments 426-480, comprising:

[0664] a) first, administering to the subject a first plurality of doses of an expression repressor or system described herein (e.g., any one of Examples 1-424), wherein optionally, each subsequent dose in the first plurality of doses is administered 5 days after a prior dose in the first plurality of doses;

[0665] b) secondly, stopping the expression repressor or system for a period of time (a "drug holiday"), for example about 2 weeks, and

[0666] c) third, administering to the subject a second plurality of doses of the expression repressor or system, wherein optionally a subsequent dose in the second plurality of doses is administered 5 days after a prior dose in the second plurality of doses.

[0667] 482. The method of embodiment 481, wherein the first plurality of doses comprises 4 doses.

[0668] 483. The method of embodiment 480 or 481, wherein the second plurality of doses comprises 2 doses.

[0669] 484. The method of any one of embodiments 481-483, wherein the subject receives no treatment at all during the drug holiday.

[0670] 485. The method of any one of embodiments 481-484, wherein the subject receives a second therapeutic agent during the drug holiday.

[0671] 486. The method of any one of embodiments 481-485, wherein the drug holiday is at least twice as long as the time between dose administrations in the first plurality of doses.

[0672] 487. The method of any one of embodiments 481-487, wherein the drug holiday is at least twice as long as the time between dose administrations in the second plurality of doses.

[0673] 488. The method of any one of embodiments 426-487, wherein the volume of the tumor decreases to undetectable levels following treatment with the expression repressor or system.

[0674] 489. The method of any one of embodiments 426-488, wherein tumor volume decreases (eg, to undetectable levels) after cessation of treatment with the expression suppressor or system.

[0675] 490. The method of any one of embodiments 425-489, wherein the cancer does not develop resistance to the expression repressor or system, or does not develop resistance to the expression repressor or system within 10, 20, 30, 40, 50, or 60 days.

[0676] 491. The method of any one of embodiments 425-490, wherein the cancer cells have a functional apoptotic pathway.

[0677] 492. The method of any one of embodiments 425-491, wherein the cancer cells have functional caspase 3.

[0678] 493. The method of embodiment 492, wherein caspase 3 is upregulated in cancer cells after administering the expression inhibitor or system to the subject.

[0679] 494. The method of any one of embodiments 425-493, wherein Ki67 is downregulated in cancer cells after administering the expression repressor or system to the subject.

[0680] 495. The method of any one of embodiments 425-494, wherein cancer cell proliferation is reduced following administration of the expression repressor or system to the subject.

[0681] 496. The method of any one of claims 425-495, wherein the method further comprises

[0682] a. contacting the cell with a second therapeutic agent or

[0683] b. administering a second therapeutic agent to the subject.

[0684] 497. The method of embodiment 496, wherein the second therapeutic agent is not an expression repressor that binds to the MYC promoter.

[0685] 498. The method of embodiment 496 or 497, wherein the second therapeutic agent is not the expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle of any one of embodiments 1-424.

[0686] 498. The method of any one of embodiments 495-497, wherein the second therapeutic agent is an expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle of any one of embodiments 1-424.

[0687] 500. The method of any one of embodiments 496-498, wherein the second therapeutic agent is one or both of immunotherapy, immune checkpoint and anti-vascular endothelial growth factor therapy, systemic chemotherapy, a tyrosine kinase inhibitor such as sorafenib, a mitogen-activated protein kinase kinase inhibitor (MEK inhibitor) such as trametinib, or a bromodomain inhibitor such as a BET inhibitor such as JQ1 or bilaricept.

[0688] 501. The method of any one of embodiments 496-500, wherein the second therapeutic agent is a tyrosine kinase inhibitor, such as sorafenib.

[0689] 502. The method of any one of embodiments 496-500, wherein the second therapeutic agent is a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1, bilaricept, or BET 672.

[0690] 503. The method of any one of embodiments 496-500, wherein the second therapeutic agent is a mitogen-activated protein kinase kinase inhibitor (MEK inhibitor), such as trametinib.

[0691] 504. The method of any one of embodiments 496-503, wherein the method further comprises administering an additional therapy to the subject.

[0692] 505. The method of embodiment 504, wherein the additional therapy comprises surgical resection orthotopic liver transplantation, radiofrequency ablation, photodynamic therapy (PDT), laser therapy, brachytherapy, radiation therapy, transcatheter arterial chemotherapy or radioembolization, or stereotactic radiotherapy.

[0693] 506. The method of any one of embodiments 496-505, wherein the second therapeutic agent is selected from a checkpoint inhibitor or a small molecule.

[0694] 507. The method of any one of embodiments 496-506, wherein the second therapeutic agent is a chemotherapeutic agent, such as a kinase inhibitor or a bromodomain inhibitor, such as a BET inhibitor.

[0695] 508. The method of embodiment 506 or 507, wherein the second therapeutic agent is selected from sorafenib, JQ1, BET672, bilaricept, or trametinib.

[0696] 509. The method of any one of embodiments 496-507, wherein the expression repressor, system or nucleic acid and the second therapeutic agent are administered simultaneously.

[0697] 510. The method of any one of embodiments 496-509, wherein the expression repressor, system or nucleic acid and the second therapeutic agent are administered sequentially.

[0698] 511. The method of any one of embodiments 504-509, wherein the additional therapy is administered concurrently.

[0699] 512. The method of any one of embodiments 504-510, wherein the additional therapy is administered sequentially.

[0700] 513. The method of any one of embodiments 496-509, wherein the second therapeutic agent is administered concurrently with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture of any one of embodiments 1-424.

[0701] 514. The method of any one of embodiments 496-513, wherein the second therapeutic agent is administered continuously with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture of any one of embodiments 1-424.

[0702] 515. The method of any one of embodiments 496-514, wherein the expression repressor, system or nucleic acid is administered intravenously and the second therapy is administered orally.

[0703] 516. The method of any preceding embodiment, wherein the cancer is a resistant or refractory cancer.

[0704] 517. The method of any preceding embodiment, wherein the cancer is resistant or refractory to a kinase inhibitor, eg, a kinase inhibitor that inhibits one or more of VEGFR, PDGFR, or RAF kinases, eg, sorafenib.

[0705] 518. The method of any preceding embodiment, wherein the subject has an amplification in a MYC super enhancer.

[0706] 519. A kit comprising a container comprising a composition comprising the expression repressor, system, one or more nucleic acids encoding the system or expression repressor, vector, lipid nanoparticle, reaction mixture, or pharmaceutical composition of any one of embodiments 1-424, and a set of instructions comprising at least one method of using the composition to modulate, e.g., reduce, MYC gene expression in a cell.

[0707] 520. The method of any one of embodiments 425-518 or the pharmaceutical composition of embodiment 424, wherein the method comprises administering a compound having the general structure of Formula (I), or the pharmaceutical composition further comprises a compound having the general structure of Formula (I):

[0708]

[0709] in:

[0710] R 1 is an alkyl group, a cycloalkyl group, or an aryl group;

[0711] R 2 is halo, alkyl or H;

[0712] R 3 、R 4 、R 5 、R 6 、R 7are each independently H or alkyl;

[0713] R 8 and R 9 together forming an optionally substituted aromatic ring;

[0714] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0715] 521. The method or pharmaceutical composition of embodiment 520, wherein R 1 is an alkyl group or a cycloalkyl group.

[0716] 522. The method or pharmaceutical composition of any one of embodiments 520 or 521, wherein R 1 = cyclopropane.

[0717] 523. The method or pharmaceutical composition of any one of embodiments 520-522, wherein R 2 It is halogenated.

[0718] 524. The method or pharmaceutical composition of any one of embodiments 520-523, wherein R 2 =Cl.

[0719] 525. The method or pharmaceutical composition of any one of embodiments 520-524, wherein R 3 、R 4 、R 5 、R 6 、R 7 Each is H.

[0720] 526. The method or pharmaceutical composition of any one of embodiments 520-525, wherein the substituted aromatic ring is a substituted phenyl ring.

[0721] 527. The method or pharmaceutical composition of any one of embodiments 520-526, wherein the compound of formula (I) comprises:

[0722]

[0723] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0724] 528. The method or pharmaceutical composition of any one of embodiments 520-527, wherein the compound of formula (I) is administered orally.

[0725] 529. The method or pharmaceutical composition of any one of embodiments 520-528, wherein the compound of formula (I) is administered at a dose of 6-10 (e.g., about 8), 10-14 (e.g., about 12) mg, or about 8-24 mg per day.

[0726] 530. The method of any one of embodiments 425-518 or the pharmaceutical composition of embodiment 424, wherein the method comprises administering a compound having the general structure of Formula (II), or the pharmaceutical composition further comprises a compound having the general structure of Formula (II):

[0727]

[0728] Where R1 is:

[0729] (1) Where X=C or N, provided that one or both X are N; and R 12 It is an electron pair, H or methyl;

[0730] (2) halogen; or

[0731] (3) methyl halogen;

[0732] R2 is H or methyl;

[0733] R3 is H or methyl;

[0734] R4 is H or methyl;

[0735] R5 is -OH or -O-methyl;

[0736] R6 is H or methyl;

[0737] R7 is H or methyl;

[0738] R8 is -(CH2) n -N(R 13 )2, where n=2-6, and R 13 =H or methyl;

[0739] R9 is H or methyl;

[0740] R 10 Yes-(CO)-R 14 , where R 14 is an alkyl group or an alkene group; and

[0741] R 11 is H or methyl,

[0742] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0743] 531. The method of embodiment 530, wherein R1 is:

[0744]

[0745] Where X=C or N, provided that one or both X are N; and R 15 It is an electron pair, H or methyl.

[0746] 532. The method of embodiment 530 or 531, wherein R1 is:

[0747]

[0748] 533. The method of any one of embodiments 530-532, wherein R1 is:

[0749]

[0750] 534. The method of any one of embodiments 530-533, wherein:

[0751] R2 is H;

[0752] R3 is H;

[0753] R4 is H;

[0754] R5 is -O-methyl;

[0755] R6 is H;

[0756] R7 is methyl;

[0757] R8 is -(CH2)2-N(methyl)2, wherein n=2-6, and R 13 =H or methyl;

[0758] R9 is H;

[0759] R 10 is -(CO)-ethylene; and

[0760] R 11 It’s H.

[0761] 535. The method of any one of embodiments 530-534, wherein formula (II) is:

[0762]

[0763] 536. The method of any one of embodiments 520-535, wherein the cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, or non-small cell lung cancer.

[0764] 537. The method of any one of embodiments 520-536, wherein the nucleic acid comprises RNA, e.g., mRNA.

[0765] 538. The method of any one of embodiments 520-537, wherein:

[0766] The first targeting moiety binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, and

[0767] The expression repressor comprises the first effector moiety, wherein the first effector moiety comprises a DNA methyltransferase.

[0768] 539. The method of embodiment 538, wherein the first targeting moiety comprises a zinc finger domain.

[0769] 540. The method of embodiment 538 or 539, wherein the first targeting moiety comprises 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 that 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.

[0770] 541. The method of any one of embodiments 538-540, wherein the first effector portion comprises MQ1 or a functional variant or fragment thereof.

[0771] 542. The method of any one of embodiments 538-541, wherein the first effector portion comprises a sequence of SEQ ID NO: 19 or 87, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0772] 543. The method of any one of embodiments 538-542, wherein the first effector portion comprises a sequence of SEQ ID NO: 129, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0773] 544. The method of any one of embodiments 538-543, wherein the RNA comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 131, 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.

[0774] 545. The method of any one of embodiments 538-544, wherein the RNA comprises a nucleotide sequence encoding the first effector portion, wherein the nucleotide sequence encoding the first effector portion comprises a sequence according to SEQ ID NO: 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.

[0775] 546. The method of any one of embodiments 538-545, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 130, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

[0776] 547. The method of any one of embodiments 538-546, wherein the RNA further encodes a second expression repressor, wherein the second expression repressor comprises:

[0777] a second targeting moiety that binds a second genomic locus, and

[0778] Second effector moiety.

[0779] 548. The method of embodiment 547, wherein the second targeting moiety binds to a second genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO:77.

[0780] 549. The method of embodiment 547 or 548, wherein the second targeting moiety comprises a zinc finger domain.

[0781] 550. The method of any one of embodiments 547-549, wherein the second targeting moiety comprises 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 that 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.

[0782] 551. The method of any one of embodiments 547-550, wherein the second effector moiety comprises KRAB or a functional variant or fragment thereof.

[0783] 552. The method of any one of embodiments 547-551, wherein the second effector portion comprises 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 that 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.

