Combination therapies involving MYC modulation

Abstract

The present disclosure relates to combination therapies. The combination therapy can include administration of an expression repressor and a kinase inhibitor. In some embodiments, the expression repressor includes a targeting moiety that binds to a MYC promoter, an anchor sequence, or a super enhancer. In some embodiments, the expression repressor includes an effector moiety that suppresses transcription or methylates DNA. The compositions and methods can be used for the treatment of cancers such as, for example, HCC.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 354,646, filed on June 22, 2022, and U.S. Provisional Patent Application No. 63 / 489,629, filed on March 10, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy created on June 19, 2023, is named O2057 - 7034WO_SL.xml and has a size of 282,044 bytes.

Background Art

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

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure provides, for example, a combination therapy comprising an expression repressor and a kinase inhibitor.

[0005] The present disclosure provides, inter alia, expression repressors and expression repressor systems that can be used, for example, to regulate, e.g., reduce, the expression of a target gene, such as MYC.

[0006] In some embodiments, the present disclosure provides a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, such as MYC, or to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising the target gene, such as MYC, or to a sequence proximal to the anchor sequence.

[0007] In some embodiments, the present disclosure relates to a nucleic acid encoding a first expression repressor, a second expression repressor, both, or a component thereof (e.g., gRNA, mRNA). In some embodiments, the nucleic acid encoding the expression repressor system is a polycistronic sequence. In some embodiments, the polycistronic sequence is a bicistronic sequence.

[0008] In some embodiments, the present disclosure relates to a method of treating a condition associated with overexpression of a target gene, such as MYC, in a subject, the method comprising administering to the subject an expression repressor or system, nucleic acid, or vector described herein, thereby treating the condition.

[0009] Any other features of any of the foregoing methods or compositions include one or more of the embodiments listed below.

[0010] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the embodiments listed below.

[0011] All publications, patent applications, patents, and other references (e.g., accession numbers in sequence databases) cited herein are hereby incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to in any table herein are hereby incorporated by reference. Unless otherwise specified, all sequence accession numbers described herein, including all tables herein, refer to database entries as of December 15, 2020. Where a gene or protein is indicated by multiple sequence accession numbers, all sequence variants are included.

[0012] Enumeration of Embodiments 1. An expression repressor comprising a targeting moiety that binds to the MYC promoter and, optionally, an effector moiety, wherein the expression repressor is capable of reducing the expression of MYC. Optionally, an effector moiety And an expression repressor comprising An expression repressor capable of reducing the expression of MYC.

[0013] 2. The expression repressor according to embodiment 1, wherein the targeting moiety 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.

[0014] 3. The expression repressor according to embodiment 1, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, 83, 85, 199, or 201.

[0015] A targeting moiety that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequences of SEQ ID NO: 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, Optionally, an effector moiety An expression repressor comprising An expression repressor capable of reducing the expression of MYC.

[0016] A targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequences of SEQ ID NO: 2, 77, 82, 83, 199, or 201 Optionally, an effector moiety An expression repressor comprising An expression repressor capable of reducing the expression of MYC.

[0017] A targeting moiety that binds to the MYC locus, An effector moiety comprising MQ1 or a fragment or variant thereof An expression repressor comprising An expression repressor capable of reducing the expression of MYC.

[0018] A targeting moiety that binds to a locus in the MYC super-enhancer region, Optionally an effector moiety, e.g., an effector moiety comprising a DNA methyltransferase, optionally an effector moiety comprising MQ1 or a fragment or variant thereof An expression repressor comprising An expression repressor capable of reducing the expression of MYC.

[0019] A targeting moiety that binds to a locus in the MYC super-enhancer region, An effector moiety comprising a transcriptional repressor, optionally comprising KRAB or a fragment or variant thereof, and An expression repressor comprising An expression repressor capable of reducing the expression of MYC.

[0020] 9. The expression repressor according to embodiment 7 or 8, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96-110, 83, 199, 201.

[0021] 10. The expression repressor according to any one of embodiments 7-9, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of GRCh37:chr8:129162465-129212140 using the hg19 reference genome.

[0022] 11. The expression repressor according to any one of embodiments 7-10, wherein the targeting moiety binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 96 or 108.

[0023] 12. The expression repressor according to any one of embodiments 7-11, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain.

[0024] 13. An expression repressor comprising a targeting moiety that binds to a locus, for example, the MYC locus, a first effector moiety comprising EZH2 or a fragment or variant thereof, a second effector moiety comprising KRAB or a fragment or variant thereof, and capable of reducing expression at the locus, for example, reducing the expression of MYC. An expression repressor.

[0025] 14. The expression repressor according to embodiment 13, wherein the targeting portion binds to a MYC promoter, a super enhancer region, or an anchor sequence.

[0026] 15. The expression repressor according to embodiment 13 or 14, wherein the targeting portion includes a TAL effector domain, a CRISPR / Cas domain, or a zinc finger domain.

[0027] 16. The expression repressor according to any one of embodiments 13 to 15, wherein the first effector portion is on the N-terminal side of the second effector, or the first effector is on the C-terminal side of the second effector portion.

[0028] 17. A targeting portion that binds to the MYC locus, the targeting portion including a zinc finger domain, optionally, an effector portion and an expression repressor, which can reduce the expression of MYC.

[0029] 18. A targeting portion including a CRISPR / Cas domain that binds to a transcription regulatory element operably linked to the MYC gene (e.g., a promoter, an enhancer, a super enhancer, or a transcription start site (TSS)) or a sequence proximate to the transcription regulatory element, for example, a CRISPR / Cas protein that is catalytically inactive; and an effector portion including MQ1 or a functional variant or fragment thereof and an expression repressor.

[0030] 19. A targeting portion including a CRISPR / Cas domain that binds to a transcription regulatory element operably linked to the MYC gene (e.g., a promoter, an enhancer, or a transcription start site (TSS)) or a sequence proximate to the transcription regulatory element, for example, a CRISPR / Cas protein that is catalytically inactive; An effector portion comprising MQ1 or a functional variant or fragment thereof and An expression repressor comprising the same.

[0031] 20. A targeting portion comprising a CRISPR / Cas domain that binds to a transcriptional regulatory element (e.g., a promoter, an enhancer, or a transcription start site (TSS)) operably linked to the MYC gene or to a sequence proximal to said transcriptional regulatory element, e.g., a CRISPR / Cas protein that is catalytically inactive; and An effector portion comprising KRAB or a functional variant or fragment thereof and An expression repressor comprising the same.

[0032] 21. A targeting portion comprising a CRISPR / Cas domain that binds to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising the MYC gene or to a sequence proximal to said anchor sequence, e.g., a CRISPR / Cas protein that is catalytically inactive; and An effector portion comprising KRAB or a functional variant or fragment thereof and An expression repressor comprising the same.

[0033] 22. A targeting portion comprising a zinc finger domain that binds to a transcriptional regulatory element (e.g., a promoter, an enhancer, or a transcription start site (TSS)) operably linked to the MYC gene or to a sequence proximal to said transcriptional regulatory element; and An effector portion comprising MQ1 or a functional variant or fragment thereof and An expression repressor comprising the same.

[0034] 23. A targeting portion comprising a zinc finger domain that binds to a transcriptional regulatory element (e.g., a promoter, an enhancer, or a transcription start site (TSS)) operably linked to the MYC gene or to a sequence proximal to said transcriptional regulatory element; and An effector portion comprising KRAB or a functional variant or fragment thereof and An expression repressor comprising the same.

[0035] 24. A targeting moiety that binds to a mouse genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 190 to 192, and Optionally, an effector moiety, and An expression repressor comprising: An expression repressor capable of reducing the expression of MYC.

[0036] 25. The expression repressor according to claim 24, wherein the effector moiety comprises a DNA methyltransferase, such as MQ1 or a fragment or variant thereof.

[0037] 26. The expression repressor according to embodiment 24 or 25, wherein the targeting moiety comprises a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.

[0038] 27. The expression repressor according to any one of embodiments 24 to 26, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain.

[0039] 28. The expression repressor according to any one of embodiments 24 to 27, wherein the expression repressor has an amino acid sequence selected from any one of SEQ ID NOs: 160 to 165, or has at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or comprises a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0040] 29. The expression repressor according to any one of embodiments 24 to 28, wherein the expression repressor is a nucleotide sequence selected from any of SEQ ID NOs: 166 to 168, or has at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or is encoded by a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0041] 30. The expression repressor according to any one of embodiments 24 to 29, wherein the targeting moiety is an amino acid sequence according to any of SEQ ID NOs: 154 to 156, or has at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0042] 31. The expression repressor according to any one of embodiments 24 to 30, wherein the targeting moiety is a nucleic acid sequence according to any of SEQ ID NOs: 157 to 159, or has at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0043] 32. The expression repressor according to any one of embodiments 24 to 31, wherein the effector moiety is a persistent effector moiety.

[0044] 33. The expression repressor according to any one of embodiments 24 to 32, wherein the effector moiety is a transient effector moiety.

[0045] 34. The expression repressor according to any one of embodiments 24 to 33, which is a fusion molecule.

[0046] 35. The expression repressor according to any one of embodiments 24 to 34, wherein the targeting portion includes a zinc finger domain, and the effector portion includes an epigenetic modification portion, for example, a DNA methyltransferase, for example, MQ1 or a fragment or variant thereof.

[0047] 36. The expression repressor according to any one of embodiments 18 to 20, 22, or 23, wherein the regulatory element is part of a cluster of regulatory elements.

[0048] 37. The expression repressor according to any one of embodiments 18 to 20, 22, or 23, wherein the regulatory element is located in a non-coding region.

[0049] 38. The expression repressor according to any one of embodiments 18 to 20, 22, or 23, wherein the regulatory element is a distal enhancer, for example, located at a position at least 1,000 nt away from a target gene promoter, for example, MYC.

[0050] 39. The expression repressor according to any one of embodiments 18 to 20, 22, 23 or 36 to 38, wherein the regulatory element increases the expression of a target gene, for example, MYC.

[0051] 40. The expression repressor according to any one of embodiments 18 to 20, 22, 23, or 36 to 39, wherein the regulatory element has one or more mutations.

[0052] 41. The expression repressor according to any one of embodiments 18 to 20, 22, 23, or 36 to 40, wherein the regulatory element has at least one disease-related single nucleotide polymorphism (SNP).

[0053] 42. The expression repressor according to any one of embodiments 18 to 20, 22, 23, or 36 to 41, wherein the transcriptional regulatory element interacts with a target gene, for example, the promoter and enhancer docking site of MYC.

[0054] 43. The expression repressor according to embodiment 42, wherein the enhancer docking site comprises the nucleotide sequence according to SEQ ID NO: 71 or 72 of the present specification, or SEQ ID NO: 73 or 74 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety).

[0055] 44. A targeting moiety comprising a zinc finger domain that binds to the anchor sequence of an anchor sequence-mediated junction (ASMC) containing the MYC gene or binds to a sequence proximal to the anchor sequence; And an effector moiety comprising KRAB or a functional variant or fragment thereof An expression repressor comprising.

[0056] 45. The expression repressor according to any one of embodiments 1 to 23 or 36 to 43, wherein the expression repressor is an amino acid sequence selected from any of SEQ ID NOs: 22 to 37, 129, 133, 134, 139 to 149, or 177 to 186 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or is at least 80, 85, 90, 95, 99, or 100% identical thereto, or has no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0057] 46. The expression repressor according to any one of embodiments 1 to 23 or 36 to 45, which is a nucleotide sequence selected from any of SEQ ID NOs: 55 to 66, 130, 189, or 193 to 197 of the present specification, or SEQ ID NOs: 67 to 70 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or is at least 80, 85, 90, 95, 99, or 100% identical thereto, or has no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0058] 47. The targeting portion is an amino acid sequence according to any one of SEQ ID NOs: 5 to 16 or 169 to 172, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and is the expression repressor according to any one of Embodiments 1 to 23 or 36 to 46.

[0059] 48. The effector portion is an amino acid sequence according to SEQ ID NO: 18, 19, or 87, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and is the expression repressor according to any one of the preceding embodiments.

[0060] 49. The effector portion is a persistent effector portion, and is the expression repressor according to any one of Embodiments 1 to 12, 17 to 19, 22, 36 to 42, or 44 to 47.

[0061] 50. The effector portion is a transient effector portion, and is the expression repressor according to any one of Embodiments 1 to 23 or 36 to 48.

[0062] 51. The effector portion includes a DNA methyltransferase, for example, MQ1 or a fragment or variant thereof, and is the expression repressor according to any one of Embodiments 1 to 12, 17 to 19, 22, 36 to 42, or 44 to 48.

[0063] 52. The effector portion includes a transcriptional repressor, for example, KRAB or a fragment or variant thereof, and is the expression repressor according to any one of Embodiments 1 to 23, 36 to 47, or 49.

[0064] 53. The expression repressor according to any of the preceding embodiments, wherein the targeting portion comprises a TAL effector domain, a CRISPR / Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.

[0065] 54. The expression repressor according to embodiment 53, wherein the CRISPR / Cas domain binds to a gRNA that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 1-4, for example, the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 1-4.

[0066] 55. The expression repressor according to embodiment 53, wherein the CRISPR / Cas domain binds to a gRNA that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96-110, for example, the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96-110.

[0067] 56. The expression repressor according to any of embodiments 53-55, wherein the CRISPR / Cas domain comprises a Cas protein or a Cpf1 protein or a variant thereof (e.g., a mutant) selected from Table 1 as described in WO 2022 / 132195 (which is incorporated herein by reference in its entirety).

[0068] 57. The expression repressor according to any of embodiments 53-56, wherein the CRISPR / Cas domain comprises a catalytically inactive CRISPR / Cas protein, such as dCas9.

[0069] 58. The zinc finger domain binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96-110. For example, the expression repressor according to embodiment 53, wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96-110.

[0070] 59. The zinc finger domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (and optionally not more than 11, 10, 9, 8, 7, 6, or 5 additional zinc fingers). The expression repressor according to any one of embodiments 17, 22, 26-53, or 57.

[0071] 60. 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. The expression repressor according to any one of embodiments 17, 22, 26-53, 57, or 58.