[0784] 553. The method of any one of embodiments 547-552, wherein the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that differs therefrom in 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.

[0785] 554. The method of any one of embodiments 547-553, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 113.

[0786] 555. The method of any one of embodiments 5209-554, wherein the nucleic acid is formulated in lipid nanoparticles (LNPs), wherein the nucleic acid is optionally encapsulated inside the LNPs.

[0787] 556. The method of any one of embodiments 520-555, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor is administered as an IV infusion, eg, every 2 weeks, eg, over 80-120 minutes.

[0788] 557. The method of any one of embodiments 520-556, wherein the nucleic acid is administered at a dose of about 0.001 mg / kg to 1.5 mg / kg or about 0.002 mg / kg to 1.5 mg / kg, e.g., every 1, 2, 3, 4, 5, 6, or 7 weeks.

[0789] 558. The method of any one of embodiments 520-557, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor and the compound having the general structure of Formula (I) are administered on different days.

[0790] 559. The method of any one of embodiments 520-558, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor and the compound having the general structure of Formula (I) are administered on the same day.

[0791] 560. The method of embodiment 559, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression suppressor is administered prior to administering the compound having the general structure of Formula (I) or Formula (II).

[0792] 561. The method of embodiment 559 or 560, wherein the compound having the general structure of Formula (I) or Formula (II) is administered between 1-24 hours after administration of the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor is completed.

[0793] definition

[0794] A / an, the: As used herein, the singular forms "a / an," and "these" include plural referents unless the context clearly dictates otherwise.

[0795] Reagent: As used herein, the term "agent" can be used to refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. As will be clear to one skilled in the art from the context, in some embodiments, the term can be used to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as will be understood by one skilled in the art from the context, in some embodiments, the term can be used to refer to a natural product, as it is found in nature and / or obtained from nature. In some embodiments, also as will be understood by one skilled in the art from the context, the term can be used to refer to one or more man-made entities, as it is designed, engineered, and / or produced by the action of the human hand and / or is not found in nature. In some embodiments, the reagent can be used in an isolated or pure form; in some embodiments, the reagent can be used in a crude form. In some embodiments, potential reagents can be provided as a collection or library, for example, which can be screened to identify or characterize active agents therein. In some embodiments, the term "agent" may refer to a compound or entity that is or comprises a polymer; in some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or one or more specific polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moieties.

[0796] 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, such as a complex. In some embodiments, the anchor sequence comprises one or more CTCF binding motifs. In some embodiments, the anchor sequence is not located within a 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 an enhancer or promoter. In some embodiments, the anchor sequence is located 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, at least 950 bp, or at least 1 kb from any transcription start site. In some embodiments, the anchor sequence is located in a region not associated with genomic imprinting, monoallelic expression, and / or monoallelic epigenetic markers. In some embodiments, the anchor sequence has one or more functions selected from the following: binding to an endogenous nucleating polypeptide (e.g., CTCF), interacting with a second anchor sequence to form an anchor sequence-mediated junction, or isolating from an enhancer other than anchor sequence-mediated junction. In some embodiments of the present disclosure, techniques are provided that can specifically target one or more specific anchor sequences without targeting other anchor sequences (e.g., sequences that may contain a nucleating agent (e.g., CTCF) binding motif in a different context); such targeted anchor sequences may be referred to as "target anchor sequences." In some embodiments, the sequence and / or activity of the target anchor sequence is regulated, while the sequence and / or activity of one or more other anchor sequences that may be present in the same system as the targeted anchor sequence (e.g., in the same cell and / or in some embodiments on the same nucleic acid molecule (e.g., the same chromosome)) are not regulated. In some embodiments, the anchor sequence comprises or is a nucleating polypeptide binding motif. In some embodiments, the anchor sequence is adjacent to the nucleating polypeptide binding motif.

[0797] Anchor sequence-mediated binding: As used herein, the term "anchor sequence-mediated binding" refers to a DNA structure, in some cases a complex, that is generated and / or maintained through physical interaction or binding of at least two anchor sequences in DNA by one or more polypeptides (e.g., nucleating polypeptides) or one or more proteins and / or nucleic acid entities (e.g., RNA or DNA) that bind to the anchor sequences to achieve spatial proximity and functional linkage between the anchor sequences (see, e.g., Figure 1).

[0798] Associated: Two events or entities are "associated" with each other, as the term is used herein, if the presence, level, form, and / or function of one event or entity is associated with another event or entity. For example, in some embodiments, a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, form, and / or function are correlated with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., in a related population). In some embodiments, two or more entities are physically "associated" with each other if they interact, directly or indirectly, so that they are physically close to each other and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently bound, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, a DNA sequence is "associated with" a target genome or transcription complex when the nucleic acid is at least partially located within the target genome or transcription complex and expression of genes in the DNA sequence is affected by the formation or disruption of the target genome or transcription complex.

[0799] Biologically active portion of an effector domain: As used herein, a "biologically active portion of an effector domain" is a portion that maintains the function (e.g., complete, partial, minimal) of an effector domain (e.g., a "minimal" or "core" domain).

[0800] Domain: As used herein, the term "domain" refers to a segment or portion of an entity. In some embodiments, a "domain" is associated with a specific structural and / or functional characteristic of an entity, such that when the domain is physically separated from the rest of its parent entity, it substantially or completely retains the specific structural and / or functional characteristic. Alternatively or additionally, in some embodiments, a domain can be or comprise a portion of an entity that, when separated from the (parent) entity and attached to a different (receiving) entity, substantially retains and / or imparts to the receiving entity one or more of the structural and / or functional characteristics that characterize it in the parent entity. In some embodiments, a domain is or comprises a segment or portion of a molecule (e.g., a small molecule, a carbohydrate, a lipid, a nucleic acid, a polypeptide, etc.). In some embodiments, a domain is or comprises a segment of a polypeptide. In some such embodiments, a domain is characterized by specific structural elements (e.g., a specific amino acid sequence or sequence motif, α-helical characteristics, β-sheet characteristics, helical coil characteristics, random coil characteristics, etc.), and / or specific functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0801] Effector moiety: As used herein, the term "effector moiety" refers to a domain that, when localized to an appropriate site in the nucleus, is capable of altering the expression of a target gene. In some embodiments, the effector moiety recruits components of the transcriptional machinery. In some embodiments, the effector moiety inhibits the recruitment of components of a transcription factor or expression inhibitor. In some embodiments, the effector moiety comprises an epigenetic modification moiety (e.g., epigenetically modifies a target DNA sequence).

[0802] Epigenetic modification portion: As used herein, "epigenetic modification portion" refers to a domain that changes: i) the structure of chromatin, such as a two-dimensional structure; and / or ii) an epigenetic marker (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing), wherein the epigenetic modification portion is appropriately positioned to the nucleic acid (e.g., by a targeting portion). In some embodiments, the epigenetic modification portion comprises an enzyme or a functional fragment or variant thereof that affects one or more epigenetic markers (e.g., increases or decreases their levels). In some embodiments, the epigenetic modification portion comprises a DNA methyltransferase, a histone methyltransferase, a CREB binding protein (CBP), or a functional fragment of any one thereof.

[0803] Expression control sequence: As used herein, the term "expression control sequence" refers to a nucleic acid sequence that increases or decreases gene transcription and includes, but is not limited to, promoters and enhancers. An "enhancer sequence" refers to a subtype of expression control sequence and increases the likelihood of gene transcription. A "silencing or repressor sequence" refers to a subtype of expression control sequence and decreases the likelihood of gene transcription.

[0804] Expression repressor: As used herein, the term "expression repressor" refers to an agent or entity having one or more functionalities that reduces the expression of a target gene in a cell and specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene, or a transcriptional control element operably linked to a target gene). An expression repressor comprises at least one targeting moiety and optionally one effector moiety.

[0805] Expression repression system: As used herein, the term "expression repression system" refers to a plurality of expression repressors that reduce the expression of a target gene in 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 nucleic acids encoding the first expression repressor and the second expression repressor) 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 nucleic acids encoding the first expression repressor and the second expression repressor) are present in separate compositions or pharmaceutical compositions. In some embodiments, the first expression repressor and the second expression repressor are present in the same cell at the same time. In some embodiments, the first expression repressor and the second expression repressor are not present in the same cell at the same time, for example, they are present sequentially. For example, the first expression repressor can be present in the cell for a first time period, and then the second expression repressor can be present in the cell for a second time period, wherein the first and second time periods can overlap or not overlap.

[0806] Fusion molecule: As used herein, the term "fusion molecule" refers to a compound comprising two or more parts (e.g., a targeting moiety and an effector moiety) that are covalently linked. Fusion molecules and their parts can comprise any combination of polypeptides, nucleic acids, polysaccharides, small molecules, or other components described herein (e.g., the targeting moiety can comprise a nucleic acid and the effector moiety can comprise a polypeptide). In some embodiments, the fusion molecule is a fusion protein, for example, comprising one or more polypeptide domains covalently linked via a peptide bond. In some embodiments, the fusion molecule is a conjugated molecule comprising a targeting moiety and an effector moiety linked by a covalent bond rather than a peptide bond or a phosphodiester bond (e.g., a targeting moiety comprising a nucleic acid and an effector moiety comprising a polypeptide linked by a covalent bond rather than a peptide bond or a phosphodiester bond). In some embodiments, the expression repressor is or comprises a fusion molecule.

[0807] Genomic complex: As used herein, the term "genomic complex" is a complex that brings together two genomic sequence elements that are separated from each other on one or more chromosomes through interactions between and among multiple proteins and / or other components (which may include genomic sequence elements). 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 comprise anchor sequence-mediated binding. In some embodiments, the genomic sequence element may be or comprise a CTCF binding motif, a promoter, and / or an enhancer. In some embodiments, the genomic sequence element comprises at least one or both of a promoter and / or a regulatory site (e.g., an enhancer). In some embodiments, complex formation is nucleated at one or more genomic sequence elements and / or nucleated by binding of one or more protein components to one or more genomic sequence elements. As will be understood by those skilled in the art, in some embodiments, the co-localization (e.g., binding) of genomic sites via complex formation alters the DNA topology at or near (in some embodiments, including between) one or more genomic sequence elements. In some embodiments, the genomic complex comprises anchor sequence-mediated binding comprising one or more loops. In some embodiments, genome complexes as described herein are nucleated by nucleating polypeptides (e.g., such as CTCF and / or cohesin). In some embodiments, genome complexes as described herein may include, for example, one or more CTCFs, cohesins, non-coding RNAs (e.g., eRNAs), transcription machinery proteins (e.g., RNA polymerases, one or more transcription factors, such as those selected from the group consisting of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc.), transcription regulators (e.g., mediators, P300s, enhancer binding proteins, repressor binding proteins, histone modifiers, etc.). In some embodiments, genome complexes as described herein include one or more polypeptide components and / or one or more nucleic acid components (e.g., one or more RNA components), which, in some embodiments, can interact with each other and / or with one or more genomic sequence elements (e.g., anchor sequences, promoter sequences, regulatory sequences (e.g., enhancer sequences)) to restrict a section of genomic DNA to a topological structure (e.g., a loop) that would not be adopted if the complex were not formed.

[0808] Moiety: As used herein, the term "moiety" refers to a defined chemical group or entity having a particular structure and / or activity, as described herein.

[0809] Modulator: As used herein, the term "modulator" refers to an agent comprising one or more targeting moieties and one or more effector moieties that are capable of altering (e.g., increasing or decreasing) the expression of a target gene (e.g., MYC).

[0810] MYC: As used herein, the term "MYC locus" refers to a portion of the human genome encoding a MYC polypeptide (e.g., a polypeptide disclosed in NCBI accession number NP002458.2 or a mutant thereof), a promoter operably linked to MYC ("MYC promoter"), and an anchor sequence forming an ASMC comprising a 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 some cases, the MYC gene is located on chromosome 8, 8q24.21. In some cases, the MYC gene starts at 128,816,862 bp from pter and ends at 128,822,856 bp from pter. In some cases, the MYC gene is approximately 6 kb. In some cases, the MYC gene encodes at least eight independent mRNA sequences—5 alternative splicing variants and 3 non-splicing variants.

[0811] 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 phosphodiester bonds. As will be clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising a single nucleic acid residue. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a 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, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone rather than phosphodiester bonds and are considered to be within the scope of the present disclosure. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds rather than phosphodiester bonds. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-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, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (such as RNA or protein). In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) based on polymerization of a complementary template, replication in a recombinant cell or system, and chemical synthesis.In some embodiments, the nucleic acid is 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 length. In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that comprises at least one element encoding a polypeptide or is the complement of a sequence encoding a polypeptide. In one embodiment, the nucleic acid has enzymatic activity.

[0812] Nucleating polypeptide: As used herein, the term "nucleating polypeptide" or "binding nucleating polypeptide" as used herein refers to a protein that is directly or indirectly associated with an anchor sequence and can interact with one or more binding nucleating polypeptides (which can interact with an anchor sequence or other nucleic acid) to form a dimer (or higher order structure) composed of two or more such binding nucleating polypeptides (which can be identical or different from each other). When binding nucleating polypeptides associated with different anchor sequences associate with each other so that the different anchor sequences remain in physical proximity to each other, the resulting structure is an anchor sequence-mediated binding. That is, the close physical proximity of a nucleating polypeptide-anchor sequence to another nucleating polypeptide-anchor sequence interaction creates an anchor sequence-mediated binding (e.g., in some cases, a DNA loop) that begins and ends at the anchor sequence. As will be immediately understood by those skilled in the art reading this specification, terms such as "nucleating polypeptide," "nucleating molecule," "nucleating protein," "binding nucleating protein," may sometimes be used to refer to a binding nucleating polypeptide. As will also be immediately understood by those skilled in the art reading this specification, an assembled collection of two or more bound nucleating polypeptides (which, in some embodiments, may include multiple copies of the same agent and / or, in some embodiments, one or more of a variety of different agents) may be referred to as a "complex," "dimer," "multimer," etc.

[0813] Operably linked: As used herein, the phrase "operably linked" refers to a juxtaposition in which the components are in a relationship that allows them to function in their intended manner. A transcriptional control element that is "operably linked" to a functional element (e.g., a gene) is associated in such a way that expression and / or activity of the functional element (e.g., a gene) is achieved under conditions compatible with the transcriptional control element. In some embodiments, the transcriptional control element that is "operably linked" is continuous (e.g., covalently linked) with the coding element of interest (e.g., a gene); in some embodiments, the operably linked transcriptional control element acts in trans on the functional element of interest (e.g., a gene) or otherwise acts at a distance therefrom. In some embodiments, being operably linked means that two nucleic acid sequences are contained on the same nucleic acid molecule. In another embodiment, being operably linked can further mean that the two nucleic acid sequences are close to each other on the same nucleic acid molecule, e.g., within 1000, 500, 100, 50, or 10 base pairs of each other or directly adjacent to each other.

[0814] Peptide, polypeptide, protein: As used herein, the terms "peptide," "polypeptide," and "protein" refer to compounds composed of amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the terms refer to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, often referred to in the art as proteins, of which there are many types.

[0815] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a modulator, e.g., a disrupting agent) formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen that, when administered to a relevant population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those suitable for the following situations: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, for example, those for oral, sublingual and systemic absorption, pills, powders, granules, pastes for tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection, as, for example, sterile solutions or suspensions, or sustained release formulations; topical administration, for example, as a cream, ointment or controlled release patch or spray applied to the skin, lungs or mouth; intravaginally or rectally, for example, as a pessary, cream or foam; sublingually; ophthalmically; transdermally; or to the nose, lungs and / or to other mucosal surfaces.

[0816] Proximal: As used herein, "proximal" refers to the proximity of two sites (e.g., nucleic acid sites) such that binding of an expression repressor at a first site and / or modification of a first site by an expression repressor will produce the same or substantially the same effect as binding and / or modification of another site. For example, a targeting moiety can bind to a first site proximal to an enhancer (a second site), and an effector moiety associated with the targeting moiety can epigenetically modify the first site such that the effect of the enhancer on target gene expression is modified substantially as if the second site (enhancer sequence) were bound and / or modified. In some embodiments, a site proximal to a target gene (e.g., an exon, intron, or splice site within a target gene), proximal to a transcriptional control element operably linked to a target gene, or proximal to an anchor sequence is less than 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 base pairs from the target gene (e.g., an exon, intron, or splice site within a target gene), the transcriptional control element, or the anchor sequence (and optionally, at least 20, 25, 50, 100, 200, or 300 base pairs from the target gene (e.g., an exon, intron, or splice site within a target gene), the transcriptional control element, or the anchor sequence).

[0817] Specificity: As used herein, the term "specificity" refers to an agent with activity that is understood by those skilled in the art to mean that the agent distinguishes between potential target entities or states. For example, in some embodiments, if an agent preferentially binds to its target in the presence of one or more competing alternative targets, the agent is said to "specifically" bind to the target. In some embodiments, the specific interaction depends on the presence of specific structural features of the target entity (e.g., epitope, cleft, binding site). It should be understood that specificity is not necessarily absolute. In some embodiments, specificity can be assessed relative to the specificity of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding agent. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding agent. In some embodiments, under conditions of binding to its target entity, the agent or entity does not detectably bind to the competitive alternative target. In some embodiments, the binding agent binds to its target entity with a higher on-rate, a lower off-rate, increased affinity, reduced dissociation, and / or increased stability compared to one or more competing alternative targets.

[0818] Specific binding: As used herein, the term "specific binding" refers to the ability to discriminate between possible binding partners in the context of binding. In some embodiments, a binding agent that interacts with a particular target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the extent of binding between a binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of the binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete for selective interactions with its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0819] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, are completed and / or proceed to perfection or achieve or avoid an absolute result. Thus, the term "substantially" may be used in some embodiments herein to capture the potential imperfections inherent in many biological and chemical phenomena.

[0820] Symptom reduction: As used herein, the phrase "symptom reduction" may be used when the degree (e.g., intensity, severity, etc.) and / or frequency of one or more symptoms of a particular disease, disorder, or condition is reduced. In some embodiments, delaying the onset of a particular symptom is considered a form of reducing the frequency of that symptom.

[0821] Target: According to the present disclosure, an agent or entity is said to "target" another agent or entity if it specifically binds to the targeted agent or entity under conditions in which they are in contact with each other. For example, in some embodiments, an 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 a substantially complementary sequence.

[0822] Target gene: As used herein, the term "target gene" means a gene that is targeted for regulation, e.g., expression. In some embodiments, the target gene is part of a targeted genomic complex (e.g., a gene whose genomic sequence is at least partly a part of a target genomic complex, e.g., within an anchor sequence-mediated binding) that is targeted by one or more regulators as described herein. In some embodiments, regulation comprises inhibiting the expression of the target gene. 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 (e.g., an expression repressor) as described herein. In some embodiments, the target gene is abnormally expressed (e.g., overexpressed) in a cell (e.g., a cell in a subject (e.g., a patient)).

[0823] Targeting moiety: As used herein, the term "targeting moiety" means an agent or entity that specifically targets (e.g., binds to) a genomic sequence element (e.g., an expression control sequence or anchor sequence). In some embodiments, the genomic sequence element is adjacent to and / or operably linked to a target gene (e.g., MYC).

[0824] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that, when administered to a subject, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, the therapeutic agent comprises an expression repression system as described herein, such as an expression repressor. In some embodiments, the therapeutic agent comprises a nucleic acid encoding an expression repression system as described herein, such as an expression repressor. In some embodiments, the therapeutic agent comprises a pharmaceutical composition as described herein.

[0825] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be understood by one of ordinary skill in the art, the effective amount of a substance can vary depending on factors such as one or more desired biological endpoints, the substance to be delivered, one or more target cells, or one or more tissues. For example, in some embodiments, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, the therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

[0826] Halo: As used herein, the term "halo" refers to a halogen selected from bromo, chloro, iodo, and fluoro.

[0827] Alkyl: As used herein, the term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double and / or triple bonds), having from one to twenty-four carbon atoms (C1-C 24 alkyl), one to twelve carbon atoms (C1-C 12 The term "alkyl" refers to a group having one to eight carbon atoms (C1-C8 alkyl), one to eight carbon atoms (C1-C8 alkyl), or one to six carbon atoms (C1-C6 alkyl) and which is attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, propenyl, butenyl, pentenyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.

[0828] Alkylene: As used herein, the term "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain connecting the rest of the molecule to a group consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e., contains one or more double bonds (alkenylene) and / or triple bonds (alkynylene)) and has, for example, one to twenty-four carbon atoms (C1-C 24 Alkylene), one to fifteen carbon atoms (C1-C 15 Alkylene), one to twelve carbon atoms (C1-C 12C1-C8 alkylene), one to eight carbon atoms (C1-C8 alkylene), one to six carbon atoms (C1-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), such as methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain can be attached to the rest of the molecule by a single bond or a double bond and to the radical group by a single bond or a double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise specifically stated in the specification, the alkylene chain can be optionally substituted.

[0829] Cycloalkyl: As used herein, the term "cycloalkyl" or "carbocycle" refers to a stable, non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems having from three to fifteen carbon atoms, preferably from three to ten carbon atoms, and which are saturated or unsaturated and attached to the remainder of the molecule by a single bond. Monocyclic radical groups may include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radical groups may include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, and the like. Unless otherwise specifically stated in this specification, cycloalkyl groups are optionally substituted.

[0830] Cycloalkylene: As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. Unless stated otherwise specifically in the specification, a cycloalkylene group may be optionally substituted.

[0831] Cycloalkenylene: Unless otherwise indicated, as used herein, the term "cycloalkenylene" refers to a divalent carbocyclic non-aromatic radical having three to ten carbon atoms (e.g., C3-C 10Non-limiting examples of cycloalkenylene groups may include cycloprop-1-ene-1,2-diyl; cycloprop-2-ene-1,1-diyl; cycloprop-2-ene-1,2-diyl; cyclobut-1-ene-1,2-diyl; cyclobut-1-ene-1,3-diyl; cyclobut-1-ene-1,4-diyl; cyclobut-2-ene-1,1-diyl; cyclobut-2-ene-1,4-diyl; cyclopent-1-ene-1,2-diyl; cyclopent-1-ene-1,3-diyl; cyclopent-1-ene-1,4-diyl; cyclopent-1-ene-1,5-diyl; cyclopent-2-ene-1,1-diyl; cyclopent-2-ene-1,4-diyl; cyclopent-2-ene-1,5-diyl. diyl; cyclopenta-3-ene-1,1-diyl; cyclopenta-1,3-diene-1,2-diyl; cyclopenta-1,3-diene-1,3-diyl; cyclopenta-1,3-diene-1,4-diyl; cyclopenta-1,3-diene-1,5-diyl; cyclopenta-1,3-diene-5,5-diyl; norbornadiene-1,2-diyl; norbornadiene-1,3-diyl; norbornadiene-1,4-diyl; norbornadiene-1,7-diyl; norbornadiene-2,3-diyl; norbornadiene-2,5-diyl; norbornadiene-2,6-diyl; norbornadiene-2,7-diyl; and norbornadiene-7,7-diyl. A cycloalkenylene group can be unsubstituted or substituted (eg, optionally substituted cycloalkenylene) as described for a cycloalkyl group.

[0832] Heterocyclyl: As used herein, the term "heterocyclyl" or "heterocycle" refers to a stable 3- to 18-membered non-aromatic ring radical consisting of two to twelve carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically stated in this specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. The example of such heterocyclic group can include but is not limited to dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidine radicals, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl.Unless otherwise specifically stated in the present specification, heterocyclic group can be optionally substituted.

[0833] Substituted: As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, cycloalkyl, or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, such as, but not limited to, a halogen atom, such as F, Cl, Br, and I; an oxo group (=O); a hydroxyl group (-OH); an alkoxy group (-OR a , where R a C1-C 12 alkyl or cycloalkyl); carboxyl group (-OC(=O)R a or -C(=O)OR a , where R a It is H, C1-C 12 alkyl or cycloalkyl); amine group (-NR a R b , where R a and R b Each independently is H, C1-C 12 alkyl or cycloalkyl); C1-C 12 In some embodiments, the substituent is C1-C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluorine. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0834] Optionally: As used herein, the term "optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that the description includes both substituted alkyl groups and alkyl groups without substitution.

[0835] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to acid addition salts and base addition salts.

[0836] Pharmaceutically acceptable acid addition salts: For example, "pharmaceutically acceptable acid addition salts" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and are formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclohexane acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, Ethylenesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0837] Pharmaceutically acceptable base addition salts: The term "pharmaceutically acceptable base addition salts" refers to salts that retain the biological effectiveness and properties of the free acids and are not biologically or otherwise undesirable. These salts are prepared by adding inorganic or organic bases to the free acids. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Suitable inorganic salts include ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, denanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, halamine, choline, betaine, phenethylbenzylamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Suitable organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0838] Stereoisomers: As used herein, the term "stereoisomer" refers to compounds made up of the same atoms joined by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0839] Tautomer: As used herein, the term "tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the described compounds.