[0072] 61. The zinc finger domain comprises 3 or 9 zinc fingers. The expression repressor according to any one of embodiments 17, 22, 26-53, or 57-59.

[0073] 62. The expression repressor according to any one of the preceding embodiments, which is a fusion molecule.

[0074] 63. A linker located between the targeting domain and the effector domain, optionally comprising a linker comprising the amino acid sequence according to SEQ ID NO: 137 or SEQ ID NO: 138. The expression repressor according to any one of the preceding embodiments.

[0075] 64. The expression repressor according to any one of Embodiments 1 to 17, 20, 21, 23, 44 to 48, 50, or 52 to 57, wherein the targeting portion includes a CRISPR / Cas domain (e.g., dCas9) that is inactive as a catalyst, and the effector portion includes a transcriptional repressor, e.g., KRAB or a fragment or variant thereof.

[0076] 65. The expression repressor according to any one of Embodiments 1 to 17, 20, 21, 23, 44 to 48, 50, 52, or 53 to 64, wherein the targeting portion includes a zinc finger domain, and the effector portion includes a transcriptional repressor, e.g., KRAB or a fragment or variant thereof.

[0077] 66. The expression repressor according to any one of Embodiments 17, 36 to 43, 45 to 47, 53, or 58 to 63, wherein the targeting portion includes a zinc finger domain, and the expression repressor does not include an effector portion.

[0078] 67. The expression repressor according to any one of Embodiments 1 to 12, 18 to 19, 22, 36 to 43, 45 to 49, 51, or 53 to 57, wherein the targeting portion includes a CRISPR / Cas domain (e.g., dCas9) that is inactive as a catalyst, and the effector portion includes an epigenetic modification portion, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.

[0079] 68. The expression repressor according to any one of Embodiments 1 to 12, 17 to 19, 22, 36 to 43, 45 to 49, 51, 53, or 58 to 63, wherein the targeting portion includes a zinc finger domain, and the effector portion includes an epigenetic modification portion, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.

[0080] 69. The expression repressor according to any of the preceding embodiments, comprising an amino acid sequence of any one of SEQ ID NOs: 22 to 34, 129, 133, 134, 139 to 149, or 177 to 186 herein, or any one of SEQ ID NOs: 34 to 37 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0081] 70. (i) The expression repressor according to any of the preceding embodiments, comprising one or more nuclear localization signal sequences (NLSs), or (ii) the expression repressor according to any of the preceding embodiments, not comprising an NLS.

[0082] 71. The expression repressor according to any of the preceding embodiments, comprising a first NLS at the N-terminus, for example, a first NLS having the sequence of SEQ ID NO: 88.

[0083] 72. The expression repressor according to any of the preceding embodiments, comprising an NLS, for example, a second NLS, at the C-terminus, for example, having the sequence of SEQ ID NO: 89.

[0084] 73. The expression repressor according to any of the preceding embodiments, wherein the first NLS and the second NLS have the same sequence.

[0085] 74. The expression repressor according to any of embodiments 71 to 73, wherein the first NLS and the second NLS have different sequences.

[0086] 75. The expression repressor according to any of the preceding embodiments, comprising an epitope tag.

[0087] 76. The expression repressor according to embodiment 75, wherein the epitope tag is an HA tag.

[0088] 77. The expression repressor according to any of the preceding embodiments, wherein the anchor array comprises the array of SEQ ID NO: 71 or 72, or an array having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0089] 78. The expression repressor according to any one of embodiments 1 to 77, wherein the anchor array comprises the array according to SEQ ID NO: 73 or 74 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or an array having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0090] 79. The expression repressor according to any of the preceding embodiments, wherein the anchor array is on the same chromosome as the MYC gene.

[0091] 80. The expression repressor according to any of the preceding embodiments, wherein the anchor array is upstream of the MYC gene (for example, upstream of the TSS or upstream of the promoter).

[0092] 81. The expression repressor according to any of the preceding embodiments, wherein the anchor array is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (for example, from the TSS or promoter of the MYC gene).

[0093] 82. The expression repressor according to any of the preceding embodiments, wherein the anchor array is 0.1 to 0.5, 0.1 to 1, 0.1 to 5, 0.1 to 10, 0.1 to 50, 0.1 to 100, 0.1 to 500, 0.1 to 1000, 0.5 to 1, 0.5 to 5, 0.5 to 10, 0.5 to 50, 0.5 to 100, 0.5 to 500, 0.5 to 1000, 1 to 5, 1 to 10, 1 to 50, 1 to 100, 1 to 500, 1 to 1000, 5 to 10, 5 to 50, 5 to 100, 5 to 500, 5 to 1000, 10 to 50, 10 to 100, 10 to 500, 10 to 1000, 50 to 100, 50 to 500, 50 to 1000, 100 to 500, 100 to 1000, or 500 to 1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene).

[0094] 83. The expression repressor according to any of embodiments 1 to 79 or 81 to 82, wherein the target sequence is downstream of the MYC gene (e.g., downstream of the TSS or downstream of the promoter).

[0095] 84. The expression repressor according to any of the preceding embodiments, wherein the targeting portion binds to a sequence at chromosomal coordinates 128746342-128746364, 128746321-128746343, 128746525-128746547, 128748014-128748036, 129188878-129188900, 129188958-129188980, 129188960-129188982, 129189067-129189089, 129189457-129189479, 129189554-129189576, 129189679-129189701, 129209511-129209533, 129209643-129209665, 129209658-129209680, 129209856-129209878, 129189452-129189474, 129189190-129189212, 129189274-129189296, 129189421-129189443, 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842 or a sequence proximate thereto.

[0096] 85. When the expression repressor binds to a target locus, such as MYC, and is measured as described in Example 7 or 28 as described in, for example, ELISA or as described in WO2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), the methylation at a site of the target locus, such as MYC, is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the methylation in the absence of the expression repressor, and optionally, the site assayed for methylation is chr8:129188693-129189048 according to the hg19 reference genome and includes, for example, the sequence according to SEQ ID NO: 123. The expression repressor according to any of the preceding embodiments.

[0097] 86. When the above-described expression repressor binds to a target locus, for example, MYC, methylation at a site of the target locus, for example, MYC, increases over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, as described in Example 28 as described in WO 2022 / 132195 pamphlet (which is hereby incorporated by reference in its entirety), the expression repressor according to any of the preceding embodiments.

[0098] 87. When the above-described expression repressor binds to the MYC locus, the expression of MYC in cells decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the expression in the absence of the above-described expression repressor, as measured, for example, by ELISA or as described in any of Examples 2 to 7 or 9 as described in WO 2022 / 132195 pamphlet (which is hereby incorporated by reference in its entirety), the expression repressor according to any of the preceding embodiments.

[0099] 88. When the above-described expression repressor binds to the MYC locus, the expression of MYC decreases appreciably over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, as measured, for example, by ELISA or as described in any of Examples 2 to 7 or 9 as described in WO 2022 / 132195 pamphlet (which is hereby incorporated by reference in its entirety), the expression repressor according to any of the preceding embodiments.

[0100] 89. When the above expression repressor binds to the MYC locus, the expression of MYC decreases to such an extent that it can be observed at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection. The expression repressor according to any of the preceding embodiments.

[0101] 90. The expression repressor according to any of embodiments 1-23 or 36-89, wherein the targeting moiety binds to a human genomic locus.

[0102] 91. The expression repressor according to any of embodiments 24-43, 49, 51, 53, 56-57, 59-62, 66-68, 70-89, wherein the targeting moiety binds to a mouse genomic locus.

[0103] 92. When the above expression repressor binds to the MYC locus, the viability of cells (e.g., cancer cells) containing the MYC locus decreases. The expression repressor according to any of the preceding embodiments.

[0104] 93. When a plurality of cells are contacted with the above expression repressor or a nucleic acid encoding the above expression repressor, the viability of the plurality of cells decreases. The expression repressor according to any of the preceding embodiments.

[0105] 94. For example, when measured by CellTiter Glo or as described in any of Examples 2-7 as described in International Publication No. 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), the viability decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the viability in the absence of the first expression repressor. The expression repressor according to any of the preceding embodiments.

[0106] 95. The expression repressor according to any of the preceding embodiments, wherein when the expression repressor is administered, apoptosis occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of the target cells (e.g., cancer cells).

[0107] 96. The expression repressor according to any of the preceding embodiments, wherein the plurality of cells includes a plurality of cancer cells and a plurality of non-cancer cells and / or a plurality of infected cells and a plurality of non-infected cells.

[0108] 97. The expression repressor according to any of the preceding embodiments, wherein when the plurality of cells is contacted with the expression repressor or a nucleic acid encoding the expression repressor, the viability of the plurality of cancer cells is reduced more significantly than the reduction in the viability of the plurality of non-cancer cells thereby.

[0109] 98. The expression repressor according to any of the preceding embodiments, wherein when the plurality of cells is contacted with the expression repressor or a nucleic acid encoding the expression repressor, the viability of the plurality of cancer cells is reduced by 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× more than the reduction in the viability of the plurality of non-cancer cells thereby.

[0110] 99. The expression repressor according to any of embodiments 92 to 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.

[0111] 100. The expression repressor according to any one of Embodiments 92 to 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.

[0112] 101. The expression repressor according to any one of the preceding embodiments, which, when contacted with a plurality of infected cells and a plurality of non-infected cells, reduces the viability of the plurality of infected cells to a greater extent than it reduces the viability of the plurality of non-infected cells.

[0113] 102. The expression repressor according to any one of the preceding embodiments, wherein the infection is a viral infection.

[0114] 103. The expression repressor according to Embodiment 102, wherein the viral infection is hepatitis, such as hepatitis B.

[0115] 104. The expression repressor according to any one of Embodiments 92 to 103, wherein the infected cells are human hepatocytes.

[0116] 105. An assay regarding the viability of cancer cells (e.g., HCC cells) using LNP delivery of the mRNA encoding the expression repressor, for example, the assay according to Example 12 as described in International Publication No. WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), when tested, the expression repressor according to any one of the preceding embodiments having an EC50 of 0.04 to 0.4, 0.04 to 0.1, 0.1 to 0.2, 0.2 to 0.3, or 0.3 to 0.4 μg / mL.

[0117] 106. An assay regarding the viability of cancer cells (e.g., lung cancer cells) using LNP delivery of the mRNA encoding the above expression repressor, for example, an assay according to Example 18 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), when tested in the assay, the expression repressor according to any of Embodiments 1 to 104 having an EC50 of 0.1 - 2.5, 0.5 - 2.2, 1.0 - 1.5, 1.2 - 2 μg / mL.

[0118] 107. An assay regarding the decrease in MYC mRNA level of cancer cells (e.g., HCC cells) using LNP delivery of the mRNA encoding the above expression repressor, for example, an assay according to Example 12 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), when tested in the assay, the expression repressor according to any of the preceding embodiments 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.

[0119] 108. An assay regarding the decrease in MYC mRNA level of cancer cells (e.g., lung cancer cells) using LNP delivery of the mRNA encoding the above expression repressor, for example, an assay according to Example 18 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), when tested in the assay, the expression repressor according to 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.

[0120] 109. The expression repressor according to any of the preceding embodiments that reduces the level of the target gene in cells, e.g., the protein encoded by MYC, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the protein level of untreated cells.

[0121] 110. An expression repressor according to any of the preceding embodiments, which is capable of reducing the tumor volume in a human subject or a mammalian model.

[0122] 111. For example, when measured, for example, on the 20th day after the start of treatment such that the above expression repressor is administered at a dose of 3 mg / kg every 5 days, in a mammalian model, for example, the tumor volume can be reduced to the same extent as or more highly than a chemotherapeutic agent. An expression repressor according to any of the preceding embodiments.

[0123] 112. For example, when measured, for example, on the 20th day after the start of treatment such that the above expression repressor is administered at 1 mg / kg, 1.5 mg / kg, or 3 mg / kg over 4 doses every 5 days followed by 3 doses every 3 days, in a mammalian model, for example, the tumor volume can be reduced compared to a PBS control. An expression repressor according to any of the preceding embodiments.

[0124] 113. An expression repressor according to any of the preceding embodiments, wherein the above tumor volume is reduced by at least about 10%, 20%, 30%, or 40% compared to a control treated with PBS, for example, on the 20th day after the start of treatment.

[0125] 114. The expression repressor according to any of embodiments 111 to 113, wherein the above chemotherapeutic agent is sorafenib or cisplatin.

[0126] 115. An expression repressor according to any of the preceding embodiments, wherein the above system is capable of reducing the tumor volume to the same extent as or more highly than a small molecule MYC inhibitor.

[0127] 116. The expression repressor according to embodiment 115, wherein the small molecule MYC inhibitor is MYCi975, and optionally the tumor volume is reduced by at least about 10%, 20%, 30%, or 40% on, for example, the 20th day after the start of treatment, compared to the control treated with MYCi975.

[0128] 117. The expression repressor according to any of the preceding embodiments, wherein there is no weight loss or the weight loss that occurs is less than 3%, 2%, or 1% compared to the start of treatment.

[0129] 118. A first expression repressor according to any of the preceding embodiments, and a second expression repressor, for example, the second expression repressor described herein, for example, the second expression repressor according to any of the preceding embodiments A system comprising.

[0130] 119. A first expression repressor comprising a first targeting moiety and optionally a first effector moiety, which binds to a transcriptional regulatory element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to the MYC gene, or binds to a sequence proximate to the transcriptional regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, which binds to an anchor sequence of an anchor sequence-mediated junction (ASMC) containing the MYC gene, or binds to a sequence proximate to the anchor sequence A system comprising.

[0131] 120. The transcriptional regulatory element comprises a promoter, and the anchor sequence comprises a CTCF binding motif, The system according to embodiment 118 or 119.

[0132] 121. The system according to any of embodiments 118 to 120, wherein the second expression repressor binds to a downstream region adjacent to the CTCF binding motif.

[0133] 122. The system according to any one of embodiments 118 to 120, wherein the second expression repressor binds to an upstream region adjacent to the CTCF binding motif.