[0840] Cationic lipids: As used herein, the term "cationic lipids" refers to lipids that are capable of being positively charged. Exemplary cationic lipids include one or more positively charged amine groups. Exemplary cationic lipids are ionizable, such that they can exist in positively charged or neutral forms depending on pH. The ionization of cationic lipids may affect the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein absorption, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev [Drug Delivery Progress Review] 32: 3-17 (1998)) and the ability to form an endosome-soluble non-double-layer structure for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther [Gene Ther] 8: 1188-1196 (2001)).

[0841] Neutral lipids: As used herein, the term "neutral lipids" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids can be synthetic or naturally derived.

[0842] Charged lipids: As used herein, the term "charged lipid" refers to any of a number of lipid species that exist in a positively or negatively charged form, regardless of pH within a useful physiological range, e.g., pH-3 to pH-9. Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium propanes (e.g., DOTAP, DOTMA), dialkyldimethylaminopropanes, ethylphosphatidylcholine, dimethylaminovinylcarbamoylsterols (e.g., DCChol).

[0843] Lipid nanoparticles: As used herein, the term "lipid nanoparticle" refers to a particle having at least one nanometer size (e.g., 1-1,000 nm) that includes a specific lipid. Lipid nanoparticles can also include nucleic acids (e.g., mRNA). In some embodiments, the lipid nanoparticle is contained in a formulation that can be used to deliver an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle comprises a nucleic acid. Such lipid nanoparticles typically comprise a nucleic acid (e.g., mRNA) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, an active agent or therapeutic agent such as a nucleic acid can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other adverse effects caused by the mechanisms of the host organism or cell, such as an adverse immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0844] This patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0845] The following detailed description of the embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, the embodiments of the present examples are shown in the accompanying drawings. However, it should be understood that the present disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the accompanying drawings.

[0846] Figure 1A Depicted is a schematic diagram of a dual-target approach based on persistent blockade of the MYC promoter using a DBD fused to a DNA methyltransferase and transient (48 / 72 h) blockade of CTCF / TF sites using either the DBD or a DBD fused to a short-lived effector.

[0847] Figure 1BDepicts guide RNA localization and chromatin context of target sites (CTCF and promoter) of the MYC gene. From top to bottom, the graph represents H3K4me3 (histone H3 Ky trimethylation) levels in HepG2 cells; H3K9me3 (histone H3 K9 trimethylation) levels (repeat 1); H3K9me3 (histone H3 K9 trimethylation) levels (repeat 2); H3K27me3 (histone H3 K27 trimethylation) levels; H3K27ac (histone H3 K27 acetylation) levels; GROseq_forward strand levels (binding of transcriptionally active RNA pol II on the forward strand); GROseq_reverse strand levels (binding of transcriptionally active RNA pol II on the reverse strand); RNAseq_rep2 levels (MYC transcript levels measured using RNAseq, replicate 2); DNA methylation levels and CTCF binding levels measured by WGBS (whole genome bisulfite sequencing). The positions of the four gRNAs are indicated by arrows. gRNA GD-28859, gRNA GD-28616, and gRNA GD-28862 target the MYC upstream anchor site 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.

[0848] Figure 1CThe schematic diagram of an exemplary bicistronic construct is shown. The 5' end of the construct has a cap structure defined by the N7-methylated guanosine of the first nucleotide connected to the mRNA by a reverse 5' to 5' triphosphate bond. In certain embodiments, the cap structure promotes protein translation and stability. The downstream of the cap structure is an untranslated region (5'UTR), which is intended to promote high-level protein translation, followed by a standard "Kozak" sequence recognized by ribosomes to start protein translation. The "Kozak" sequence is followed by a CDS, which is a single continuous sequence comprising a first expression repressor and a second expression repressor separated by a tPT2A "ribosome skipping" sequence (joint), the first expression repressor comprising a first targeting moiety and a first effector moiety, and the second expression repressor comprising a second targeting moiety and a second effector moiety. Without wishing to be bound by theory, when the ribosome arrives at the tPT2A joint, it begins to translate the joint into amino acids. The first 18 amino acids produced from the P2A joint are retained at the C-terminus of the first expression repressor (e.g., comprising ZF DBD and MQ1), and then the ribosome releases it. The ribosome then continues to move forward until it reaches the T2A joint, where the first 17 residues are translated and released. Next, the second polypeptide is translated, which comprises a single amino acid, followed by the beginning of a second expression repressor (e.g., comprising a second ZF DBD and KRAB). The CDS is followed by a 3'UTR, which is intended to help high-level translation and stabilize the mRNA. Finally, the 3' end of the mRNA is a poly A tail. In certain embodiments, the poly A tail promotes protein translation and mRNA stability.

[0849] Figure 2A and Figure 2B The figure shows the efficacy of the combination of bicistronic ZF9-MQ_ZF3-KRAB and lenvatinib. Figure 2A The graph shows the viability of treated Hep3B cells relative to untreated cells. Figure 2B The graph shows the viability of SK-HEP-1 cells after treatment relative to untreated cells. This study was performed as described in Example 1.

[0850] Figure 3A and Figure 3B The figure shows the efficacy of the combination of bicistronic ZF9-MQ_ZF3-KRAB with AKT and ERK inhibitors. Figure 3A The graph shows the viability of SK-HEP-1 cells after treatment with the ERK inhibitor ulixertinib relative to untreated cells. Figure 3B The graph shows the viability of SK-HEP-1 cells after treatment with the AKT inhibitor MK-2206 relative to untreated cells. Single = AKT or ERK inhibitor alone, respectively; SNC = short noncoding (control). This study was performed as described in Example 2.

[0851] Figure 4A and Figure 4B The bar graph shows the efficacy of the combination of bicistronic ZF9-MQ_ZF3-KRAB and osimertinib. Figure 4A The bar graph shows the concentration of Myc protein (normalized to no treatment) in PC9 cells after 48 h of treatment with PBS (no treatment), osimertinib, bicistronic ZF9-MQ_ZF3-KRAB, and the combination of bicistronic ZF9-MQ_ZF3-KRAB and osimertinib. Figure 4B The bar graph shows PC9 cell viability 72 hours after treatment. * indicates p < or = 0.05; ** indicates p < or = 0.01; *** indicates p < or = 0.001. This study was performed as described in Example 3.

[0852] Figure 5A and Figure 5B The bar graph shows the efficacy of the combination of bicistronic ZF9-MQ_ZF3-KRAB and trametinib. Figure 5A The bar graph shows the concentration of Myc protein (normalized to no treatment) in H1975 cells after 48 h of treatment with PBS (no treatment), trametinib, bicistronic ZF9-MQ_ZF3-KRAB, and the combination of bicistronic ZF9-MQ_ZF3-KRAB and trametinib. Figure 5B The bar graph shows H1975 cell viability 72 hours after treatment. * indicates p < or = 0.05; ** indicates p < or = 0.01; *** indicates p < or = 0.001. This study was performed as described in Example 3.

[0853] Figure 6 Figure 2 shows the decreased survival probability with osimertinib treatment. Univariate Kaplan-Meier analysis of MYC overexpression and time to progression. Entries with time to progression greater than the 99th percentile and less than 30 days have been omitted.

[0854] Figure 7A The graph shows the cell viability of H1975 cells and two osimertinib-resistant cell lines derived from H1975 (OsimR_1.25 and OsimR_1.5) after 72 hours of osimertinib treatment. This study was performed as described in Example 7.

[0855] Figure 7B The graph shows the cell viability of H1975 cells and two osimertinib-resistant cell lines derived from H1975 (OsimR_1.25 and OsimR_1.5) after treatment with the expression repressor 72 hours later. This study was performed as described in Example 7.

[0856] Figure 8AProtein matrix gel images of MYC protein levels after treatment with osimertinib or MR-32380 were evaluated. This study was performed as described in Example 8.

[0857] Figure 8B The graph shows MYC protein levels in H1975 cells and two osimertinib-resistant cell lines derived from H1975 (OsimR_1.25 and OsimR_1.5) after treatment with vehicle (control), osimertinib, or MR-32380. This study was performed as described in Example 8.

[0858] Figure 9 Western blot images show MYC protein levels in PC9 cells harboring a sensitizing EGFR mutation (del19) treated with osimertinib or MR-32054 alone or in combination. This study was performed as described in Example 9.

[0859] Figure 10 The bar graph shows MYC protein levels (normalized to actin, no treatment) in PC9 cells harboring a sensitizing EGFR mutation (del19) treated with osimertinib or MR-32054 alone or in combination. This study was performed as described in Example 9.

[0860] Figure 11 The bar graph shows cell viability in H1975 cells harboring sensitizing mutations (EGFR L858R, T790M) treated with osimertinib or MR-32054 alone or in combination. This study was performed as described in Example 10.

[0861] Figure 12 The graph shows the Bliss synergy scores of H1975 cells with sensitizing mutations (EGFR L858R, T790M) treated with osimertinib or MR-32054 alone or in combination. All values ​​shown are between 0 and 40. This study was performed as described in Example 10.

[0862] Figure 13 The bar graph shows cell viability in PC9 cells harboring a sensitizing EGFR mutation (del19) treated with osimertinib or MR-32054 alone or in combination. This study was performed as described in Example 11.

[0863] Figure 14 The graph shows the Bliss synergy scores of PC9 cells with a sensitizing EGFR mutation (del19) treated with osimertinib or MR-32054 alone or in combination. All values ​​shown are between 0 and 40. This study was performed as described in Example 11.

[0864] Figure 15The figure shows the efficacy of the combination of bicistronic ZF9-MQ_ZF3-KRAB and osimertinib. The combination of bicistronic ZF9-MQ_ZF3-KRAB and osimertinib reduced tumor growth more than either component alone. This study was performed as described in Example 12.

[0865] Figure 16 The graph shows body weight (% change from baseline) in the H1975 subcutaneous tumor model with sensitizing EGFR mutations (L858R, T790M). This study was performed as described in Example 12. DETAILED DESCRIPTION

[0866] The present disclosure provides techniques for regulating (e.g., reducing) the expression of a target gene (e.g., MYC) in a cell (e.g., in a subject or patient) by using the expression repressors or systems described herein.

[0867] Many different diseases and syndromes, including cancer, autoimmune diseases, cardiovascular disease, and obesity, can be caused by misregulation of gene expression. In particular, overexpression of transcription factors has long been known to drive tumorigenesis, and recent studies have shown that overexpressed oncogenic transcription factors can alter core cellular autoregulatory circuits.

[0868] MYC is a transcription factor and master cell regulator that is frequently dysregulated in more than 50% of human cancers and plays a central role in almost every aspect of the tumorigenic process. In addition to early response genes, MYC generally upregulates gene expression. MYC is the most commonly amplified oncogene, and elevated expression of its gene product is associated with tumor aggressiveness and poor clinical outcome. Elevated levels of c-MYC can promote tumorigenesis in a variety of tissues. Most tumor cells depend on the transcription factor c-MYC for growth and proliferation. MYC overexpression is also associated with chronic liver diseases, such as viral and alcohol-related liver diseases. MYC overexpression levels vary in specific cancer subtypes. Without wishing to be bound by theory, it is believed that regulating, for example, reducing MYC levels in a subject (e.g., overall or in one or more specific target tissues) with a MYC misregulation disorder can alleviate or eliminate the symptoms of the MYC misregulation disorder.

[0869] Expression repressor

[0870] As described herein, present disclosure provides, in part, an expression repressor for regulating, for example, reducing the expression of a target gene, such as MYC. In certain embodiments, the expression repressor may include a targeting portion and an optional effector portion in conjunction with a target gene promoter (e.g., MYC promoter). In certain embodiments, the targeting portion specifically binds to a target DNA sequence, such as a MYC DNA sequence, thereby localizing the function of the expression repressor to the DNA sequence. In certain embodiments, the expression repressor includes a targeting portion and an effector portion. In certain embodiments, the expression repressor includes a targeting portion and a plurality of effector portions, such as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0871] In some embodiments, the expression repressor comprises one or more targeting moieties, e.g., as described in International Application No. WO / 2022 / 132195 (incorporated herein by reference in its entirety).

[0872] In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety, for example, covalently linked by a peptide bond. In some embodiments, the targeting moiety and the effector moiety are located on the same polypeptide chain, for example, connected by one or more peptide bonds and / or linkers. In some embodiments, the expression repressor is or comprises a fusion molecule, for example, the fusion molecule comprises a targeting moiety and an effector moiety connected by a peptide bond and / or linker. In some embodiments, the expression repressor comprises a targeting moiety at the N-terminus of the effector moiety arranged on the same polypeptide chain. In some embodiments, the expression repressor comprises a targeting moiety at the C-terminus of the effector moiety arranged 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 by 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, for example, by a peptide bond (for example, the targeting moiety and the plurality of effector moieties are all connected by a series of covalent bonds, although each individual moiety may not share a covalent bond with each other effector moiety).

[0873] In other embodiments, the expression repressor comprises a non-covalently linked targeting moiety and an effector moiety, for example as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0874] Typically, expression repressor as herein described is in conjunction with (for example, by targeting moiety) target gene (for example MYC) proximal and / or can be operably connected to the genomic sequence element of target gene (for example MYC).In certain embodiments, the combination of expression repressor and genomic sequence element regulates (for example reduces) target gene (for example MYC).For example, the combination of expression repressor and genomic sequence element comprising raising transcription machinery component or suppressing its raising effector part can regulate (for example reduce) target gene (for example MYC) expression.As a further example, the combination of expression repressor comprising effector part (for example epigenetic modification part) with enzymatic activity can regulate (for example reduce) target gene (for example MYC) expression by the local enzymatic activity of effector part.As a further example, the combination of expression repressor and genomic sequence element and the local enzymatic activity of expression repressor all contribute to the expression of final regulation (for example reduce) target gene (for example MYC).

[0875] In some embodiments, the expression repressor comprises an effector portion, wherein the effector portion comprises a protein selected from the group consisting of: 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 functional variants or fragments thereof.

[0876] In certain embodiments, the present disclosure provides nucleic acid sequences encoding expression repressors, expression repressor systems, targeting moieties and / or effector moieties as described herein. Technicians know that the nucleic acid sequence of RNA is identical to the corresponding DNA sequence, except that thymine (T) is usually replaced by uracil (U). It should be understood that when the nucleotide sequence is represented by a DNA sequence (e.g., comprising A, T, G, C), the present disclosure also provides a corresponding RNA sequence (e.g., comprising A, U, G, C), wherein "U" replaces "T". Conventional symbols are 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 referred to as the 5' direction.

[0877] Those skilled in the art will appreciate that due to the degeneracy of the genetic code, a large number of nucleotide sequences encoding expression repressors comprising a targeting moiety and / or effector moiety as described herein can be generated, some of which have similarity, e.g., 90%, 95%, 96%, 97%, 98%, or 99% identity, to the nucleic acid sequences disclosed herein. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at each position in the nucleic acid molecules of the present invention where arginine is specified by a codon, the codon can be changed to any of the corresponding codons described above without altering the encoded polypeptide.

[0878] In some embodiments, the nucleic acid sequence encoding the expression repressor comprising the targeting moiety and the effector moiety can be a portion or all of a codon-optimized coding region, optimized for mammalian, e.g., human, codon usage. In some embodiments, the nucleic acid sequence encoding the targeting moiety and the effector moiety is codon-optimized to increase protein expression and / or 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 than the protein level when encoded by the non-codon-optimized nucleic acid sequence.

[0879] Expression repression system

[0880] The expression repression system of the present disclosure may include two or more expression repressors. In some embodiments, the expression repression system includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more expression repressors (and optionally no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2). In some embodiments, the expression repression system targets two or more different sequences (e.g., the 1st and 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th and / or other DNA sequences, and optionally no more than the 20th, 19th, 18th, 17th, 16th, 15th, 14th, 13th, 12th, 11th, 10th, 9th, 8th, 6th, 5th, 4th, 3rd or 2nd sequence). In some embodiments, the expression repression system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors does not detectably bind, e.g., does not bind, to another member of the plurality of expression repressors. In some embodiments, the expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor does not detectably bind, e.g., does not bind, to the second expression repressor.

[0881] In certain embodiments, the expression repression system of the present disclosure comprises two or more expression repressors, wherein the expression repressors are present together in a composition, a pharmaceutical composition or a mixture. In certain embodiments, the expression repression system of the present disclosure comprises two or more expression repressors, wherein one or more expression repressors do not mix with at least one other expression repressor. For example, the expression repression system may comprise a first expression repressor and a second expression repressor, wherein the presence of the first expression repressor in the nucleus does not overlap with the presence of the second expression repressor in the nucleus of the same cell, and wherein the expression repression system achieves a reduction in MYC gene expression by the non-overlapping presence of the first and second expression repressors. In certain embodiments, compared with the reduction in MYC gene expression achieved by the first or second expression repressor alone, the expression repression system achieves a greater reduction in MYC gene expression.

[0882] In some embodiments, the expression repressors of the expression repressor system each comprise a different targeting moiety (e.g., the first, second, third or additional expression repressors each comprise a targeting moiety that is different from one another). For example, the expression repressor system may comprise a first expression repressor and a second expression repressor, wherein the first expression repressor comprises a first targeting moiety (e.g., a Cas9 domain, a TAL effector domain, or a Zn finger domain), and the second expression repressor comprises a second targeting moiety that is different from the first targeting moiety (e.g., a Cas9 domain, a TAL effector domain, or a Zn finger domain). In some embodiments, different can refer to comprising different types of targeting moieties, e.g., the first targeting moiety comprises a Cas9 domain and the second DNA targeting moiety comprises a Zn finger domain. In other embodiments, different can refer to comprising different 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) and the second targeting moiety comprises a second Cas9 domain (e.g., from a second species). In an embodiment, when the expression repressor system comprises two or more targeting moieties of the same type, e.g., two or more Cas9 or ZF domains, the targeting moieties specifically bind to two or more different sequences. For example, in an expression repressor system comprising two or more Cas9 domains, the two or more Cas9 domains can be selected or altered so that they only apparently bind to a gRNA corresponding to their target sequence (e.g., without apparently binding to a gRNA corresponding to the target of another Cas9 domain). In another example, in an expression repressor system comprising two or more effector moieties, the two or more effector moieties can be selected or altered so that they only apparently bind to their target sequence (e.g., without apparently binding to the target sequence of another effector moiety).

[0883] 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 additional expression repressors each bind to a different DNA sequence). For example, the expression repressor system may comprise 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 second DNA sequence. In some embodiments, different can mean that at least one position is different between the DNA sequence bound by one expression repressor and the DNA sequence bound by another expression repressor, or that there is at least one position in the DNA sequence bound by one expression repressor that is not present in the DNA sequence bound by another expression repressor.

[0884] In some embodiments, the first DNA sequence may be located on a first genomic DNA strand and the 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.

[0885] In some embodiments, the expression repression system comprises two or more (e.g., two) expression repressors and the plurality (e.g., two) expression repressors comprise targeting moieties that bind to different DNA sequences. In such embodiments, the first targeting moiety can bind to a first DNA sequence and the second DNA targeting moiety can bind to a second DNA sequence, wherein the first and second DNA sequences are different and do not overlap.

[0886] In some embodiments, the expression repressors of the expression repressor system each comprise a different effector portion (e.g., the first, second, third or additional expression repressors each comprise an effector portion that is different from one another). For example, an expression repressor system may comprise a first expression repressor and a second expression repressor, wherein the first expression repressor comprises a first effector portion (e.g., comprising a DNA methyltransferase or a functional fragment thereof), and the second expression repressor comprises a second effector portion that is different from the first effector portion (e.g., comprising a transcription repressor (e.g., KRAB) or a functional fragment thereof). In some embodiments, different can mean comprising different types of effector portions. In other embodiments, different can refer to comprising different variants of the same type of effector portion, for example, the first effector portion comprises a first DNA methyltransferase (e.g., having a first site specificity or amino acid sequence) and the second effector portion comprises a second DNA methyltransferase (e.g., having a second site specificity or amino acid sequence).

[0887] In some embodiments, the expression repressor system comprises a first expression repressor comprising a first effector portion and a second expression repressor comprising a second effector portion, wherein the first effector portion comprises a protein selected from the group consisting of: MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, S IRT2, 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, and the second effector portion comprises a different protein selected from the group consisting of: 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, SIR T6, 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 functional variants or fragments thereof.

[0888] In some embodiments, the first or second effector portion comprises a DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any one thereof) and the other effector portion comprises a transcriptional repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or any one thereof). In some embodiments, the first or second effector portion comprises a histone methyltransferase activity and the other effector portion comprises a 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 one thereof). In some embodiments, the first or second effector portion comprises a histone methyltransferase activity and the other effector portion comprises a DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any one thereof). In some embodiments, the first or second effector moiety comprises DNA methyltransferase activity and the other effector moiety comprises transcriptional repressor activity. In some embodiments, the first or second effector moiety comprises histone methyltransferase activity and the other effector moiety comprises transcriptional repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any one thereof).

[0889] In certain embodiments, two or more (for example, all) expression repressors of expression repressor system are not covalently associated with each other, for example, each expression repressor is not covalently associated with any other expression repressor.In another embodiment, two or more expression repressors of expression repressor system are covalently associated with each other.In one embodiment, expression repressor system comprises the first expression repressor and the second expression repressor that are arranged on the same polypeptide, for example, as fusion molecule, for example, is connected with optional joint by peptide bond.In certain embodiments, peptide is self-cleavage peptide, for example T2A self-cleavage peptide.In one embodiment, expression repressor system comprises and is connected by non-peptide bond, for example the first expression repressor and the second expression repressor that are puted together.

[0890] connector

[0891] As disclosed herein, expression repressors or expression repressor systems may comprise one or more linkers. A linker may connect a targeting moiety to an effector moiety, an effector moiety to another effector moiety, or a targeting moiety to another targeting moiety. The linker may be a chemical bond, such as one or more covalent bonds 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 can be between 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 in length, or greater than or equal to 2, 5, 10, 15, 20, 25, or 30 amino acids in length (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length). In some embodiments, linkers can be used to separate a first domain or portion from a second domain or portion, e.g., to separate a DNA-targeting moiety from an effector moiety. In some embodiments, for example, a linker can be positioned between a DNA-targeting moiety and an effector moiety, e.g., to provide molecular flexibility of the secondary and tertiary structures. The linker may comprise a flexible, rigid and / or cleavable linker as described herein. In some embodiments, the linker comprises at least one glycine, alanine, and serine amino acid to provide flexibility. In some embodiments, the linker is a hydrophobic linker, for example comprising a negatively charged sulfonate group, a polyethylene glycol (PEG) group, or a pyrophosphate diester group. In some embodiments, the linker is cleavable to selectively release the moiety (e.g., polypeptide) from the modulator, but is sufficiently stable to prevent premature cleavage.

[0892] In some embodiments, one or more portions and / or domains of the expression repressors described herein are connected to one or more linkers. In some embodiments, the expression repressor may comprise a linker between the targeting moiety and the effector moiety. In some embodiments, the linker may 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 may comprise a first linker between the first targeting moiety and the first effector moiety, and a second linker between the second targeting moiety and the second effector moiety. In some embodiments, the first and second linkers may be the same. In some embodiments, the first and second linkers may be different. In some embodiments, the first linker may comprise an amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity thereto, and the second linker may comprise an amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity thereto.

[0893] As known to those skilled in the art, the most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linkers). Flexible linkers can be useful for connecting domains / parts that require a certain degree of mobility or interaction, and can include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, and thus reduce adverse interactions between the linker and the part / domain. In some embodiments, the linker is a GS linker or a variant thereof, such as G4S (SEQ ID NO: 207).

[0894] Rigid linkers are useful for maintaining a fixed distance between domains / parts and maintaining their independent functions. Rigid linkers can also be useful when the spatial separation of domains is crucial to maintaining the stability or biological activity of one or more components in the fusion. Rigid linkers can have alpha helical structures or proline-rich sequences (XP). n , wherein X represents any amino acid, preferably Ala, Lys or Glu.

[0895] Cleavable linkers can release free functional domains in vivo. In some embodiments, linkers can be cleaved under specific conditions (e.g., in the presence of a reducing agent or protease). In vivo cleavable linkers can utilize the reversible nature of disulfide bonds. One example includes a thrombin-sensitive sequence (e.g., PRS) between two Cys residues. In vitro thrombin treatment of CPRSC results in cleavage of the thrombin-sensitive sequence, while the reversible disulfide bond remains intact. Such linkers are known and 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 linkers in fusion proteins can also be performed by proteases, which are expressed in vivo under certain conditions, in specific cells or tissues, or in certain restricted cell compartments. The specificity of many proteases provides for slow cleavage of the linker in a restricted compartment. In some embodiments, the cleavable linker can be a self-cleaving linker, such as a T2A peptide linker. In some embodiments, the linker may comprise a "ribosome skipping" sequence, such as a tPT2A linker.