[0134] 123. The first expression repressor includes 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: 73 or 74 as described in SEQ ID NOs: 2, 3, 4, 71, 72, 75-86, or 200-206 of the present specification, or the pamphlet of International Publication No. 2022 / 132195 (which is incorporated herein by reference in its entirety); and the second expression repressor includes 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: 73 or 74 as described in SEQ ID NOs: 2, 3, 4, 71, 72, 75-86, or 200-206 of the present specification, or the pamphlet of International Publication No. 2022 / 132195 (which is incorporated herein by reference in its entirety). The system according to any one of embodiments 118 to 122.

[0135] 124. The first expression repressor includes a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of any one of the sequences of SEQ ID NOs: 96-110. The system according to any one of embodiments 118 to 123.

[0136] 125. The first expression repressor includes a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 83; and the second expression repressor includes a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 77. The system according to any one of Embodiments 118 to 124.

[0137] 126. A first expression repressor comprising a first targeting moiety and optionally a first effector moiety, the first expression repressor binding to a promoter operably linked to the MYC gene or binding to a sequence proximal to the promoter, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, the second expression repressor binding to an enhancer of the MYC gene (e.g., a super-enhancer) A system comprising.

[0138] 127. 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: 204, and the second expression repressor comprises a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of either the sequence of SEQ ID NO: 199 or 201, The system according to Embodiment 126.

[0139] 128. A system for reducing MYC expression, a) A first expression repressor, i) A first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with differences 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 from it and ii) A first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with differences 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 from it and a first expression repressor, comprising b) a second expression repressor, comprising i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7169, 171, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom; and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom; and a second expression repressor, comprising a system comprising.

[0140] 129. The system according to embodiment 128, wherein the first expression repressor further comprises a first nuclear localization signal, such as the SV40 NLS, such as a sequence according to SEQ ID NO: 135 or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, which is located, for example, on the N-terminal side of the first targeting moiety.

[0141] 130. The system according to embodiment 128 or 129, wherein the first expression repressor further comprises a second nuclear localization signal, such as the nucleoplasmin NLS, such as a sequence according to SEQ ID NO: 136 or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, which is located, for example, on the C-terminal side of the first effector moiety.

[0142] 131. The system according to any one of embodiments 128 to 130, wherein the second expression repressor further includes, for example, a first nuclear localization signal located on the N-terminal side of the second targeting portion, for example, the SV40 NLS, for example, the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0143] 132. The system according to any one of embodiments 128 to 131, wherein the second expression repressor further includes, for example, a second nuclear localization signal located on the C-terminal side of the second effector portion, for example, the nucleoplasmin NLS, for example, the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0144] 133. The system according to any one of embodiments 128 to 132, wherein the first expression repressor further includes a first linker located between the first targeting portion and the first effector portion, and optionally the first linker has the amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0145] 134. The system according to any one of embodiments 128 to 133, wherein the second expression repressor further includes a second linker located between the second targeting portion and the second effector portion, and optionally the second linker has the amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0146] 135. The system according to any one of embodiments 128 to 134, wherein the first expression repressor further includes an amino acid sequence on the C-terminal side of the first effector portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, for example, the sequence according to SEQ ID NO: 126 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0147] 136. The system according to any one of embodiments 128 to 132, wherein the second expression repressor has an amino acid sequence on the N-terminal side of the second targeting portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, or further includes amino acid P.

[0148] 137. The system according to any one of embodiments 128 to 136, wherein the first expression repressor has the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0149] 138. The system according to any one of embodiments 128 to 137, wherein the second expression repressor has the amino acid sequence according to SEQ ID NO: 24, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0150] 139. The system according to any one of embodiments 128 to 137, wherein the second targeting portion includes the amino acid sequence according to SEQ ID NO: 169, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0151] 140. The system according to any one of embodiments 128 to 137, wherein the second targeting portion includes the amino acid sequence according to SEQ ID NO: 171, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0152] 141. The system according to any one of embodiments 128 to 140, wherein the second expression repressor has the amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0153] 142. The system according to any one of embodiments 128 to 140, wherein the second expression repressor has the amino acid sequences according to SEQ ID NO: 179 and 185, or a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto.

[0154] A nucleic acid encoding the first expression repressor and the second repressor of the system according to any one of Embodiments 128 to 142.

[0155] A nucleic acid encoding a system for reducing MYC expression, a) A first region encoding a first expression repressor, wherein the first expression repressor is i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom; and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom comprising the first region; b) A second region encoding a second expression repressor, wherein the second expression repressor is i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7 or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom; and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom comprising the second region and comprising the nucleic acid.

[0156] 145. The nucleic acid according to embodiment 144, wherein the first region is on the 5'-side of the second region.

[0157] 146. The nucleic acid according to embodiment 144, wherein the first region is on the 3'-side of the second region.

[0158] 147. The nucleic acid according to embodiment 145 or 146, further comprising a nucleotide sequence encoding a first nuclear localization signal, such as an SV40 NLS, such as the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, wherein the first region is located, for example, on the N-terminal side of the first targeting moiety.

[0159] 148. The nucleic acid according to any one of embodiments 145 to 147, further comprising a nucleotide sequence encoding a second nuclear localization signal, such as a nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, wherein the first region is located, for example, on the C-terminal side of the first effector moiety.

[0160] 149. The nucleic acid according to any one of embodiments 145 to 148, further comprising a nucleotide sequence encoding a first nuclear localization signal, such as an SV40 NLS, such as the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, wherein the second region is located, for example, on the N-terminal side of the second targeting moiety.

[0161] 150. The nucleic acid according to any one of embodiments 145 to 149, further comprising a nucleotide sequence encoding a second nuclear localization signal, such as a nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, wherein the second region is located, for example, on the C-terminal side of the second effector moiety.

[0162] 151. The first region is a first linker located between the first targeting portion and the first effector portion, and optionally further comprises a nucleotide sequence encoding a first linker having the amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any one of embodiments 145 to 150.

[0163] 152. The second region is a second linker located between the second targeting portion and the second effector portion, and optionally further comprises a nucleotide sequence encoding a second linker having the amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any one of embodiments 145 to 151.

[0164] 153. The first region is an amino acid sequence on the C-terminal side of the first effector portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, for example, the sequence according to SEQ ID NO: 126 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any one of embodiments 145 to 152.

[0165] 154. The second region is an amino acid sequence on the N-terminal side of the second targeting portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, or a nucleotide sequence encoding amino acid P. The nucleic acid according to any one of embodiments 145 to 153.

[0166] 155. The first expression repressor has the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any one of embodiments 145 to 154.

[0167] 156. The nucleic acid according to any one of embodiments 145 to 155, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0168] 157. The nucleic acid according to any one of embodiments 145 to 156, wherein the first region includes a nucleotide sequence encoding the first targeting portion, and the nucleotide sequence encoding the first targeting portion has a sequence according to SEQ ID NO: 46 or 131, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the number of positions different therefrom does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0169] 158. The nucleic acid according to any one of embodiments 145 to 157, wherein the first region includes a nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion has a sequence according to SEQ ID NO: 52 or 132, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the number of positions different therefrom does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0170] 159. The nucleic acid according to any one of embodiments 145 to 158, wherein the second region includes a nucleotide sequence encoding the second targeting portion, and the nucleotide sequence encoding the second targeting portion has a sequence according to SEQ ID NO: 40, or at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the number of positions different therefrom does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0171] 160. The nucleic acid according to any one of embodiments 145 to 159, wherein the first region contains a nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion is the sequence according to SEQ ID NO: 51, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or contains a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0172] 161. The nucleic acid according to any one of embodiments 145 to 160, wherein the first region is the nucleotide sequence according to SEQ ID NO: 63 or 130, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or contains a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the polyA sequence is optional.

[0173] 162. The nucleic acid according to any one of embodiments 145 to 161, wherein the second region is the nucleotide sequence according to SEQ ID NO: 57, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or contains a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the polyA sequence is optional.

[0174] 163. A nucleic acid encoding a system for reducing MYC expression, a) a first region encoding a first expression repressor, wherein the first expression repressor i) a first targeting portion having the amino acid sequence according to SEQ ID NO: 13 or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence in which the positions different from it do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, ii) an amino acid sequence according to SEQ ID NO: 19 or 87, or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and a first effector portion comprising a first region, b) a second region encoding a second expression repressor, wherein the second expression repressor i) has a second targeting portion having an amino acid sequence according to SEQ ID NO: 169 or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and ii) has a second effector portion having an amino acid sequence according to SEQ ID NO: 18, or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom comprising a second region and a nucleic acid comprising

[0175] 164. A nucleic acid encoding a system for reducing MYC expression, a) a first region encoding a first expression repressor, wherein the first expression repressor i) has a first targeting portion having an amino acid sequence according to SEQ ID NO: 13 or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and ii) the first effector portion having an amino acid sequence according to SEQ ID NO: 19 or 87, or having a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and a first region comprising b) a second region encoding a second expression repressor, wherein the second expression repressor i) has a second targeting portion having an amino acid sequence according to SEQ ID NO: 171, or having a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and ii) has a second effector portion having an amino acid sequence according to SEQ ID NO: 18, or having a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom a second region comprising a nucleic acid comprising

[0176] 165. The nucleic acid according to embodiment 163 or 164, wherein the first region is located on the 5'-side of the second region.

[0177] 166. The nucleic acid according to embodiment 163 or 164, wherein the first region is located on the 3'-side of the second region.

[0178] 167. The nucleic acid according to any one of embodiments 163 to 166, further comprising a nucleotide sequence encoding a first nuclear localization signal, such as an SV40 NLS, such as a sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, wherein the first region is located, for example, on the N-terminal side of the first targeting portion.

[0179] 168. The nucleic acid according to any one of Embodiments 163 to 167, wherein the first region further includes a second nuclear localization signal, for example, located on the C-terminal side of the first effector portion, such as the nucleoplasmin NLS, for example, a nucleotide sequence encoding the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0180] 169. The nucleic acid according to any one of Embodiments 163 to 168, wherein the second region further includes a first nuclear localization signal, for example, located on the N-terminal side of the second targeting portion, such as the SV40 NLS, for example, a nucleotide sequence encoding the sequence according to SEQ ID NO: 135 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0181] 170. The nucleic acid according to any one of Embodiments 163 to 169, wherein the second region further includes a second nuclear localization signal, for example, located on the C-terminal side of the second effector portion, such as the nucleoplasmin NLS, for example, a nucleotide sequence encoding the sequence according to SEQ ID NO: 136 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0182] 171. The nucleic acid according to any one of Embodiments 163 to 170, wherein the first region is a first linker located between the first targeting portion and the first effector portion, and optionally further includes a nucleotide sequence encoding a first linker having the amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0183] 172. The nucleic acid according to any one of Embodiments 163 to 171, wherein the second region is a second linker located between the second targeting portion and the second effector portion, and optionally further includes a nucleotide sequence encoding a second linker having the amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0184] 173. The nucleic acid according to any one of Embodiments 163 to 171, wherein the first region further comprises a nucleotide sequence encoding an amino acid sequence located on the C-terminal side of the first effector portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, for example, the sequence according to SEQ ID NO: 126 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0185] 174. The nucleic acid according to any one of Embodiments 163 to 173, wherein the second region further comprises an amino acid sequence located on the N-terminal side of the second targeting portion, for example, a sequence of up to 30, 25, 20, or 18 amino acids, or a nucleotide sequence encoding amino acid P.

[0186] 175. The nucleic acid according to any one of Embodiments 163 to 174, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0187] 176. The nucleic acid according to any one of Embodiments 144 to 175, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0188] 177. The nucleic acid according to any one of Embodiments 144 to 176, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 179 or 185, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0189] 178. The first expression repressor includes the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, and the second expression repressor has the amino acid sequence according to SEQ ID NO: 24, 141, 177, 179, 183, or 185, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid according to any one of Embodiments 144 to 177.

[0190] 179. The first region includes the nucleotide sequence encoding the first targeting portion, and the nucleotide sequence encoding the first targeting portion is the sequence according to SEQ ID NO: 46 or 131 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position. The nucleic acid according to any one of Embodiments 144 to 178.

[0191] 180. The first region includes the nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion is the sequence according to SEQ ID NO: 52 or 132 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position. The nucleic acid according to any one of Embodiments 144 to 179.

[0192] 181. The second region is the nucleotide sequence according to SEQ ID NO: 173 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence in which the positions different therefrom do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position, and the polyA sequence is optional. The nucleic acid according to any one of Embodiments 144 to 180.

[0193] 182. The nucleic acid according to any one of embodiments 144 to 181, wherein the second region is the nucleotide sequence according to SEQ ID NO: 175 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the polyA sequence is optional.

[0194] 183. The nucleic acid according to any one of embodiments 144 to 182, wherein the second region includes the nucleotide sequence encoding the second effector portion, and the nucleotide sequence encoding the second effector portion is the sequence according to SEQ ID NO: 51 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0195] 184. The nucleic acid according to any one of embodiments 144 to 183, wherein the first region is the nucleotide sequence according to SEQ ID NO: 63 or 130 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the polyA sequence is optional.

[0196] 185. The nucleic acid according to any one of embodiments 144 to 184, wherein the second region is the nucleotide sequence according to SEQ ID NO: 189 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or includes a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the polyA sequence is optional.

[0197] 186. The nucleic acid according to any one of embodiments 144 to 185, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 194, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or comprises a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the polyA sequence is optional.