[0896] Examples of molecules suitable for use in linkers as described herein include negatively charged sulfonic acid groups; lipids, such as poly(--CH2--) hydrocarbon chains, such as polyethylene glycol (PEG) groups, unsaturated variants thereof, hydroxylated variants thereof, amidated or other N-containing variants thereof; non-carbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecules capable of covalently linking two or more components of an expression repressor. Non-covalent linkers (e.g., hydrophobic lipid globules to which a polypeptide is linked) may also be included, for example, through a hydrophobic region of a polypeptide or a hydrophobic extension of a polypeptide, such as a series of residues rich in leucine, isoleucine, valine, or possibly alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues. The components of the expression repressor can be linked using charge-based chemistry such that a positively charged component of the expression repressor is linked to a negative charge on another component.

[0897] Targeting moiety

[0898] The present disclosure provides, for example, an expression repressor comprising a targeting moiety that specifically targets, for example, a genomic sequence element (e.g., promoter, TSS, or anchor sequence) in conjunction with a target gene, near the target gene, and / or operably connected to a target gene. The targeting moiety can specifically bind to a DNA sequence, such as a DNA sequence associated with a target gene (e.g., MYC). Any molecule or compound that specifically binds to a DNA sequence can be used as a targeting moiety.

[0899] In certain embodiments, targeting moiety targets (for example, in conjunction with) the component of genome complex (for example, ASMC).In certain embodiments, targeting moiety targets (for example, in conjunction with) the expression control sequence (for example, promoter or enhancer) that is operably connected with target gene.In certain embodiments, targeting moiety targets (for example, in conjunction with) target gene or a part for target gene.The target of targeting moiety can be referred to as the component of its targeting.The component of targeting can be any genomic sequence element or target gene itself that is operably connected with target gene, includes but not limited to promoter, enhancer, anchor sequence, exon, intron, UTR coding sequence, splice site or transcription start site.In certain embodiments, targeting moiety specifically binds one or more target anchor sequences (for example, in cell) and is not in conjunction with non-targeted anchor sequence (for example, in same cell).

[0900] In some embodiments, the disclosure provides an expression repressor comprising: a targeting portion that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19 or 20 nucleotides of the sequence of SEQ ID NO: 1, 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, or 75, 76, 78, 79, 80, 81, 84, 85, or 86, wherein the expression repressor is capable of reducing the expression of MYC.

[0901] In some embodiments, the present disclosure provides an expression repressor comprising: a targeting portion that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of a sequence of SEQ ID NO: 2 or 77, 82, or 83, and wherein the expression repressor is capable of reducing expression of a target gene (e.g., MYC). In some embodiments, the expression repressor comprises an effector portion.

[0902] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety, wherein the targeting moiety binds to a genomic locus within 1400 nt upstream or downstream of SEQ ID NO:4.

[0903] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of a sequence of SEQ ID NO: 4, 77, 82, or 83.

[0904] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, 96, or 108.

[0905] In some embodiments, the targeting moiety can be or comprise 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 comprises an effector moiety. In some embodiments, the expression repressor comprises a plurality of targeting moieties, wherein each targeting moiety does not detectably bind (e.g., does not bind) another targeting moiety. In some embodiments, the expression repression system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors comprises a targeting moiety, wherein each targeting moiety does not detectably bind, e.g., does not bind to another targeting moiety. In some embodiments, the expression repression system comprises a first expression repressor comprising a first targeting moiety and a second expression repressor comprising a second targeting moiety, wherein the first targeting moiety does not detectably bind, e.g., does not bind to the second targeting moiety. In some embodiments, the expression repression system comprises a first expression repressor comprising a first targeting moiety and a second expression repressor comprising a second targeting moiety, wherein the first targeting moiety does not detectably bind, e.g., does not bind to another first targeting moiety, and the second targeting moiety does not detectably bind, e.g., does not bind to another second targeting moiety. In some embodiments, targeting moieties used in the compositions and methods described herein are functional (eg, bind to a DNA sequence) in a monomeric state (eg, a non-dimeric state).

[0906] In some embodiments, the targeting moiety can be or comprise a CRISPR / Cas domain, a TAL effector domain, a Zn finger domain, or a nucleic acid molecule. In some embodiments, the targeting moiety is or comprises a DNA binding domain from a nuclease. Exemplary nucleic acid targeting moieties are described, for example, in International Application WO / 2022 / 132195 (incorporated herein by reference in its entirety).

[0907] As disclosed herein, expression repressors or systems comprising repressors may comprise nucleic acids, such as 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, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via phosphodiester bonds. As will be clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising a single nucleic acid residue. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is or comprises greater than 50% ribonucleotides and is referred to herein as ribonucleic acid (RNA). In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a 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, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone rather than phosphodiester bonds and are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds rather than phosphodiester bonds. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-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, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In certain embodiments, nucleic acid has a nucleotide sequence encoding a functional gene product (such as RNA or protein). In certain embodiments, nucleic acid comprises one or more introns. In certain embodiments, nucleic acid is prepared by separation from a natural source, by enzymatic synthesis (in vivo or in vitro) based on the polymerization of a complementary template, by replication in a recombinant cell or system, and by chemical synthesis. As used herein, "recombinant" refers to any nucleic acid that is non-naturally occurring when used to describe nucleic acid.In some embodiments, the nucleic acid is 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 length. In some embodiments, the length of the nucleic acid can be about 2nt to about 5000nt, about 10nt to about 100nt, about 50nt to about 150nt, about 100nt to about 200nt, about 150nt to about 250nt, about 200nt to about 300nt, about 250nt to about 350nt, about 300nt to about 500nt, about 10nt to about 1000nt, about 50nt to about 1000nt, about 100nt to about 1000nt, about 1000nt to about 2000nt, about 2000nt to about 3000nt, about 3000nt to about 4000nt, about 4000nt to about 5000nt, or any range therebetween. In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that comprises at least one element encoding a polypeptide or is the complement of a sequence encoding a polypeptide. In one embodiment, the nucleic acid has enzymatic activity.

[0908] CRISPR / Cas domains

[0909] In some embodiments, the targeting moiety is or comprises a CRISPR / Cas domain, for example, as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference). The CRISPR / Cas protein may comprise a CRISPR / Cas effector and optionally one or more other domains. The CRISPR / Cas domain typically has structural and / or functional similarities to proteins (e.g., Cas proteins) involved in clustered regulatory interspaced short palindromic repeats (CRISPR) systems. The CRISPR / Cas domain optionally comprises a guide RNA, for example, a single guide RNA (sgRNA). In some embodiments, the gRNA included in the CRISPR / Cas domain is non-covalently bound to the CRISPR / Cas domain.

[0910] In some embodiments, the Cas protein is modified to inactivate the nuclease, such as a nuclease-deficient Cas. In some embodiments, the Cas protein is a Cas9 protein. While on a specific DNA sequence targeted by a gRNA, wild-type Cas9 produces a double-strand break (DSB), many CRISPR endonucleases with modified functionality are available, for example: the "nickase" version of Cas9 only produces a single-strand break; catalytically inactive Cas9 ("dCas9") does not cut the target DNA. In some embodiments, the binding of dCas to a DNA sequence can interfere with transcription at the site through steric hindrance. In some embodiments, the DNA targeting portion is or comprises a catalytically inactive Cas, such as a dCas. Many catalytically inactive Cas proteins are known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, such as D10A and H840A mutations.

[0911] TAL effector domain

[0912] In some embodiments, the DNA targeting portion is or comprises a TAL effector domain, for example, as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference). A TAL effector domain, such as a TAL effector domain that specifically binds to a DNA sequence, comprises a plurality of TAL effector repeats or fragments thereof, and optionally additional portions of one or more naturally occurring TAL effector repeats (e.g., the N-terminus and / or C-terminus of a plurality of TAL effector domains), wherein each TAL effector repeat recognizes a nucleotide. The TAL effector protein may comprise a TAL effector and optionally one or more other domains. Many TAL effector domains are known to those skilled in the art and are commercially available, for example, from Thermo Fisher Scientific.

[0913] Zn finger domain

[0914] In some embodiments, the DNA targeting moiety is or comprises a Zn finger domain. The Zn finger domain comprises a Zn finger, such as a naturally occurring Zn finger or an engineered Zn finger, or a fragment thereof. Many Zn fingers are known to those skilled in the art and are commercially available, for example, from Sigma-Aldrich. In general, a Zn finger domain comprises a plurality of Zn fingers, wherein each Zn finger recognizes three nucleotides. The Zn finger protein may comprise a Zn finger domain and optionally one or more other domains. In some embodiments, the Zn finger domain or Zn finger is a Zn finger domain or Zn finger as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0915] In addition, as disclosed in these and other references, zinc fingers and / or multi-finger zinc finger domains can be linked together using any suitable linker sequence (including, for example, a linker of 5 or more amino acids in length). See also U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences of 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. Additionally, enhancing the binding specificity of zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02 / 077227.

[0916] In certain embodiments, the DNA targeting moiety comprises a Zn finger domain comprising an engineered zinc finger that binds to a target DNA sequence (in a sequence-specific manner). In some embodiments, the Zn finger domain comprises one Zn finger or a fragment thereof. In some embodiments, the Zn finger domain comprises a plurality of Zn fingers (or fragments thereof), such as 2, 3, 4, 5, 6 or more Zn fingers (and optionally, no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 Zn fingers). In some embodiments, the Zn finger domain comprises at least three Zn fingers. In some embodiments, the Zn finger domain comprises four, five or six Zn fingers. In some embodiments, the Zn finger domain comprises 8, 9, 10, 11 or 12 Zn fingers. In some embodiments, a Zn finger domain comprising three Zn fingers recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger domain comprising four Zn fingers recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger domain comprising six Zn fingers recognizes a target DNA sequence comprising 18 to 21 nucleotides.

[0917] In some embodiments, the targeting domain comprises a two-handed zinc finger protein. A two-handed zinc finger protein is a protein in which two clusters of zinc fingers are separated by an intervening amino acid, such that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two-handed zinc finger binding protein is SIP1, in which a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three zinc fingers is located at the carboxyl terminus (see Remade et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these domains is capable of binding to a unique target sequence, and the spacing between the two target sequences can comprise many nucleotides.

[0918] In some embodiments, the expression repressor comprises a targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a Zn finger domain comprising a Zn finger (ZFN) that binds to a target sequence (e.g., a promoter or transcription start site (TSS) sequence operably linked to a target gene (e.g., MYC), e.g., a sequence proximal to a transcriptional regulatory element, e.g., an anchor sequence comprising an anchor sequence-mediated junction (ASMC) of a target gene (e.g., MYC), e.g., a sequence proximal to the anchor sequence). In some embodiments, the ZFN can be engineered to carry an epigenetic effector molecule to the target site. In some embodiments, the targeting moiety comprises a Zn finger domain comprising 2, 3, 4, 5, 6, 7, or 8 zinc fingers. The amino acid sequences of exemplary targeting moieties disclosed herein are listed in Table 4. The nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 5. In some embodiments, the expression repressors or systems described herein comprise a targeting moiety having a sequence listed in Table 4, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical thereto. In some embodiments, the nucleic acids described herein comprise a sequence listed in Table 5, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical thereto.

[0919] Table 4: Amino acid sequences of exemplary targeting moieties

[0920]

[0921]

[0922] Table 5: Nucleotide sequences of exemplary targeting moieties

[0923]

[0924]

[0925]

[0926]

[0927]

[0928] In some embodiments, the expression repressor comprises a targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a Zn finger domain comprising a Zn finger (ZFN) that binds to a target sequence in the mouse genome (e.g., a promoter or transcription start site (TSS) sequence operably linked to a target gene (e.g., MYC), e.g., a sequence proximal to a transcriptional regulatory element, e.g., an anchor sequence comprising an anchor sequence-mediated junction (ASMC) of a target gene (e.g., MYC), e.g., a sequence proximal to the anchor sequence). In some embodiments, the ZFN can be engineered to carry an epigenetic effector molecule to the target site. In some embodiments, the targeting moiety comprises a Zn finger domain comprising 2, 3, 4, 5, 6, 7, or 8 zinc fingers. The amino acid sequences of exemplary targeting moieties disclosed herein are listed in Table 14. The nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 15. In some embodiments, the expression repressors or systems described herein comprise a targeting moiety having a sequence listed in Table 14, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical thereto. In some embodiments, the nucleic acids described herein comprise a sequence listed in Table 15, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical thereto.