[0198] 187. The nucleic acid according to any one of embodiments 144 to 186, wherein the first region comprises a nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion has at least 80, 85, 90, 95, 99, or 100% identity to the sequence according to SEQ ID NO: 52 or 132, or comprises a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0199] 188. The nucleic acid according to any one of embodiments 144 to 187, wherein the first region comprises a nucleotide sequence encoding the first targeting portion, and the nucleotide sequence encoding the first targeting portion has at least 80, 85, 90, 95, 99, or 100% identity to the sequence according to SEQ ID NO: 46 or 131, or comprises a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0200] 189. The nucleic acid according to any one of embodiments 144 to 188, wherein the second region comprises a nucleotide sequence encoding the second effector portion, and the nucleotide sequence encoding the second effector portion has at least 80, 85, 90, 95, 99, or 100% identity to the sequence according to SEQ ID NO: 51, or comprises a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0201] 190. The nucleic acid according to any one of Embodiments 144 to 189, wherein the second region comprises the nucleotide sequence according to SEQ ID NO: 189, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or has a sequence in which the number of positions different from that sequence does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0202] 191. The nucleic acid according to any one of Embodiments 144 to 190, wherein the second region comprises the nucleotide sequence according to SEQ ID NO: 194, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or has a sequence in which the number of positions different from that sequence does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0203] 192. The nucleic acid according to any one of Embodiments 144 to 191, having the nucleotide sequence according to SEQ ID NO: 93 or 112, or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence in which the number of positions different from that sequence does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0204] 193. The nucleic acid according to any one of Embodiments 144 to 192, having the nucleotide sequence according to SEQ ID NO: 196 or 197, or having at least 80, 85, 90, 95, 99, or 100% identity thereto, or having a sequence in which the number of positions different from that sequence does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0205] 194. The system or nucleic acid according to any one of Embodiments 118 to 193, wherein the first expression repressor comprises the first effector portion.

[0206] 195. The system or nucleic acid according to any one of embodiments 118 to 194, wherein the second expression repressor comprises the second effector portion.

[0207] 196. The system or nucleic acid according to any one of embodiments 118 to 195, wherein the first effector portion has an amino acid sequence different from that of the second effector portion.

[0208] 197. The system or nucleic acid according to any one of embodiments 118 to 196, wherein the first effector portion is a persistent effector portion.

[0209] 198. The system or nucleic acid according to any one of embodiments 118 to 125 or 144 to 197, wherein the first effector portion is a transient effector portion.

[0210] 199. The system or nucleic acid according to any one of embodiments 118 to 198, wherein the first effector portion is an epigenetic modification portion.

[0211] 200. The system or nucleic acid according to any one of embodiments 118 to 143, 163 to 197 or 199, wherein the first effector portion comprises a histone methyltransferase.

[0212] 201. The system or nucleic acid according to embodiment 200, wherein the first effector portion comprises a protein selected from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any of these, for example, a SET domain of any of these.

[0213] 202. The system or nucleic acid according to any one of embodiments 118 to 143, 163 to 197, or 199, wherein the first effector portion comprises a histone demethylase (e.g., lysine demethylase).

[0214] 203. The system or nucleic acid according to embodiment 202, wherein the first effector portion comprises a protein selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or any functional variant or fragment thereof).

[0215] 204. The system or nucleic acid according to any one of embodiments 118 - 143, 163 - 197, or 199, wherein the first effector portion comprises a histone deacetylase.

[0216] 205. The system or nucleic acid according to embodiment 204, wherein the first effector portion comprises a protein selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or any functional variant or fragment thereof.

[0217] 206. The system or nucleic acid according to any one of embodiments 118 - 197 or 200, wherein the first effector portion comprises a DNA methyltransferase.

[0218] 207. The system or nucleic acid according to embodiment 206, wherein the first effector portion comprises a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof.

[0219] 208. The system or nucleic acid according to any one of embodiments 118 - 143, 160 - 196, or 198, wherein the first effector portion is a transcriptional repressor portion and comprises, for example, a transcriptional repressor.

[0220] 209. The system or nucleic acid according to embodiment 198 or 199, wherein the first effector portion comprises a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these.

[0221] 210. The system or nucleic acid according to any one of embodiments 118 to 209, wherein the first effector portion promotes epigenetic modification of the transcriptional regulatory element or a sequence adjacent thereto.

[0222] 211. The system or nucleic acid according to any one of embodiments 118 to 210, wherein the first effector portion catalyzes epigenetic modification of the transcriptional regulatory element or a sequence adjacent thereto.

[0223] 212. The system or nucleic acid according to any one of embodiments 118 to 125, 194, or 197 to 211, wherein the second expression repressor does not contain an effector portion.

[0224] 213. The system or nucleic acid according to any one of embodiments 118 to 212, wherein the second effector portion is a transient effector portion.

[0225] 214. The system or nucleic acid according to any one of embodiments 118 to 125 or 194 to 211, wherein the second effector portion is a persistent effector portion.

[0226] 215. The system or nucleic acid according to any one of embodiments 118 to 211 or 214, wherein the second effector portion is an epigenetic modification portion.

[0227] 216. The system or nucleic acid according to any one of embodiments 118 to 125, 194 to 211, or 214 to 215, wherein the second effector portion comprises a histone methyltransferase.

[0228] 217. The system or nucleic acid according to embodiment 216, wherein the second effector portion comprises SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a protein selected from a functional variant or fragment of any of these, for example, a protein selected from any of the SET domains of these.

[0229] 218. The system or nucleic acid according to any one of embodiments 118-125, 194-211, or 214-215, wherein the second effector portion comprises a histone demethylase (e.g., lysine demethylase).

[0230] 219. The system or nucleic acid according to embodiment 218, wherein the second effector portion comprises KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a protein selected from a functional variant or fragment of any of these.

[0231] 220. The system or nucleic acid according to any one of embodiments 118-125, 194-211, or 214-215, wherein the second effector portion comprises a histone deacetylase.

[0232] 221. The system or nucleic acid according to embodiment 220, wherein the second effector portion comprises HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a protein selected from a functional variant or fragment of any of these.

[0233] 222. The system or nucleic acid according to any one of embodiments 118-125, 194-211, or 214-215, wherein the second effector portion comprises a DNA methyltransferase.

[0234] 223. The system or nucleic acid according to embodiment 222, wherein the second effector portion comprises a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any of these.

[0235] 224. The system or nucleic acid according to any one of embodiments 118 to 211 or 213, wherein the second effector portion is a transcriptional repressor portion.

[0236] 225. The system or nucleic acid according to embodiment 224, wherein the second effector portion promotes epigenetic modification of the anchor sequence or a sequence adjacent thereto.

[0237] 226. The system or nucleic acid according to embodiment 223 or 224, wherein the second effector portion binds to one or more endogenous epigenetic modification proteins or one or more endogenous transcriptional modification proteins.

[0238] 227. The system or nucleic acid according to any one of embodiments 223 to 226, wherein the second effector portion comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these.

[0239] 228. The first effector portion is a persistent effector portion, and the second effector portion is a transient effector portion, The system or nucleic acid according to any one of embodiments 118 to 197, 199 to 207, 210 to 211, 213, or 224 to 227.

[0240] 229. The system or nucleic acid according to embodiment 228, wherein the first effector portion is an epigenetic modification portion.

[0241] 230. The system or nucleic acid according to embodiment 227 or 228, wherein the second effector portion is a transcriptional repressor portion.

[0242] 231. The first effector portion includes a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any of these, or a combination of any of these, and the second effector portion includes a transcriptional repressor or a functional variant or fragment of any of these. The system or nucleic acid according to any one of embodiments 227 to 230.

[0243] 232. The first effector portion includes a histone methyltransferase, a histone demethylase, a histone deacetylase, a DNA methyltransferase, a functional variant or fragment of any of these, or a combination of any of these, and the second expression repressor does not include the second effector portion. The system or nucleic acid according to any one of embodiments 118 to 125, 194, 197, 199 to 207, 210 to 212, or 190.

[0244] The first effector portion includes SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, any functional variant or fragment thereof, or any combination thereof, and The second effector portion includes KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, any functional variant or fragment thereof, or any combination thereof, The system or nucleic acid according to any one of Embodiments 118-125, 199-207, 210-211, 213-214, or 224-231.

[0245] 234. The first effector portion includes a DNA methyltransferase, and The second effector portion includes a transcriptional repressor, The system or nucleic acid according to any one of Embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, or 233.

[0246] 235. The first effector portion includes a DNA methyltransferase, and The second expression repressor does not include the second effector portion, The system or nucleic acid according to any one of Embodiments 118 to 125, 194, 197, 200, 206 to 207, 210 to 212, or 232.

[0247] 236. The system or nucleic acid according to any one of Embodiments 118 to 125, 200, 206 to 207, 210 to 235, wherein the first effector portion comprises MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof.

[0248] 237. The system or nucleic acid according to any one of Embodiments 118 to 211, 214, 224 to 234, or 236, wherein the second effector portion comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or any functional variant or fragment thereof.

[0249] 238. The system or nucleic acid according to any one of Embodiments 118 to 211, 199, 206 to 207, 210 to 211, 213, 224 to 234, or 236 to 237, wherein the first effector portion comprises MQ1 or any functional variant or fragment thereof, and the second effector comprises KRAB or any functional variant or fragment thereof. The system or nucleic acid according to any one of Embodiments 118 to 211, 199, 206 to 207, 210 to 211, 213, 224 to 234, or 236 to 237.

[0250] 239. The system or nucleic acid according to any one of Embodiments 118 to 125, 194, 197, 199 to 207, or 210 to 212, 229, 232, 235, or 236, wherein the first effector portion comprises MQ1 or any functional variant or fragment thereof, and the second expression repressor does not comprise the second effector portion. The system or nucleic acid according to any one of Embodiments 118 to 125, 194, 197, 199 to 207, or 210 to 212, 229, 232, 235, or 236.

[0251] 240. The above-described first expression repressor is an amino acid sequence selected from any of SEQ ID NOs: 22-33, 129, 133, 134, 139-149, 177-180, or 183-186 described herein, or SEQ ID NOs: 34-37 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the system or nucleic acid according to any one of Embodiments 118-200.

[0252] 241. The above-described second expression repressor is an amino acid sequence selected from any of SEQ ID NOs: 22-33, 129, 133, 134, 139-149, 177-180, or 183-186 described herein, or SEQ ID NOs: 34-37 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the system or nucleic acid according to any one of Embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240.

[0253] 242. The above-described first expression repressor comprises the amino acid sequence of SEQ ID NO: 30, 129, 133, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the above-described second expression repressor comprises the amino acid sequence of SEQ ID NO: 24, 134, 141, 177, 179, 183, or 185, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom. The system or nucleic acid according to any one of Embodiments 118 to 198, 200, 206 to 211, 213 to 216, 222 to 223, 236 to 237, or 240 to 241.

[0254] 243. The above-described first expression repressor is encoded by the first nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the above-described second expression repressor is encoded by the second nucleotide sequence of SEQ ID NO: 57, 189, or 194, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom. The system or nucleic acid according to any one of Embodiments 118 to 198, 200, 206 to 211, 213 to 216, 222 to 223, 236 to 237, or 240 to 242.

[0255] 244. The above-described first and second repressors are encoded by the nucleic acid sequence of SEQ ID NO: 93, 94, 112, 113, 196, or 197, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and are the systems or nucleic acids according to any one of Embodiments 118 to 198, 200, 206 to 211, 213 to 216, 222 to 223, 236 to 237, or 240 to 243.

[0256] 245. The system or nucleic acid according to Embodiment 244, comprising the amino acid sequence of SEQ ID NO: 91, 92, 121, 122, 181, 182, 187, or 188, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0257] 246. The above-described first expression repressor, from the N-terminus to the C-terminus: (i) a first nuclear localization signal, for example, SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (ii) a first targeting portion, for example, a zinc finger binding domain, for example, ZF9; for example, the sequence according to SEQ ID NO: 13; (iii) a first effector portion, for example, DNA methyltransferase, for example, MQ1; for example, the sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, for example, nucleoplasmin NLS; for example, the sequence according to SEQ ID NO: 136 and comprising; and the above-described second expression repressor, from the N-terminus to the C-terminus: (v) a third nuclear localization signal, for example, SV40NLS; for example, the sequence according to SEQ ID NO: 135; (vi) a second targeting portion, for example, a zinc finger binding domain, for example, ZF3; for example, the sequence according to SEQ ID NO: 7; (vii) a second effector portion, such as KRAB, such as the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, such as the nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 A system or nucleic acid according to any of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, comprising.

[0258] 247. The first expression repressor is, from the N-terminus to the C-terminus: (i) a first nuclear localization signal, such as the SV40 NLS; such as the sequence according to SEQ ID NO: 135; (ii) a first targeting portion, such as a zinc finger binding domain, such as ZF9; such as the sequence according to SEQ ID NO: 13; (iii) a first effector portion, such as DNA methyltransferase, such as MQ1; such as the sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, such as the nucleoplasmin NLS; such as the sequence according to SEQ ID NO: 136 comprising; and the second expression repressor is, from the N-terminus to the C-terminus: (v) a third nuclear localization signal, such as SV40NLS; such as the sequence according to SEQ ID NO: 135; (vi) a second targeting portion, such as a zinc finger binding domain, such as ZF54; such as the sequence according to SEQ ID NO: 169; (vii) a second effector portion, such as KRAB, such as the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, such as the nucleoplasmin NLS, such as the sequence according to SEQ ID NO: 136 A system or nucleic acid according to any of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, comprising.

[0259] 248. The above-described first expression repressor has, from the N-terminus to the C-terminus: (i) a first nuclear localization signal, for example, SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (ii) a first targeting portion, for example, a zinc finger binding domain, for example, ZF9; for example, the sequence according to SEQ ID NO: 13; (iii) a first effector portion, for example, DNA methyltransferase, for example, MQ1; for example, the sequence according to SEQ ID NO: 19 or 87; (iv) a second nuclear localization signal, for example, nucleoplasmin NLS; for example, the sequence according to SEQ ID NO: 136 and includes; and the above-described second expression repressor has, from the N-terminus to the C-terminus: (v) a third nuclear localization signal, for example, SV40 NLS; for example, the sequence according to SEQ ID NO: 135; (vi) a second targeting portion, for example, a zinc finger binding domain, for example, ZF67; for example, the sequence according to SEQ ID NO: 171; (vii) a second effector portion, for example, KRAB, for example, the sequence according to SEQ ID NO: 18; and (viii) a fourth nuclear localization signal, for example, nucleoplasmin NLS, for example, the sequence according to SEQ ID NO: 136 The system or nucleic acid according to any one of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, which includes the above.

[0260] 249. The system according to any one of embodiments 118-248, which can more highly reduce the expression of MYC as compared to the above-described first expression repressor alone or the above-described second expression repressor alone.