[0929] Table 14: Amino acid sequences of exemplary mouse-specific targeting moieties

[0930]

[0931] Table 15: Nucleotide sequences of exemplary mouse-specific targeting moieties

[0932]

[0933]

[0934] Effector part

[0935] In some embodiments, the expression repressors of the present disclosure comprise one or more effector moieties. In some embodiments, when used as part of an expression repressor or expression repression system described herein, the effector moiety reduces the expression of a target gene in a cell.

[0936] In some embodiments, the effector moiety has a function unrelated to the binding of the targeting moiety. For example, the effector moiety can target (e.g., bind) a genomic sequence element or genomic complex component proximal to the genomic sequence element targeted by the targeting moiety, or recruit a transcription factor. As a further example, the effector moiety can comprise an enzymatic activity, such as a genetic modification function.

[0937] In some embodiments, the effector portion comprises an epigenetic modification portion. In some embodiments, the effector portion comprises a DNA modification function, such as a DNA methyltransferase. In some embodiments, the effector portion is or comprises a protein selected from the group consisting of MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any one thereof.

[0938] In some embodiments, the effector portion comprises a transcriptional repressor. In some embodiments, the transcriptional repressor blocks the recruitment of factors that stimulate or promote transcription (e.g., transcription of a target gene). In some embodiments, the transcriptional repressor recruits factors that inhibit, for example, transcription (e.g., transcription of a target gene). In some embodiments, the effector portion, e.g., a transcriptional repressor, is or comprises a protein selected from the group consisting of KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any one thereof.

[0939] In some embodiments, the effector moiety promotes epigenetic modification, e.g., directly or indirectly. For example, the effector moiety can indirectly promote epigenetic modification by recruiting endogenous proteins that epigenetically modify chromatin. The effector moiety can directly promote epigenetic modification by catalyzing epigenetic modification, wherein the effector moiety has enzymatic activity and places epigenetic markers directly on chromatin.

[0940] In some embodiments, the effector portion comprises a histone modification function, such as a histone methyltransferase, a histone demethylase, or a histone deacetylase activity. In some embodiments, the effector portion is or comprises a protein selected from the group consisting of KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional variant or fragment of any one thereof. In some embodiments, the effector portion is or comprises a protein selected from the group consisting of 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 one thereof.

[0941] In some embodiments, the effector portion comprises a protein having a function as described herein. In some embodiments, the effector portion is or comprises a protein selected from the group consisting of: KRAB (e.g., a protein encoded by NP_056209.2 or NM_015394.5); a SET domain (e.g., a SET domain of: SETDB1 (e.g., a protein encoded by NP_001353347.1 or NM_001366418.1); EZH2 (e.g., a protein encoded by NP_004447.2 or NM_004456.5); G9A (e.g., a protein encoded by NP_001350618.1 or NM_001363689.1); or SUV39H1 (e.g., a protein encoded by NP_003164.1 or NM_001366418.1). NM_003173.4); histone demethylase LSD1 (e.g., a protein according to NP_055828.2 or encoded by NM_015013.4); FOG1 (e.g., the N-terminal residues of FOG1) (e.g., a protein according to NP_722520.2 or encoded by NM_153813.3); or KAP1 (e.g., a protein according to NP_005753.1 or NM_005762.3); a functional fragment or variant of any of them, or a polypeptide having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the above sequences.

[0942] In some embodiments, the effector portion is or comprises a protein selected from the group consisting of: DNMT3A (e.g., human DNMT3A) (e.g., a protein according to NP_072046.2 or encoded by NM_022552.4); DNMT3B (e.g., a protein according to NP_008823.1 or encoded by NM_006892.4); DNMT3L (e.g., a protein according to NP_787063.1 or encoded by NM_175867.3); a DNMT3A / 3L complex, bacterial MQ1 (e.g., according to CAA35058.1 or P15840.3); a functional fragment of any one thereof, or a polypeptide having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the above sequences.

[0943] In another aspect, the present disclosure relates to an expression repressor or polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moieties, wherein the one or more effector moieties are or comprise a Krueppel-associated box (KRAB), e.g., a protein encoded by NP_056209.2, or NM_015394.5, or a functional variant or fragment thereof. In some embodiments, the KRAB is a synthetic KRAB construct. In some embodiments, the KRAB comprises the amino acid sequence of SEQ ID NO: 18: DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 18)

[0944] In some embodiments, the KRAB effector portion is encoded by the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 51, 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.

[0945] GACGCCAAGAGCCTGACCGCCTGGAGCCGGACCCTGGTGACCTTCAAGGACGTGTTCGTGGACTTCACCCGGGAGGAGTGGAAGCTGCTGGACACCGCCCAGCAGATCCTGTACCGGAACGTGATGCTGGAGAACTACAAGAACCT GGTGAGCCTGGGCTACCAGCTGACCAAGCCCGACGTGATCCTGCGGCTGGAGAAGGGCGAGGAGCCCTGGCTGGTGGAGCGGGAGATCCACCAGGAGACCCACCCCGACAGCGAGACCGCCTTCGAGATCAAGAGCAGCGTG(SEQ IDNO:51)

[0946] In some embodiments, the KRAB used in the polypeptides or expression repressors described herein is a variant, e.g., comprising one or more mutations relative to the KRAB sequence of SEQ ID NO: 18. In some embodiments, the KRAB variant comprises one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 18.

[0947] In some embodiments, the polypeptide or expression repressor is a fusion protein comprising an effector moiety that is or comprises KRAB and a DNA targeting moiety.

[0948] In another aspect, the present disclosure relates to expression repressors or polypeptides comprising one or more (e.g., one) targeting moiety and one or more effector moieties, wherein the one or more effector moieties are or comprise MQ1, e.g., bacterial MQ1, or functional variants or fragments thereof. In some embodiments, MQ1 is Mollicutes spiroplasma MQ1. In some embodiments, MQ1 is Spiroplasma monobiae MQ1. In some embodiments, MQ1 is MQ1 from strain ATCC 33825 and / or corresponding to Uniprot ID P15840. In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, an effector domain described herein comprises 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 in 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.

[0949] SKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSWNSKNPVSNGYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRGSGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLHKK NEEELNQWKQKLESLGYQNSIEVLNAADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKSNINKASL IGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDSQDGKRVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGG(SEQ ID NO:19)

[0950] MSKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSWNSKNPVSNGYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRGSGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLHKK NEEELNQWKQKLESLGYQNSIEVLNAADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKSNINKASL IGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDSQDGKRVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGG(SEQ ID NO:87)

[0951] In some embodiments, MQ1 is encoded by the nucleotide sequence of SEQ ID NO: 52 or 132. In some embodiments, the nucleic acids described herein comprise the sequence of SEQ ID NO: 52, 132, or a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical thereto, or that 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.

[0952]

[0953]

[0954] In some embodiments, the MQ1 used in the polypeptides or expression repressors described herein is a variant, e.g., comprising one or more mutations relative to wild-type MQ1 (SEQ ID NO: 19). In some embodiments, the MQ1 variant comprises one or more amino acid substitutions, deletions, or insertions relative to wild-type MQ1, e.g., MQ1 of SEQ ID NO: 19. In some embodiments, the MQ1 variant comprises a K297P substitution. In some embodiments, the MQ1 variant comprises an N299C substitution. In some embodiments, the MQ1 variant comprises an E301Y substitution. In some embodiments, the MQ1 variant comprises a Q147L substitution (e.g., and has reduced DNA methyltransferase activity relative to wild-type MQ1). In some embodiments, the MQ1 variant comprises a K297P, N299C, and E301Y substitution (e.g., and has reduced DNA binding affinity relative to wild-type MQ1). In some embodiments, the MQ1 variant comprises a Q147L, K297P, N299C, and E301Y substitution (e.g., and has reduced DNA methyltransferase activity and DNA binding affinity relative to wild-type MQ1).

[0955] In some embodiments, the one or more effector moieties are or comprise DNMT1, e.g., human DNMT1, or a functional variant or fragment thereof. In some embodiments, DNMT1 is human DNMT1, e.g., corresponding to gene ID 1786, e.g., corresponding to UniProt ID P26358.2. In some embodiments, DNMT1 comprises the amino acid sequence of SEQ ID NO: 20 as described in International Application WO / 2022 / 132195 (incorporated herein by reference in its entirety). In some embodiments, the effector domains described herein comprise a sequence according to, e.g., SEQ ID NO: 20 as described in International Application WO / 2022 / 132195 (incorporated herein by reference in its entirety), or a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity thereto, or a sequence that differs from it by 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. In some embodiments, DNMT1 is encoded by the nucleotide sequence of SEQ ID NO: 53, e.g., as described in International Application WO / 2022 / 132195, the entire contents of which are incorporated herein by reference, 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.

[0956] Exemplary effector moieties can include, but are not limited to, any effector moiety as described in International Application WO / 2022 / 132195 (the entire contents of which are incorporated herein by reference).

[0957] Other parts

[0958] The expression repressor may further comprise one or more additional moieties (e.g., in addition to the one or more targeting moieties and the one or more effector moieties). In some embodiments, the additional moiety is selected from a labeling moiety or a monitoring moiety, a cleavable moiety (e.g., a cleavable moiety located between the DNA targeting moiety and the effector moiety or located at the N-terminus or C-terminus of the polypeptide), a small molecule, a membrane translocating polypeptide, or a pharmaceutical agent moiety.

[0959] Exemplary expression repressors

[0960] The following exemplary expression repressors are presented for illustrative purposes only and are not intended to be limiting.

[0961] In some embodiments, the expression repressor comprises a targeting portion comprising a Zn finger domain and an effector portion comprising a KRAB (e.g., a KRAB domain). In some embodiments, the expression repressor is encoded by a nucleic acid sequence (e.g., a nucleic acid encoding an expression repressor (e.g., a cDNA)) of any one of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 189, 194, 195, and 196. The nucleic acid sequences of these exemplary expression repressors are disclosed in Table 6. In some embodiments, the nucleic acids described herein comprise a nucleic acid sequence of any one of SEQ ID NOs: 55-60, 189, 194-196, or a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity thereto or differing therefrom by 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. In some embodiments, the nucleic acid sequence comprises a poly A sequence, while in other embodiments, the nucleic acid lacks a poly A sequence.

[0962] Table 6: Nucleotide sequences of exemplary ZF-KRAB effectors

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969] In some embodiments, the expression repressor comprises a targeting portion and an effector portion, the targeting portion comprising a Zn finger domain (e.g., having an amino acid sequence according to any one of SEQ ID NOs: 5-10 or 169-172), the effector portion comprising KRAB (e.g., amino acid sequence SEQ ID NO: 18), such as a KRAB domain. In some embodiments, the expression repressor described herein comprises an amino acid sequence of any one of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 139-144, 177-180, or 183-186. The protein sequences of these exemplary expression repressors are disclosed in Table 7. In some embodiments, the expression repressor described herein comprises the amino acid sequence of any one of SEQ ID NOs: 22-27, 139-144, 177-180, 183-186, 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.

[0970] Table 7: Amino acid sequences of exemplary zinc finger-KRAB effectors

[0971]

[0972]

[0973]

[0974]

[0975] In some embodiments, the expression repressor comprises a targeting portion comprising a Zn finger domain (e.g., a domain encoded by the nucleotide sequence of any one of SEQ ID NOs: 44-49 or 115) and an effector portion comprising MQ1, e.g., bacterial MQ1 (e.g., encoded by the nucleotide sequence of SEQ ID NO: 52). In some embodiments, the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 116, 117, 118, or 130. The nucleic acid sequences of these exemplary expression repressors are disclosed in Table 8. In some embodiments, the nucleic acids described herein comprise the nucleic acid sequence of any one of SEQ ID NOs: 61-66, 116-118, 130, or a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical thereto, or that 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. In some embodiments, the nucleic acid sequence comprises a poly A sequence, while in other embodiments, the nucleic acid lacks a poly A sequence. For example, in some embodiments, a nucleic acid described herein comprises a sequence according to any of SEQ ID NOs: 61-66, 116-118, or 130 (or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a sequence that 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) but lacks a 3' poly A sequence or comprises a 3' poly A sequence of shorter length.