[0261] The system according to any one of embodiments 128 - 194, or 242 - 249, which can reduce the expression of MYC to a higher degree compared to any of the expression repressors of SEQ ID NO: 22, 23, 25 - 29, 31 - 33, or SEQ ID NO: 34 - 37 as described in WO 2022 / 132195 pamphlet (which is hereby incorporated by reference in its entirety).

[0262] 251. The system according to any one of embodiments 118 - 250, which can reduce the tumor volume of, for example, a human subject or a mammalian model.

[0263] 252. For example, in a model system as described in Example 15 of WO 2022 / 132195 pamphlet, when measured, for example, on the 20th day after the start of treatment such that the above - mentioned expression repressor is administered at a dose of 3 mg / kg every 5 days, for example, in a mammalian model, the system according to any one of embodiments 128 - 193 or 242 - 209, which can reduce the tumor volume to the same extent or to a higher degree compared to a chemotherapeutic agent.

[0264] 253. For example, in a model system as described in Example 14 of WO 2022 / 132195 pamphlet, when measured, for example, on the 15th day after the start of treatment such that the above - mentioned expression repressor is administered at a dose of 6 mg / kg every 5 days, for example, in a mammalian model, the system according to any one of embodiments 128 - 193 or 242 - 252, which can reduce the tumor volume to a higher degree compared to a chemotherapeutic agent.

[0265] 254. For example, on the 20th day after the start of treatment, the system according to any one of embodiments 128 - 193 or 242 - 253, wherein the above - mentioned tumor volume is reduced by at least about 10%, 20%, 30%, 40%, 50%, or 60% compared to a control treated with PBS.

[0266] 255. The system according to embodiment 254, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0267] 256. The system according to any one of embodiments 128 - 193 or 242 - 253, which can reduce tumor volume to the same extent as or more highly than a small molecule MYC inhibitor.

[0268] 257. The system according to embodiment 256, wherein the small molecule MYC inhibitor is MYCi975, and optionally the tumor volume is reduced by at least about 10%, 20%, 30%, or 40% on, for example, the 20th day after the start of treatment, compared to a control treated with the above - mentioned MYCi975.

[0269] 258. The system according to any one of embodiments 118 - 257, wherein no weight loss occurs or the weight loss that occurs is less than 3%, 2%, or 1% compared to the start of treatment.

[0270] 259. The system or nucleic acid according to 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.

[0271] 260. The system or nucleic acid according to 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.

[0272] 261. The system or nucleic acid according to any one of embodiments 118 - 260, wherein the first targeting moiety comprises a CRISPR / Cas domain (for example, a first CRISPR / Cas domain).

[0273] The system or nucleic acid according to any one of embodiments 118 to 261, wherein the second targeting portion comprises a second CRISPR / Cas domain (for example, a second CRISPR / Cas domain).

[0274] 263. The system or nucleic acid according to embodiment 262, wherein (i) the first CRISPR / Cas domain binds to a first guide RNA, (ii) the second CRISPR / Cas domain binds to a second guide RNA, or (iii) both (i) and (ii).

[0275] 264. The first CRISPR / Cas domain does not bind to the second guide RNA, or binds with a K of at least 10, 20, 50, 100, 1000, or 10,000 nM D and the second CRISPR / Cas domain does not bind to the first guide RNA, or binds with a K of at least 10, 20, 50, 100, 1000, or 10,000 nM D The system or nucleic acid according to embodiment 262 or 263.

[0276] 265. The system or nucleic acid according to any one of embodiments 260 to 264, wherein the first CRISPR / Cas domain comprises an amino acid sequence different from that of the second CRISPR / Cas domain.

[0277] 266. The system or nucleic acid according to any one of embodiments 260 to 265, wherein the first or second CRISPR / Cas domain comprises the amino acid sequence of a Cas protein or a Cpf1 protein or a variant thereof (for example, a mutant) selected from Table 1 as described in WO 2022 / 132195 pamphlet.

[0278] 267. The system or nucleic acid according to any one of embodiments 260 to 266, wherein the first CRISPR / Cas domain comprises the amino acid sequence of a Cas protein or a Cpf1 protein or a variant thereof (e.g., a mutant) selected from Table 1 as described in WO 2022 / 132195, and the second CRISPR / Cas domain comprises the amino acid sequence of a different Cas protein or Cpf1 protein or a variant thereof (e.g., a mutant) selected from Table 1 as described in WO 2022 / 132195.

[0279] 268. The system or nucleic acid according to any one of embodiments 118 to 260, wherein the first targeting moiety comprises a zinc finger domain (e.g., a first zinc finger domain).

[0280] 269. The system or nucleic acid according to any one of embodiments 118 to 260 or 268, wherein the second targeting moiety comprises a zinc finger domain (e.g., a second zinc finger domain).

[0281] 270. The system or nucleic acid according to any one of embodiments 118 to 261 or 268 to 269, wherein the first targeting moiety comprises a first zinc finger domain and the second targeting moiety comprises a second zinc finger domain.

[0282] 271. The system or nucleic acid according to any one of embodiments 268 to 270, wherein the first zinc finger domain and the second zinc finger domain bind to the same genomic locus, for example, having the same amino acid sequence.

[0283] 272. The system or nucleic acid according to any one of embodiments 268 to 271, wherein the first zinc finger domain and the second zinc finger domain have different amino acid sequences or bind to different genomic loci.

[0284] 273. The above-described first zinc finger molecule contains at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally not more than 11, 10, 9, 8, 7, 6, or 5 additional zinc fingers), and the system or nucleic acid according to any one of Embodiments 118 to 261 or 267 to 272.

[0285] 274. The above-described first zinc finger molecule contains 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 zinc fingers, and the system or nucleic acid according to any one of Embodiments 267 to 273.

[0286] 275. The above-described first zinc finger domain contains 3 or 9 zinc fingers, and the system or nucleic acid according to any one of Embodiments 268 to 274.

[0287] 276. The above-described second zinc finger domain contains at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally not more than 11, 10, 9, 8, 7, 6, or 5 additional zinc fingers), and the system or nucleic acid according to any one of Embodiments 268 to 275.

[0288] 277. The above-described second zinc finger domain contains 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 zinc fingers, and the system or nucleic acid according to any one of Embodiments 268 to 276.

[0289] 278. The system or nucleic acid according to any one of embodiments 268-277, wherein the second zinc finger domain comprises three or nine zinc fingers.

[0290] 279. The system or nucleic acid according to any one of embodiments 118-278, wherein the first targeting moiety comprises a TAL effector domain (e.g., a first TAL effector domain).

[0291] 280. The system or nucleic acid according to any one of embodiments 118-260 or 279, wherein the second targeting moiety comprises a TAL effector domain (e.g., a second TAL effector domain).

[0292] 281. The system or nucleic acid according to embodiment 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, not more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).

[0293] 282. The system or nucleic acid according to any one 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.

[0294] 283. The system or nucleic acid according to any one 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, not more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats).

[0295] 284. The system or nucleic acid according to any one 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.

[0296] 285. The system or nucleic acid according to any one of embodiments 118-284, wherein the first targeting moiety comprises a nucleic acid (e.g., a first nucleic acid).

[0297] 286. The system according to any one of embodiments 129-285, wherein the second targeting moiety comprises a nucleic acid (e.g., a second nucleic acid).

[0298] 287. The system or nucleic acid according to any one of embodiments 129-286, wherein the first targeting moiety comprises a polypeptide (e.g., a first polypeptide).

[0299] 288. The system or nucleic acid according to any one of embodiments 129-287, wherein the second targeting moiety comprises a polypeptide (e.g., a second polypeptide).

[0300] 289. The system according to embodiment 287 or 288, wherein the nucleic acid is covalently bound to the polypeptide.

[0301] 290. The system according to embodiment 288 or 289, wherein the nucleic acid is non-covalently bound to the polypeptide.

[0302] 291. The system or nucleic acid according to any one of embodiments 275 to 290, wherein the nucleic acid comprises a sequence complementary to the transcription regulatory element or a sequence proximate thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatch compared to the transcription regulatory element or a sequence proximate thereto.

[0303] 292. The system or nucleic acid according to any one of embodiments 275 to 291, wherein the nucleic acid comprises a sequence complementary to the anchor sequence or a sequence proximate thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatch compared to the anchor sequence or a sequence proximate thereto.

[0304] 293. The system according to any one of embodiments 275 to 292, wherein the nucleic acid comprises DNA, peptide nucleic acid (PNA), peptide-oligonucleotide conjugate, locked nucleic acid (LNA), bridged nucleic acid (BNA), polyamide, triple-stranded forming oligonucleotide, antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecule.

[0305] 294. The system according to any one of embodiments 275 to 293, wherein the nucleic acid comprises gRNA.

[0306] 295. The system according to any one of embodiments 275 to 294, wherein the nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity to any one of SEQ ID NOs: 1 to 4, or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions different therefrom.

[0307] 296. The system according to any one of embodiments 275 to 295, wherein the first nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 1 to 4, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the second nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 1 to 4, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0308] 297. The system according to any one of embodiments 275 to 295, wherein the first nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 96 to 110, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the second nucleic acid comprises a sequence having at least 80, 85, 90, 95, 99, or 100% identity with any one of SEQ ID NOs: 96 to 110, or the number of positions different from it does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0309] 298. The system according to any one of embodiments 118 to 297, wherein the transcription regulatory element comprises a promoter.

[0310] 299. The system according to any one of embodiments 118 to 298, wherein the transcription regulatory element comprises an enhancer; for example, a super enhancer.

[0311] 300. The system according to any one of embodiments 118 to 299, wherein the anchor sequence comprises a CTCF binding motif.

[0312] 301. The system according to any one of embodiments 118 to 300, wherein the anchor sequence comprises a YY1 binding motif; for example, a YY1 binding motif as described in International Publication No. WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety).

[0313] 302. The system according to any one of Embodiments 118 to 301, wherein the anchor array includes the sequence of SEQ ID NO: 71 or 72, or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0314] 303. The system according to any one of Embodiments 118 to 302, wherein the anchor array includes the sequence according to SEQ ID NO: 73 or 74 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or a sequence having no more than 8, 7, 6, 5, 4, 3, 2, or 1 modification compared thereto.

[0315] 304. The system according to any one of Embodiments 118 to 303, wherein the anchor array is on the same chromosome as the MYC gene.

[0316] 305. The system according to any one of Embodiments 118 to 304, wherein the anchor array is upstream of the MYC gene (e.g., upstream of the TSS or upstream of the promoter).

[0317] 306. The system according to any one of Embodiments 118 to 305, wherein the anchor array is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene).

[0318] 307. The system according to any one of embodiments 118 to 306, wherein the anchor array is 0.1 to 0.5, 0.1 to 1, 0.1 to 5, 0.1 to 10, 0.1 to 50, 0.1 to 100, 0.1 to 500, 0.1 to 1000, 0.5 to 1, 0.5 to 5, 0.5 to 10, 0.5 to 50, 0.5 to 100, 0.5 to 500, 0.5 to 1000, 1 to 5, 1 to 10, 1 to 50, 1 to 100, 1 to 500, 1 to 1000, 5 to 10, 5 to 50, 5 to 100, 5 to 500, 5 to 1000, 10 to 50, 10 to 100, 10 to 500, 10 to 1000, 50 to 100, 50 to 500, 50 to 1000, 100 to 500, 100 to 1000, or 500 to 1000 kilobases away from the MYC gene (for example, from the TSS or promoter of the MYC gene).

[0319] 308. The system according to any one of embodiments 118 to 303 or 305 to 307, wherein the anchor array is on a chromosome different from the MYC gene.

[0320] 309. The system according to any one of embodiments 118 to 308, wherein the second targeting moiety binds to the anchor array or a sequence proximate to the anchor array with sufficient affinity to compete for binding with an endogenous polypeptide (for example, CTCF or YY1).

[0321] 310. The system according to any one of embodiments 118 to 309, wherein the first targeting moiety binds to a sequence at chromosomal coordinates 128746342 to 128746364, 128746321 to 128746343, or 128746525 to 128746547, or a sequence proximate thereto.

[0322] 311. The system according to any one of embodiments 118 to 309, wherein the first targeting moiety binds to a sequence at chromosomal coordinates 128746405 to 128746425, 128748069 to 128748089, 129188825 to 129188845, or 129188822 to 129188842, or a sequence proximate thereto.

[0323] 312. The system according to any one of Embodiments 118 to 311, wherein the second targeting portion binds to a sequence at chromosomal coordinates 128748014 to 128748036, or a sequence adjacent thereto.

[0324] 313. The system according to any one of Embodiments 118 to 311, wherein the second targeting portion binds to a sequence at chromosomal coordinates 128746405 to 128746425, 128748069 to 128748089, 129188825 to 129188845, or 129188822 to 129188842, or a sequence adjacent thereto.

[0325] 314. The system according to any one of Embodiments 118 to 314, wherein the first expression repressor is a fusion molecule.

[0326] 315. The system according to any one of Embodiments 118 to 314, wherein the second expression repressor is a fusion molecule.

[0327] 316. The system according to any one of Embodiments 118 to 315, wherein the first expression repressor includes a linker.

[0328] 317. The system according to any one of Embodiments 118 to 316, wherein the second expression repressor includes a linker.

[0329] 318. The first expression repressor includes a first CRISPR / Cas molecule, for example, a targeting portion including a CRISPR / Cas protein that is inactive as a first catalyst, and an effector portion including an epigenetic modification portion; and The second expression repressor includes a second CRISPR / Cas molecule, for example, a targeting portion including a CRISPR / Cas protein that is inactive as a second catalyst, and an effector portion optionally including a transcriptional repressor. The system according to any one of Embodiments 118 to 267 or 285 to 317.

[0330] 319. The first expression repressor includes a targeting portion containing a first zinc finger domain and an effector portion containing an epigenetic modification portion; and The second expression repressor includes a targeting portion containing a second zinc finger domain and, optionally, an effector portion containing a transcriptional repressor. The system according to any one of embodiments 118-260, 268-278, or 285-317.

[0331] 320. The first expression repressor includes a targeting portion containing a CRISPR / Cas molecule, for example, a catalytically inactive CRISPR / Cas protein, and an effector portion containing an epigenetic modification portion; and The second expression repressor includes a targeting portion containing a zinc finger domain and, optionally, an effector portion containing a transcriptional repressor. The system according to any one of embodiments 118-120, 262, 268, or 275-318.

[0332] 321. The first expression repressor includes a targeting portion containing a zinc finger domain and an effector portion containing an epigenetic modification portion; and The second expression repressor includes a targeting portion containing a CRISPR / Cas domain, for example, a catalytically inactive CRISPR / Cas protein, and, optionally, an effector portion containing a transcriptional repressor. The system according to any one of embodiments 118-260, 268, or 275-318.

[0333] 322. The zinc finger domain (e.g., the first or second zinc finger domain) contains 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 zinc fingers, e.g., 3 or 9 zinc fingers, and the system according to any one of Embodiments 260, 268 to 278, or 275 to 318.

[0334] 323. The epigenetic modification part contains DNA methyltransferase, and the system according to any one of Embodiments 322.

[0335] 324. The epigenetic modification part contains MQ1 or its functional variant or fragment, and the system according to any one of Embodiments 118 to 323.

[0336] 325. The second expression repressor contains an effector part containing a transcriptional repressor, and the system according to any one of Embodiments 118 to 324.

[0337] 326. The transcriptional repressor contains KRAB or its functional variant or fragment, and the system according to any one of Embodiments 118 to 323.

[0338] 327. The system according to any one of Embodiments 118 to 326, wherein the first expression repressor comprises the amino acid sequence of any one of SEQ ID NOs: 28 to 33, 145 to 149, 151, or 152 in the present specification, or SEQ ID NOs: 35 to 37 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or an amino acid sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0339] 328. The system according to any one of Embodiments 118 to 327, wherein the second expression repressor comprises the amino acid sequence of any one of SEQ ID NOs: 22 to 27, 139 to 144, 150, 177 to 180, 183 to 186 in the present specification, or SEQ ID NO: 34 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), or an amino acid sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0340] 329. The system according to any one of Embodiments 118 to 328, wherein when the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto, the expression of MYC in the cell decreases.

[0341] 330. For example, when measured by QPCR or ELISA, the expression decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the expression in the absence of the first expression repressor. The system according to Embodiment 327.

[0342] When the first expression repressor binds to the transcription regulatory element, the expression of MYC decreases to an extent that can be observed, for example, over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, as measured by, for example, QPCR or ELISA. The system according to embodiment 326 or 327.

[0343] When the first expression repressor binds to the transcription regulatory element, the expression of MYC decreases to an extent that can be observed at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection. The system according to any one of embodiments 329 to 331.

[0344] When the second expression repressor binds to the anchor sequence or a sequence in proximity thereto, the expression of MYC in the cell decreases. The system according to any one of embodiments 328 to 332.

[0345] When expression is measured by, for example, QPCR or ELISA, it decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% as compared to the expression in the absence of the second expression repressor. The system according to embodiment 333.

[0346] When the second expression repressor binds to the anchor sequence or a sequence in proximity thereto, the expression of MYC decreases to an extent that can be observed, for example, over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions. The system according to embodiment 333 or 334.

[0347] 336. When the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC decreases to such an extent that it can be observed 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection. The system according to any one of embodiments 334 to 335.

[0348] 337. When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto, and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC in the cell decreases. The system according to any one of embodiments 329 to 336.

[0349] 338. When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto, and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the expression of MYC decreases to such an extent that it can be observed 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours after transfection. The system according to any one of embodiments 329 to 337.

[0350] 339. For example, when measured by QPCR or ELISA, the expression decreases by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the expression in the absence of the first and second expression repressors. The system according to embodiment 337 or 338.

[0351] When the first expression repressor binds to the transcription regulatory element or a sequence proximate thereto, and the second expression repressor binds to the anchor sequence or a sequence proximate thereto, the expression of MYC decreases such that it can be observed, for example, when measured by QPCR or ELISA, 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. The system according to any one of embodiments 329 to 339.

[0352] 341. The decrease in expression caused by the first expression repressor binding to the transcription regulatory element or a sequence proximate thereto and the second expression repressor binding to the anchor sequence or a sequence proximate thereto is greater than the decrease in expression caused individually by the first expression repressor binding to the transcription regulatory element or a sequence proximate thereto or the second expression repressor binding to the anchor sequence or a sequence proximate thereto. The system according to any one of embodiments 329 to 340.

[0353] 342. When the first expression repressor binds to the transcription regulatory element or a sequence proximate thereto, and the second expression repressor binds to the anchor sequence or a sequence proximate thereto, the expression decreases by 1.05× (i.e., 1.05-fold), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× more than when either the first expression repressor binds to the transcription regulatory element or a sequence proximate thereto or the second expression repressor binds to the anchor sequence or a sequence proximate thereto, when measured by, for example, QPCR or ELISA. The system according to embodiment 341.

[0354] 343. The system according to any one of embodiments 329 to 342, wherein the decrease in expression caused by the first expression repressor binding to the transcription regulatory element or a sequence proximate thereto and the second expression repressor binding to the anchor sequence or a sequence proximate thereto persists for a longer time (e.g., over a greater number of hours, days, or cell divisions) compared to the decrease in expression caused individually by the first expression repressor binding to the transcription regulatory element or a sequence proximate thereto or the second expression repressor binding to the anchor sequence or a sequence proximate thereto.

[0355] 344. When the first expression repressor binds to the transcription regulatory element or a sequence proximate thereto and the second expression repressor binds to the anchor sequence or a sequence proximate thereto, the expression decreases by 1.05× (i.e., 1.05-fold), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× longer (e.g., as measured in number of hours, days, or cell divisions) compared to either the first expression repressor binding to the transcription regulatory element or a sequence proximate thereto or the second expression repressor binding to the anchor sequence or a sequence proximate thereto, as measured by, for example, QPCR or ELISA. The system according to embodiment 343.

[0356] When the first expression repressor binds to the promoter or a sequence adjacent thereto and the second expression repressor binds to the super enhancer or a sequence adjacent thereto, the expression of MYC decreases to a degree that can be observed, for example, when measured by QPCR or ELISA, 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. The system according to any one of Embodiments 329 to 344.

[0357] 346. The decrease in expression caused by the first expression repressor binding to the promoter or a sequence adjacent thereto and the second expression repressor binding to the super enhancer or a sequence adjacent thereto is greater than the decrease in expression caused individually by the first expression repressor binding to the promoter or a sequence adjacent thereto or by the second expression repressor binding to the super enhancer or a sequence adjacent thereto. The system according to any one of Embodiments 329 to 345.

[0358] 347. When the first expression repressor binds to the promoter or a sequence adjacent thereto and the second expression repressor binds to the super enhancer or a sequence adjacent thereto, the expression decreases by 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× more than when either the first expression repressor binds to the promoter or a sequence adjacent thereto or the second expression repressor binds to the super enhancer or a sequence adjacent thereto, as compared to either case, when measured by, for example, QPCR or ELISA. The system according to Embodiment 346.

[0359] 348. The system according to any one of embodiments 329 to 347, wherein the decrease in expression caused by the first expression repressor binding to the promoter or an array adjacent thereto and the second expression repressor binding to the super enhancer or an array adjacent thereto persists for a longer period of time (e.g., over a greater number of hours, days, or cell divisions) compared to the decrease in expression caused individually by the first expression repressor binding to the promoter or an array adjacent thereto or the second expression repressor binding to the super enhancer or an array adjacent thereto.

[0360] 349. When the first expression repressor binds to the promoter or an array adjacent thereto and the second expression repressor binds to the super enhancer or an array adjacent thereto, the expression decreases by 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× longer (e.g., as measured by the number of hours, days, or cell divisions) compared to either the first expression repressor binding to the promoter or an array adjacent thereto or the second expression repressor binding to the super enhancer or an array adjacent thereto, as measured, for example, by QPCR or ELISA. The system according to embodiment 348.

[0361] 350. The system according to any one of embodiments 329 to 349, wherein the expression decreases to such an extent that it can be observed indefinitely (e.g., over a period longer than can be experimentally measured).

[0362] 351. The system according to any one of embodiments 329 to 350, wherein when the first expression repressor binds to the transcriptional regulatory element or an array adjacent thereto, the viability of the cell containing the transcriptional regulatory element or an array adjacent thereto decreases.

[0363] 352. When a plurality of cells are contacted with the first expression repressor or a nucleic acid encoding the first expression repressor, the viability of the plurality of cells is decreased, and optionally, the plurality of cells include cancerous and non-cancerous cells and / or infected and non-infected cells, and the system according to any one of Embodiments 329 to 351.

[0364] 353. For example, when measured by CellTiter Glo, the viability is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the viability in the absence of the first expression repressor, and the system according to Embodiment 352.

[0365] 354. By administering the first expression repressor, apoptosis occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells), and the system according to any one of Embodiments 329 to 353.

[0366] 355. When the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, the viability of cells containing the anchor sequence or a sequence adjacent thereto is decreased, and the system according to any one of Embodiments 329 to 354.

[0367] 356. When a plurality of cells are contacted with the second expression repressor or a nucleic acid encoding the second expression repressor, the viability of the plurality of cells is decreased, and the system according to any one of Embodiments 329 to 355.

[0368] 357. When the second expression repressor binds to the super enhancer or a sequence adjacent thereto, the viability of cells containing the transcription regulatory element or a sequence adjacent thereto is decreased, and the system according to any one of Embodiments 329 to 356.

[0369] When a plurality of cells are contacted with the nucleic acid encoding the second expression repressor or the first expression repressor, the viability of the plurality of cells is decreased, and optionally, the plurality of cells include cancerous and non-cancerous cells and / or infected and non-infected cells, the system according to any one of embodiments 329 to 357.

[0370] 359. For example, when measured by CellTiter Glo, the viability is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to the viability in the absence of the second expression repressor, the system according to embodiment 358.

[0371] 360. By administering the second expression repressor, apoptosis occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells), the system according to any one of embodiments 329 to 359.

[0372] 361. When the first expression repressor binds to the transcription regulatory element or a sequence proximate thereto, and the second expression repressor binds to the anchor sequence or a sequence proximate thereto, the viability of the cells containing the anchor sequence or a sequence proximate thereto is decreased, the system according to any one of embodiments 329 to 360.

[0373] 362. When the first expression repressor binds to the promoter or a sequence proximate thereto, and the second expression repressor binds to the super enhancer or a sequence proximate thereto, the viability of the cells is decreased, the system according to any one of embodiments 329 to 361.

[0374] 363. When a plurality of cells are contacted with the system or the nucleic acid encoding the system, the viability of the plurality of cells is decreased, the system according to any one of embodiments 329 to 362.

[0375] 364. For example, when measured by CellTiter Glo, the viability is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% as compared to the viability in the absence of the above system, the system according to Embodiments 329 to 363.

[0376] 365. The reduction in viability caused by the above first expression repressor binding to the above transcription regulatory element or a sequence adjacent thereto, and the above second expression repressor binding to the above anchor sequence or a sequence adjacent thereto, is greater than the reduction in viability caused individually by the above first expression repressor binding to the above transcription regulatory element or a sequence adjacent thereto or the above second expression repressor binding to the above anchor sequence or a sequence adjacent thereto, the system according to any one of Embodiments 329 to 364.

[0377] 366. The reduction in viability caused by the above first expression repressor binding to the above promoter or a sequence adjacent thereto, and the above second expression repressor binding to the above super enhancer or a sequence adjacent thereto, is greater than the reduction in viability caused individually by the above first expression repressor binding to the above promoter or a sequence adjacent thereto or the above second expression repressor binding to the above super enhancer or a sequence adjacent thereto, the system according to any one of Embodiments 329 to 365.

[0378] When the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto and the second expression repressor binds to the anchor sequence or a sequence adjacent thereto, for example, when measured by CellTiter Glo, the viability is 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× lower than when either the first expression repressor binds to the transcription regulatory element or a sequence adjacent thereto or the second expression repressor binds to the anchor sequence or a sequence adjacent thereto alone. The system according to embodiment 366.

[0379] When the first expression repressor binds to the promoter or a sequence adjacent thereto and the second expression repressor binds to the super enhancer or a sequence adjacent thereto, for example, when measured by CellTiter Glo, the viability is 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× lower than when either the first expression repressor binds to the promoter or a sequence adjacent thereto or the second expression repressor binds to the super enhancer or a sequence adjacent thereto alone. The system according to embodiment 366 or 367.

[0380] By administering the first expression repressor and the second expression repressor, apoptosis occurs in at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells). The system according to any one of embodiments 329 to 368.

[0381] 370. The system according to any one of embodiments 329 to 369, wherein the plurality of cells includes a plurality of cancer cells and a plurality of non-cancer cells.

[0382] 371. The system according to embodiment 370, wherein when the plurality of cells are contacted with the system or a nucleic acid encoding the system, the viability of the plurality of cancer cells is reduced more significantly than the reduction in the viability of the plurality of non-cancer cells thereby caused.

[0383] 372. The system according to embodiment 370 or 371, wherein when the plurality of cells are contacted with the system or a nucleic acid encoding the system, the viability of the plurality of cancer cells is reduced by 1.05× (i.e., 1.05 times), 1.1×, 1.15×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 20×, 50×, or 100× greater than the reduction in the viability of the plurality of non-cancer cells thereby caused.

[0384] 373. The expression repressor or system according to any preceding embodiment, wherein when assayed according to Example 29 as described in WO 2022 / 132195 (which is incorporated herein by reference in its entirety), the viability of non-cancer cells (e.g., primary hepatocytes) does not decrease by more than 5, 10, 15, or 20%.

[0385] 374. The expression repressor or system according to embodiment 320, wherein the viability is assayed 72 hours after contacting the cells with the expression repressor or system.

[0386] 375. The expression repressor or system according to embodiment 374, wherein the assay includes contacting the non-cancer cells with the expression repressor or system at 2.5, 2, 1.25, 1, 0.6, or 0.5 μg / ml.