[0976] Table 8: Nucleotide sequences of exemplary ZF-MQ1 effectors

[0977]

[0978]

[0979]

[0980]

[0981]

[0982]

[0983]

[0984]

[0985] In some embodiments, the expression repressor comprises a targeting moiety comprising a Zn finger domain (e.g., comprising an amino acid sequence of any one of SEQ ID NOs: 11-14) and an effector moiety comprising MQ1, e.g., bacterial MQ1 (e.g., SEQ ID NO: 19). In some embodiments, the expression repressor comprises an amino acid sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 129, and 145-149. The protein sequences of these exemplary expression repressors are disclosed in Table 9. In some embodiments, the expression repressor described herein comprises an amino acid sequence of any one of SEQ ID NOs: 28-33, 129, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a sequence that differs from it by 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.

[0986] Table 9 Amino acid sequences of exemplary ZF-MQ1 effectors

[0987]

[0988]

[0989]

[0990]

[0991] In some embodiments, the expression repressor comprises a targeting moiety comprising a Zn finger domain (e.g., having an amino acid sequence of any one of SEQ ID NOs: 11-14) and an effector moiety comprising MQ1, e.g., bacterial MQ1 (e.g., SEQ ID NO: 87).

[0992] In some embodiments, the expression repressor comprises a targeting portion comprising a Zn finger domain (e.g., a domain encoded by the nucleotide sequence of any one of SEQ ID NOs: 166-168) and an effector portion comprising MQ1, e.g., bacterial MQ1 (e.g., encoded by the nucleotide sequence of SEQ ID NO: 52). In some embodiments, the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 157, 158, or 159. The nucleic acid sequences of these exemplary expression repressors are disclosed in Table 16. In some embodiments, the nucleic acid described herein comprises the nucleic acid sequence of any one of SEQ ID NOs: 166-168, or a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity thereto or differing therefrom by 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. In some embodiments, the nucleic acid sequence comprises a poly A sequence, while in other embodiments, the nucleic acid lacks a poly A sequence. For example, in some embodiments, a nucleic acid described herein comprises a sequence according to any one of SEQ ID NOs: 166-168 (or a sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a sequence that 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) but lacks a 3' poly A sequence or comprises a 3' poly A sequence of shorter length.

[0993] Table 16: Nucleotide sequences of exemplary mouse-specific ZF-MQ1 effectors

[0994]

[0995]

[0996]

[0997] In some embodiments, the expression repressor comprises a targeting moiety comprising a Zn finger domain (e.g., comprising the amino acid sequence of any one of SEQ ID NOs: 154-156) and an effector moiety comprising MQ1, e.g., bacterial MQ1 (e.g., SEQ ID NO: 19). In some embodiments, the expression repressor comprises the amino acid sequence of any one of SEQ ID NOs: 160-165. The protein sequences of these exemplary expression repressors are disclosed in Table 17. In some embodiments, the expression repressor described herein comprises the amino acid sequence of any one of SEQ ID NOs: 160-165, or a sequence having at least 80%, 85%, 90%, 95%, 99% or 100% identity thereto, or a sequence that 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.

[0998] Table 17: Amino acid sequences of exemplary ZF-MQ1 effectors

[0999]

[1000]

[1001]

[1002] In some embodiments, the present disclosure provides an expression repressor system comprising a first targeting moiety containing a first ZF, a first effector moiety containing a DNA methyltransferase, such as MQ1, or a functional fragment thereof, a second targeting moiety containing a second ZF, and a second effector moiety containing a KRAB, such as a KRAB domain. In some embodiments, the expression repressor system is encoded by a first nucleic acid encoding the first targeting moiety and the first effector moiety (wherein expression is driven by a first promoter or IRES) and a second nucleic acid encoding the second targeting moiety and the second effector moiety (wherein expression is driven by a second promoter or IRES). In some embodiments, a monocistronic seq...

Claims

1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (1) A nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a targeting moiety that binds to the MYC promoter, and (b) optionally, an effector moiety, wherein the expression repressor is capable of reducing the expression of MYC; and (2) Compounds having the general structure of formula (I): in: R 1 is alkyl, cycloalkyl or aryl; R 2 is halo, alkyl or H; R 3 , R 4 , R 5 , R 6 , R 7 are each independently H or alkyl; R 8 and R 9 together form an optionally substituted aromatic ring; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

2. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (1) A nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a first targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of a sequence of any one of SEQ ID NOs: 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, and (b) optionally, a first effector moiety, wherein the expression repressor is capable of reducing the expression of MYC; and (2) Compounds having the general structure of formula (I): in: R 1 is alkyl, cycloalkyl or aryl; R 2 is halo, alkyl or H; R 3 , R 4 , R 5 , R 6 , R 7 are each independently H or alkyl; R 8 and R 9 together form an optionally substituted aromatic ring; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

3. The method of claim 1 or 2, wherein R 1 is an alkyl group or a cycloalkyl group.

4. A method as claimed in any one of the preceding claims, wherein R 1 =Cyclopropane.

5. A method as claimed in any one of the preceding claims, wherein R 2 It is halogenated.

6. A method as claimed in any one of the preceding claims, wherein R 2 =Cl.

7. A method as claimed in any one of the preceding claims, wherein R 3 , R 4 , R 5 , R 6 , R 7 Each is H.

8. A method as claimed in any one of the preceding claims, wherein the substituted aromatic ring is a substituted phenyl ring.

9. The method of any one of the preceding claims, wherein the compound of formula (I) comprises: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

10. The method of any one of the preceding claims, wherein the compound of formula (I) is administered orally.

11. The method of any one of the preceding claims, wherein the compound of formula (I) is administered at a dose of 6-10 (eg, about 8) or 10-14 (eg, about 12) mg per day.

12. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (1) A nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a targeting moiety that binds to the MYC promoter, and (b) optionally, an effector moiety, wherein the expression repressor is capable of reducing the expression of MYC; and (2) Compounds having the general structure of formula (II): Where R1 is: (1) Where X = C or N, provided that one or both X are N; and R 12 It is an electron pair, H or methyl; (2) halogen; or (3) Methyl halogen; R2 is H or methyl; R3 is H or methyl; R4 is H or methyl; R5 is -OH or -O-methyl; R6 is H or methyl; R7 is H or methyl; R8 is -(CH2) n -N(R 13 )2, where n = 2-6, and R 13 =H or methyl; R9 is H or methyl; R 10 Yes-(CO)-R 14 , where R 14 is an alkyl or alkene; and R 11 is H or methyl; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

13. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (1) A nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a first targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of a sequence of any one of SEQ ID NOs: 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, and (b) optionally, a first effector moiety, wherein the expression repressor is capable of reducing the expression of MYC; and (2) Compounds having the general structure of formula (II): Where R1 is: (1) Where X = C or N, provided that one or both X are N; and R 12 It is an electron pair, H or methyl; (2) halogen; or (3) Methyl halogen; R2 is H or methyl; R3 is H or methyl; R4 is H or methyl; R5 is -OH or -O-methyl; R6 is H or methyl; R7 is H or methyl; R8 is -(CH2) n -N(R 13 )2, where n = 2-6, and R 13 =H or methyl; R9 is H or methyl; R 10 Yes-(CO)-R 14 , where R 14 is an alkyl or alkene; and R 11 is H or methyl; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

14. The method of claim 12 or 13, wherein R1 is: Where X = C or N, provided that one or both X are N; and R 15 It is an electron pair, H or methyl.

15. The method of any one of claims 12-14, wherein R1 is:

16. The method of any one of claims 12-15, wherein R1 is:

17. The method of any one of claims 12 to 16, wherein: R2 is H; R3 is H; R4 is H; R5 is -O-methyl; R6 is H; R7 is methyl; R8 is -(CH2)2-N(methyl)2, wherein n=2-6, and R 13 =H or methyl; R9 is H; R 10 is -(CO)-ethylene; and R 11 It's H.

18. The method of any one of claims 12-17, wherein formula (II) is:

19. The method of any one of claims 12-18, wherein the compound of formula (II) is administered orally.

20. The method of any one of claims 12-19, wherein the compound of formula (II) is administered at a dose of 40-100 (eg, about 80) mg per day.

21. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (I) a nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a targeting moiety that binds to the MYC promoter, and (b) optionally, an effector moiety, wherein the expression repressor is capable of reducing the expression of MYC; and (II) ERK inhibitors (eg, uritinib), AKT inhibitors (eg, MK-2206), or MEK inhibitors (eg, trametinib).

22. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (I) a nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a first targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of a sequence of any one of SEQ ID NOs: 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, and (b) optionally, a first effector moiety, wherein the expression repressor is capable of reducing the expression of MYC; and (II) ERK inhibitors (eg, uritinib), AKT inhibitors (eg, MK-2206), or MEK inhibitors (eg, trametinib).

23. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: A nucleic acid (eg, RNA, eg, mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a targeting moiety that binds to the MYC promoter, and (b) optionally, an effector moiety, Among them, the expression repressor can reduce the expression of MYC; The cancer was resistant to osimertinib.

24. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: A nucleic acid (eg, RNA, eg, mRNA) encoding an expression repressor, wherein the expression repressor comprises: (a) a first targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of a sequence of any one of SEQ ID NOs: 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, and (b) optionally, a first effector moiety, The cancer was resistant to osimertinib.

25. The method of any one of the preceding claims, wherein the cancer is lung cancer, such as NSCLC.

26. The method of any one of the preceding claims, wherein the cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, or secondary liver cancer.

27. The method of any one of claims 1-26, wherein the nucleic acid comprises RNA, such as mRNA.

28. The method of any one of claims 2 to 27, wherein: The first targeting moiety binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, and The expression repressor comprises the first effector moiety, wherein the first effector moiety comprises a DNA methyltransferase.

29. The method of claim 28, wherein the first targeting moiety comprises a zinc finger domain.

30. The method of claim 28 or 29, wherein the first targeting moiety comprises 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 that 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.

31. The method of any one of claims 28-30, wherein the first effector moiety comprises MQ1 or a functional variant or fragment thereof.

32. The method of any one of claims 28-31, wherein the first effector portion comprises a sequence of SEQ ID NO: 19 or 87, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

33. The method of any one of claims 28-32, wherein the first effector portion comprises the sequence of SEQ ID NO: 129, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that 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.

34. The method of any one of claims 28, wherein the RNA comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 131, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto, or that differs therefrom by 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.

35. The method of any one of claims 28-34, wherein the RNA comprises a nucleotide sequence encoding the first effector portion, wherein the nucleotide sequence encoding the first effector portion comprises a sequence according to SEQ ID NO: 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.

36. The method of any one of claims 28 to 35, wherein the RNA comprises the nucleotide sequence according to SEQ ID NO: 130, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that 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.

37. The method of any one of claims 28-36, wherein the RNA further encodes a second expression repressor, wherein the second expression repressor comprises: a second targeting moiety that binds a second genomic locus, and The second effector moiety.

38. The method of claim 37, wherein the second targeting moiety binds to a second genomic locus comprising at least 14, 15, 16, 17, 18, 19 or 20 nucleotides of the sequence of SEQ ID NO:

77.

39. The method of claim 37 or 38, wherein the second targeting moiety comprises a zinc finger domain.

40. The method of any one of claims 37-39, wherein the second targeting moiety comprises 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 that 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.

41. The method of any one of claims 37-40, wherein the second effector moiety comprises KRAB or a functional variant or fragment thereof.

42. The method of any one of claims 37-41, wherein the second effector portion comprises 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.

43. The method of any one of claims 37-42, wherein the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24, or a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto or that 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.

44. The method of any one of claims 37-43, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 113, 196 or 208.

45. The method of any one of the preceding claims, wherein the nucleic acid is formulated in a lipid nanoparticle (LNP), wherein optionally the nucleic acid is encapsulated inside the LNP.

46. ​​The method of any one of the preceding claims, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor is administered as an IV infusion, eg, every 2 weeks, eg, over 80-120 minutes.

47. The method of any of the preceding claims, wherein the nucleic acid is administered at a dose of about 0.001 mg / kg to 1.5 mg / kg or about 0.002 mg / kg to 1.5 mg / kg, e.g., every 1, 2, 3, 4, 5, 6 or 7 weeks.

48. The method of any one of the preceding claims, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor and the compound having the general structure of Formula (I) or Formula (II) are administered on different days.

49. The method of any one of the preceding claims, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor and the compound having the general structure of Formula (I) or Formula (II) are administered on the same day.

50. The method of claim 49, wherein the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor is administered prior to administering the compound having the general structure of Formula (I) or Formula (II).

51. The method of claim 49 or 50, wherein the compound having the general structure of Formula (I) or Formula (II) is administered between 1-24 hours after administration of the nucleic acid (eg, RNA, eg, mRNA) encoding the expression repressor is completed.

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