[0387] 376. When contacted with a plurality of infected cells and a plurality of non-infected cells, it reduces the viability of the plurality of infected cells more than it reduces the viability of the plurality of non-infected cells, and / or it reduces the viability of the plurality of cancerous cells more than it reduces the viability of the plurality of non-cancerous cells, the system according to any one of embodiments 352 to 375.

[0388] 377. 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, the system according to any one of embodiments 352 to 376.

[0389] 378. The cancer cells are lung cancer cells, gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or liver cancer cells, the system according to any one of embodiments 352 to 377.

[0390] 379. The cells are human lung epithelial cells or human lung fibroblasts, the system according to any one of embodiments 352 to 378.

[0391] 380. The infection is a viral infection, the system according to any one of embodiments 352 to 379.

[0392] 381. The viral infection is hepatitis, for example, hepatitis B, the expression repressor according to embodiment 380.

[0393] 382. The infected cells are human hepatocytes, the system according to any one of embodiments 378 to 381.

[0394] 383. The viral infection is a chronic infection, the system according to any one of embodiments 352 to 382.

[0395] 384. A first amino acid region comprising a sequence encoding the first expression repressor of the system according to any one of embodiments 118 to 383; a second amino acid region comprising an array encoding the second expression repressor described in the system according to any one of embodiments 118 to 383, and a fusion protein comprising the same.

[0396] 385. The fusion protein according to embodiment 384, which is a third amino acid region located between the first amino acid region and the second amino acid region and comprising the third amino acid region.

[0397] 386. The fusion protein according to embodiment 385, wherein the third amino acid region comprises a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a T2A self-cleaving peptide sequence, for example, the sequence according to SEQ ID NO: 120.

[0398] 387. The fusion protein according to embodiment 386, wherein the third amino acid region comprises a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a tandem 2A peptide sequence, for example, a tPT2A sequence, for example, the sequence according to SEQ ID NO: 124.

[0399] 388. The fusion protein according to embodiment 385, wherein the peptide sequence comprises a T2A peptide sequence and a P2A peptide sequence.

[0400] 389. The first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and the second expression repressor comprises the amino acid sequence according to SEQ ID NO: 24 or 142, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The fusion protein according to any one of embodiments 384 to 388.

[0401] 390. The first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and The above-described second expression repressor comprises the amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The fusion protein according to any one of Embodiments 384 to 388.

[0402] 391. The above-described first expression repressor comprises the amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and The above-described second expression repressor comprises the amino acid sequence according to SEQ ID NO: 179 or 185, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The fusion protein according to any one of Embodiments 384 to 388.

[0403] 392. The amino acid sequence of SEQ ID NO: 91, 92, 121, or 122, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the fusion protein according to any one of Embodiments 384 to 391.

[0404] 393. The amino acid sequence of SEQ ID NO: 181, 182, 187, or 188, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the fusion protein according to any one of Embodiments 384 to 392.

[0405] 394. A nucleic acid comprising a sequence encoding the system according to any one of Embodiments 118 to 393.

[0406] 395. A nucleic acid comprising a sequence encoding the system according to Embodiment 394.

[0407] A first region comprising a sequence encoding the first expression repressor of the system according to any one of Embodiments 118 to 393; and A second region comprising a sequence encoding the second expression repressor of the system according to any one of Embodiments 118 to 393 The nucleic acid according to Embodiment 394 or 395, comprising

[0408] 397. A nucleic acid according to any one of Embodiments 394 to 396, comprising a third region located between the first region and the second region.

[0409] 398. The nucleic acid according to any one of Embodiments 394 to 397, wherein the third region encodes a ribosome skipping sequence.

[0410] 399. The nucleic acid according to Embodiment 397 or 398, wherein the third region encodes a tPT2A peptide sequence, for example, the sequence according to SEQ ID NO: 124.

[0411] 400. The nucleic acid according to any one of Embodiments 397 to 399, wherein the third region encodes a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a T2A self-cleaving peptide sequence, for example, the sequence according to SEQ ID NO: 95.

[0412] 401. The nucleic acid according to any one of Embodiments 397 to 400, wherein the third region encodes a protease cleavage peptide sequence, for example, a self-cleaving peptide sequence, for example, a tandem 2A peptide sequence, for example, a tPT2A peptide sequence, for example, the sequence according to SEQ ID NO: 124.

[0413] 402. The first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, 129 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and The second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24, 142, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto. The nucleic acid according to any one of Embodiments 394 to 401.

[0414] 403. The above first expression repressor contains the amino acid sequence according to SEQ ID NO: 30, 129 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto; and The above second expression repressor contains the amino acid sequence according to SEQ ID NO: 177, 179, 183, or 185 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, The nucleic acid according to any one of Embodiments 394 to 401.

[0415] 404. The nucleic acid according to any one of Embodiments 394 to 403, encoding the amino acid sequence of SEQ ID NO: 91, 92, 121, 122 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0416] 405. The nucleic acid according to any one of Embodiments 394 to 404, encoding the amino acid sequence of SEQ ID NO: 181, 182, 187, 188, or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0417] 406. The nucleic acid according to any one of Embodiments 394 to 405, containing the nucleotide sequence of SEQ ID NO: 93, 94, 112, or 113 or a sequence having at least 80, 85, 90, 95, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0418] The nucleic acid according to any one of Embodiments 394 to 406, comprising the nucleotide sequences of SEQ ID NOs: 196 and 197, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0419] A nucleic acid comprising a sequence encoding a repressor or a repressor system according to any one of Embodiments 1 to 407.

[0420] The nucleic acid according to any one of Embodiments 394 to 408, which is RNA, such as mRNA.

[0421] The nucleic acid according to any one of Embodiments 394 to 409, which comprises, for example, N7-methylated guanosine linked, for example, by an inverted 5'→5' triphosphate bond to the 5' end of the above RNA.

[0422] The nucleic acid according to any one of Embodiments 394 to 410, which comprises a 5'UTR.

[0423] The nucleic acid according to any one of Embodiments 394 to 411, which comprises, for example, a Kozak sequence between the above 5'UTR and the above sequence encoding the repressor.

[0424] A first nucleic acid comprising a sequence encoding the first repressor of the system according to any one of Embodiments 118 to 393; A second nucleic acid, which is a second repressor and comprises a sequence encoding the second repressor of the system according to any one of Embodiments 118 to 393 A system comprising the same.

[0425] 414. The above-described first nucleic acid has the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second nucleic acid having the nucleotide sequence of SEQ ID NO: 57, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the system according to Embodiment 413.

[0426] 415. The above-described first nucleic acid has the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second nucleic acid having the nucleotide sequence of SEQ ID NO: 189, or 194, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the system according to Embodiment 414.

[0427] 416. The above-described first nucleic acid has the nucleotide sequence of SEQ ID NO: 189, 194, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, and the second nucleic acid having the nucleotide sequence of SEQ ID NO: 63, 130, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom, the system according to Embodiment 415.

[0428] 417. The nucleic acid or system according to any one of embodiments 394 to 416, wherein the nucleic acid comprises mRNA.

[0429] 418. The system according to any one of the preceding embodiments, or a vector comprising the nucleic acid encoding the expression repressor.

[0430] 419. A lipid nanoparticle comprising the system, nucleic acid, mRNA, or vector according to any one of the preceding embodiments.

[0431] 420. The lipid nanoparticle according to embodiment 419, comprising an ionizable lipid, such as a cationic lipid, such as MC3, SSOP.

[0432] 421. The lipid nanoparticle according to embodiment 419 or 420, further comprising one or more of a neutral lipid, an ionizable amine-containing lipid, a biodegradable alkyn lipid, steroids, phospholipids, polyunsaturated lipids, structural lipids (such as sterols), PEG, cholesterol, or a polymer-conjugated lipid.

[0433] 422. A reaction mixture comprising the expression repressor, system, nucleic acid, vector, or lipid nanoparticle according to any one of the preceding embodiments.

[0434] 423. The reaction mixture according to embodiment 422, further comprising cells.

[0435] 424. A pharmaceutical composition comprising the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or reaction mixture according to any one of the preceding embodiments.

[0436] 425. A method for reducing the expression of the MYC gene in a cell, comprising: contacting the cell (e.g., a cancer cell) with the expression repressor, system, one or more nucleic acids encoding the system or the expression repressor, vector, lipid nanoparticle, or pharmaceutical composition according to any one of embodiments 1 to 424. comprising A method for reducing the expression of the MYC gene in said cells thereby.

[0437] 426. A method for treating cancer in a subject in need of cancer treatment, administering to said subject an expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 424 comprising A method for treating said cancer in said subject thereby.

[0438] 427. A method for reducing tumor growth in a subject in need of reducing tumor growth, administering to said subject an expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of Embodiments 1 to 424 comprising A method for reducing the tumor size in said subject thereby.

[0439] 428. The method according to Embodiment 427, wherein said reduction in tumor growth comprises a reduction in tumor volume compared to the tumor volume at the start of treatment.

[0440] 429. The method according to Embodiment 428, wherein said reduction in tumor growth in said subject is greater compared to an untreated subject.

[0441] 430. A method for increasing or restoring the sensitivity of cancer to a kinase inhibitor, such as a VEGFR1-3 inhibitor (e.g., lenvatinib), a tyrosine kinase inhibitor, such as sorafenib, an ERK inhibitor, such as ulixertinib, an AKT inhibitor, such as MK-2206, ARQ092, or TAS-117, a MEK inhibitor, such as trametinib or cobimetinib, or an EGFR TKI, such as osimertinib, erlotinib, afatinib, or gefitinib, the method comprising administering an expression repressor or system described herein to a subject having cancer.

[0442] 431. When the expression repressor or system is administered, for example, in an assay for cancer cell viability, such as the assay according to Example 38 as described in WO 2022 / 132195 (which is hereby incorporated by reference in its entirety), the IC of the kinase inhibitor 50 is reduced by 10%, 20%, 30%, or 40%, the method according to Embodiment 430.

[0443] 432. The method according to Embodiment 430 or 431, wherein the kinase inhibitor inhibits one or more (e.g., all) of VEGFR, PDGFR, MEK, ERK, AKT, EGFR, or RAF kinase.

[0444] 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 the cancer an expression repressor, system, or nucleic acid described herein (e.g., any of Embodiments 1 to 423), and optionally, when the expression repressor or system is administered, for example, in an assay for cancer cell viability, such as the assay according to Example 39 as described in WO 2022 / 132195 (which is hereby incorporated by reference in its entirety), the IC of the bromodomain inhibitor 50 is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, the method.

[0445] 434. The method according to Embodiment 433, wherein the bromodomain inhibitor is JQ1, BET672, or vilabresib, or comprises the same.

[0446] 435. A method of increasing or restoring cancer sensitivity to a MEK inhibitor, such as trametinib, comprising administering to a subject having said cancer an expression repressor, system, or nucleic acid described herein (e.g., any of embodiments 1-423), and optionally, when administering said expression repressor or system, in an assay such as an assay according to Example 51 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety), the IC 50 of said MEK inhibitor decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0447] 436. The method according to any of embodiments 427-435, wherein said decrease in tumor growth of said subject is greater than or equal to the decrease in tumor size when said subject is treated with a chemotherapeutic agent or a small molecule MYC inhibitor.

[0448] 437. The method according to embodiment 436, wherein said chemotherapeutic agent is sorafenib or cisplatin.

[0449] 438. The method according to embodiment 437, wherein said small molecule MYC inhibitor is MYCi975.

[0450] 439. A method of reducing the tumor size of a subject in need thereof, comprising administering to said subject an expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition of 1-424, and said decrease in tumor size is greater than or equal to the decrease in tumor size when said subject is treated with a chemotherapeutic agent.

[0451] 440. The method of 439, wherein said chemotherapeutic agent is sorafenib or cisplatin.

[0452] The method according to any of the preceding embodiments, which does not cause any significant side effects in the subject as compared to when treated with a chemotherapeutic agent or a small molecule MYC inhibitor.

[0453] 442. The method according to any of embodiments 436 - 441, wherein the chemotherapeutic agent is sorafenib or cisplatin.

[0454] 443. The method according to embodiment 442, wherein the small molecule MYC inhibitor is MYCi975.

[0455] 444. The method according to any of embodiments 426 - 443, wherein the cancer is stage I, stage II, stage III, or stage IV cancer.

[0456] 445. The method according to any of the preceding embodiments, wherein the weight of the subject remains substantially the same before and after treatment.

[0457] 446. The method according to any of the preceding embodiments, wherein no weight loss occurs in the subject, or the weight loss that occurs in the subject is less than 3%, 2%, or 1% compared to the start of treatment.

[0458] 447. The method according to any of the preceding embodiments, wherein no decrease or increase in body weight occurs in the subject after treatment compared to the body weight of the subject before treatment.

[0459] 448. A method for treating a liver disease in a subject in need thereof, comprising administering an expression repressor to the subject, the expression repressor comprising a targeting moiety that binds to the MYC locus (e.g., a transcription region of MYC, a MYC promoter, or an anchor sequence of an anchor sequence mediated junction (ASMC) containing the MYC gene, or a sequence proximal to the anchor sequence), and optionally an effector moiety, such as an effector moiety described herein; thereby treating the liver disease in the subject.

[0460] 449. Further comprising administering a second expression repressor to the subject, wherein the second expression repressor comprises a targeting moiety that binds to an anchor sequence of an anchor sequence-mediated junction (ASMC) comprising a target gene, such as MYC, and optionally a second effector moiety, such as an effector moiety described herein; for example, KRAB; The method according to embodiment 447, thereby treating the liver disease of the subject.

[0461] 450. A method of treating a liver disease of a subject in need thereof, administering to the subject an expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of embodiments 1 to 424 comprising, A method of treating the liver disease of the subject thereby.

[0462] 451. The method according to embodiment 450, wherein the liver disease is a chronic liver disease.

[0463] 452. The method according to embodiment 450 or 451, wherein the liver disease is virus-related or alcohol-related.

[0464] 453. The method according to any one of embodiments 450 to 452, wherein the liver disease is hepatitis or hepatocellular carcinoma.

[0465] 454. The method according to embodiment 453, wherein the hepatocellular carcinoma is selected from HCC subtype S1, HCC subtype S2, or HCC subtype S3.

[0466] 455. The method according to embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S1.

[0467] 456. The method according to embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S2.

[0468] The method according to any one of embodiments 450 to 456, wherein the liver disease is caused by hepatitis B virus or hepatitis C virus.

[0469] 458. A method of treating a lung disease in a subject in need thereof, administering to the subject an expression repressor comprising a targeting moiety that binds to the MYC locus (e.g., the transcriptional region of MYC, the MYC promoter, or the anchor sequence of an anchor sequence-mediated junction (ASMC) comprising the MYC gene or a sequence proximal to the anchor sequence) and optionally an effector moiety, e.g., an effector moiety described herein comprising; thereby treating the lung disease in the subject.

[0470] 459. Administering to the subject a second expression repressor comprising a targeting moiety that binds to a genomic locus located in the super enhancer region of a target gene, e.g., MYC, and optionally a second effector moiety, e.g., an effector moiety described herein; e.g., KRAB further comprising; thereby treating the lung disease in the subject, the method according to embodiment 458.

[0471] 460. A method of treating a lung disease in a subject in need thereof, administering to the subject an expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition according to any one of embodiments 1 to 424 comprising, thereby treating the lung disease in the subject.

[0472] 461. The method according to embodiment 459 or 460, wherein the lung disease is cancer, e.g., lung cancer, e.g., lung carcinoma, e.g., non-small cell lung carcinoma or small cell lung carcinoma.

[0473] The method according to any one of embodiments 425-461, wherein contacting or administering comprises intravenous administration to a subject.

[0474] The method according to any one of embodiments 425-462, wherein contacting or administering comprises intratumoral delivery (e.g., injection).

[0475] The method according to any one of embodiments 425-463, wherein the cancer is characterized by an increase in MYC expression compared to a reference level (e.g., compared to MYC expression in reference cells, e.g., other non-cancerous cells of the subject that are otherwise similar).

[0476] The method according to any one of embodiments 426-464, wherein the cancer is characterized by partial or complete duplication of the MYC gene.

[0477] The method according to 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 lymphoma), cervical cancer, or gastric cancer.

[0478] The method according to any one of embodiments 426-466, wherein the cancer is a human chorionic gonadotropin (hCG)-secreting cancer.

[0479] The method according to any one of embodiments 426-467, wherein the cancer is hepatocellular carcinoma.

[0480] The method according to any one of embodiments 426-468, wherein the cancer is refractory cancer, e.g., refractory hepatocellular carcinoma.

[0481] The method according to any one of embodiments 426-469, wherein the cancer is non-small cell lung carcinoma or small cell lung carcinoma.

[0482] 471. The method according to any one of embodiments 426 to 470, wherein the cancer overexpresses alpha-fetoprotein (AFP) (e.g., compared to the AFP expression of reference cells, e.g., non-cancerous cells that are otherwise similar to the subject).

[0483] 472. The method according to any one of embodiments 431 to 471, wherein the cancer cells are characterized by, for example, containing the MYC gene or the presence of a super-enhancer containing the anchor sequence-mediated junction containing the MYC gene, and optionally the cancer is selected from liver cancer, colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, or endometrial cancer.

[0484] 473. The method according to embodiment 472, wherein the expression repressor (e.g., the second expression repressor) binds to the anchor sequence of the anchor sequence-mediated junction (ASMC) containing the MYC gene or to a sequence proximate to the anchor sequence.

[0485] 474. The method according to any one of embodiments 426 to 473, wherein the cancer cells are characterized by not containing the MYC gene or the absence of a super-enhancer containing the anchor sequence-mediated junction containing the MYC gene.

[0486] 475. The method according to embodiment 474, wherein the expression repressor (e.g., the first expression repressor) binds to the MYC promoter.

[0487] 476. The method according to any one of embodiments 426 to 475, wherein the cancer comprises cells containing the MYC gene or a super-enhancer containing the anchor sequence-mediated junction containing the MYC gene and cells not containing the MYC gene or a super-enhancer containing the anchor sequence-mediated junction containing the MYC gene.

[0488] The method according to any one of embodiments 426-476, comprising cells characterized by an increase in MYC expression compared to a reference level (e.g., compared to MYC expression in reference cells, e.g., other non-cancerous cells of the same kind in the subject), and cells not characterized by an increase in MYC expression compared to a reference level (e.g., compared to MYC expression in reference cells, e.g., other non-cancerous cells of the same kind in the subject), e.g., cells having normal MYC expression.

[0489] The method according to any one of embodiments 426-477, wherein the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition is administered as a monotherapy.

[0490] The method according to any one of embodiments 426-478, comprising administering a plurality of doses of the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition to the subject, e.g., at least 2, 3, 4, 5, or 6 doses.

[0491] The method according to any one of embodiments 426-479, comprising administering a plurality of doses of the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition to the subject at 5-day intervals.

[0492] a) First, administering to the subject a first plurality of doses of an expression repressor or system described herein (e.g., according to any one of embodiments 1-424), optionally, each subsequent dose in the first plurality being administered 5 days after the previous dose in the first plurality; b) Second, withholding the expression repressor or system for a period (a "drug holiday"), e.g., about 2 weeks, and c) Third, administering to the subject a second plurality of doses of the expression repressor or system, optionally, each subsequent dose in the second plurality being administered 5 days after the previous dose in the second plurality The method according to any one of embodiments 426-480.

[0493] 482. The method according to embodiment 481, wherein the first plurality of dosages includes four dosages.

[0494] 483. The method according to embodiment 480 or 481, wherein the second plurality of dosages includes two dosages.

[0495] 484. The method according to any one of embodiments 481 to 483, wherein the subject does not receive administration of any therapeutic agent during the drug withdrawal period.

[0496] 485. The method according to any one of embodiments 481 to 484, wherein the subject receives administration of a second therapeutic agent during the drug withdrawal period.

[0497] 486. The method according to any one of embodiments 481 to 485, wherein the drug withdrawal period is at least twice as long as the time between administrations of the dosages in the first plurality of dosages.

[0498] 487. The method according to any one of embodiments 481 to 487, wherein the drug withdrawal period is at least twice as long as the time between administrations of the dosages in the second plurality of dosages.

[0499] 488. The method according to any one of embodiments 426 to 487, wherein after treatment with the expression repressor or system, the volume of the tumor is reduced to a non-detectable level.

[0500] 489. The method according to any one of embodiments 426 to 488, wherein after cessation of treatment with the expression repressor or system, the tumor volume shrinks (e.g., to a non-detectable level).

[0501] 490. The method according to any one of embodiments 425 to 489, wherein the cancer does not become resistant to the expression repressor or system, or does not become resistant to the expression repressor or system within a period of 10, 20, 30, 40, 50, or 60 days.

[0502] 491. The method according to any one of embodiments 425 to 490, wherein the cancer cells have a functional apoptosis pathway.

[0503] 492. The method according to any one of embodiments 425 to 491, wherein the cancer cells have functional caspase 3.

[0504] 493. The method according to embodiment 492, wherein caspase 3 is upregulated in cancer cells upon administration of the expression repressor or system to the subject.

[0505] 494. The method according to any one of embodiments 425 to 493, wherein Ki67 is downregulated in cancer cells upon administration of the expression repressor or system to the subject.

[0506] 495. The method according to any one of embodiments 425 to 494, wherein cancer cell proliferation is reduced upon administration of the expression repressor or system to the subject.

[0507] 496. a. Contacting the cells with a second therapeutic agent or b. Administering the second therapeutic agent to the subject The method according to any one of embodiments 425 to 495, further comprising.

[0508] 497. The method according to embodiment 496, wherein the second therapeutic agent is not an expression repressor that binds to the MYC promoter.

[0509] 498. The method according to embodiment 496 or 497, wherein the second therapeutic agent is not an expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle according to any one of embodiments 1 to 424.

[0510] 498. The method according to any one of embodiments 495 to 497, wherein the second therapeutic agent is an expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle according to any one of embodiments 1 to 424.

[0511] 500. The method according to any one of embodiments 496 to 498, wherein the second therapeutic agent is one or both of immunotherapy, immune checkpoint therapy, and anti-vascular endothelial growth factor therapy, systemic chemotherapy, tyrosine kinase inhibitor, such as sorafenib, mitogen-activated protein kinase kinase inhibitor (MEK inhibitor), such as trametinib, or bromodomain inhibitor, such as BET inhibitor, such as JQ1 or vilabresib.

[0512] 501. The method according to any one of embodiments 496 to 500, wherein the second therapeutic agent is a tyrosine kinase inhibitor, such as sorafenib.

[0513] 502. The method according to any one of embodiments 496 to 500, wherein the second therapeutic agent is a bromodomain inhibitor, such as BET inhibitor, such as JQ1, vilabresib, or BET 672.

[0514] 503. The method according to any one of embodiments 496 to 500, wherein the second therapeutic agent is a mitogen-activated protein kinase kinase inhibitor (MEK inhibitor), such as trametinib.

[0515] 504. The method according to any one of embodiments 496 to 503, further comprising administering an additional therapy to the subject.

[0516] 505. The method according to embodiment 504, wherein the additional therapy includes surgical resection, orthotopic liver transplantation, radiofrequency ablation, photodynamic therapy (PDT), laser therapy, brachytherapy, radiotherapy, transcatheter arterial chemoembolization or radioembolization, or stereotactic radiotherapy.

[0517] 506. The method according to any one of embodiments 496 to 505, wherein the second therapeutic agent is selected from checkpoint inhibitors or small molecules.

[0518] The method according to any one of embodiments 496 to 506, wherein the second therapeutic agent is a chemotherapeutic agent, such as a kinase inhibitor or a bromodomain inhibitor, such as a BET inhibitor.

[0519] The method according to embodiment 506 or 507, wherein the second therapeutic agent is selected from sorafenib, JQ1, BET672, vilabresib, or trametinib.

[0520] The method according to any one of embodiments 496 to 507, wherein the expression repressor, system, or nucleic acid and the second therapeutic agent are administered simultaneously.

[0521] The method according to any one of embodiments 496 to 509, wherein the expression repressor, system, or nucleic acid and the second therapeutic agent are administered sequentially.

[0522] The method according to any one of embodiments 504 to 509, wherein the additional therapy is administered simultaneously.

[0523] The method according to any one of embodiments 504 to 510, wherein the additional therapy is administered sequentially.

[0524] The method according to any one of embodiments 496 to 509, wherein the second therapeutic agent is administered simultaneously with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture according to any one of embodiments 1 to 424.

[0525] The method according to any one of embodiments 496 to 513, wherein the second therapeutic agent is administered continuously with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture according to any one of embodiments 1 to 424.

[0526] The method according to any one of embodiments 496 to 514, wherein the expression repressor, system, or nucleic acid is administered intravenously and the second therapy is administered orally.

[0527] 516. The method according to any of the preceding embodiments, wherein the cancer is a resistant or refractory cancer.

[0528] 517. The method according to any of the preceding embodiments, wherein the cancer is resistant or refractory to a kinase inhibitor, such as a kinase inhibitor that inhibits one or more of VEGFR, PDGFR, or RAF kinase, such as sorafenib.

[0529] 518. The method according to any of the preceding embodiments, wherein the subject has an amplification of the MYC super-enhancer.

[0530] 519. A kit comprising a container containing a composition comprising an expression repressor, a system, one or more nucleic acids encoding the system or the expression repressor, a vector, a lipid nanoparticle, a reaction mixture, or a pharmaceutical composition according to any of embodiments 1 to 424, and a set of instructions comprising at least one method of regulating, such as reducing, the expression of the MYC gene in a cell by the composition.

[0531] 520. The general structure of formula (I): [Chemical formula] (wherein, 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) A compound having; The method according to any one of Embodiments 425 to 518, which comprises administering the compound or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, or the pharmaceutical composition according to Embodiment 424, which further comprises the same.

[0532] 521.R 1 The method or pharmaceutical composition according to Embodiment 520, wherein R is alkyl or cycloalkyl.

[0533] 522.R 1 The method or pharmaceutical composition according to Embodiment 520 or 521, wherein R is cyclopropane.

[0534] 523.R 2 The method or pharmaceutical composition according to any one of Embodiments 520 to 522, wherein R is halo.

[0535] 524.R 2 The method or pharmaceutical composition according to any one of Embodiments 520 to 523, wherein R is Cl.

[0536] 525.R 3 , R 4 , R 5 , R 6 , R 7 The method or pharmaceutical composition according to any one of Embodiments 520 to 524, wherein each of R is H.

[0537] 526. The method or pharmaceutical composition according to any one of Embodiments 520 to 525, wherein the substituted aromatic ring is a substituted phenyl ring.

[0538] 527. The compound of the above formula (I) is

Chemical formula

[0539] The method or pharmaceutical composition according to any one of embodiments 520 to 527, wherein the compound of formula (I) is administered orally.

[0540] 529. The method or pharmaceutical composition according to any one of embodiments 520 to 528, wherein the compound of formula (I) is administered at a dose of 6 to 10 (e.g., about 8), 10 to 14 (e.g., about 12) mg, or about 8 to 24 mg per day.

[0541] 530. General structure of formula (II):

Chemical formula

Chemical formula

[0542] 531. R1 is

Chemical formula

[0543] 532. R1 is

Chemical formula

[0544] 533. R1 is

Chemical formula

[0545] 534. 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 to 6, and R 13 = H or methyl); R9 is H; R 10 is -(CO)-ethylene; and R 11 is H), the method according to any one of embodiments 530 to 533.

[0546] 535. The method according to any one of embodiments 530 to 534, wherein formula (II) is

Chemical formula

[0547] 536. The method according to any one of embodiments 520 to 535, wherein the cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, or non-small cell lung cancer.

[0548] 537. The method according to any one of embodiments 520 to 536, wherein the nucleic acid comprises RNA, for example, mRNA.

[0549] 538. 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, and the first effector moiety comprises a DNA methyltransferase. The method according to any one of embodiments 520 to 537.

[0550] 539. The method according to embodiment 538, wherein the first targeting moiety comprises a zinc finger domain.

[0551] 540. The first targeting moiety comprises the amino acid sequence according to SEQ ID NO: 13 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 different position therefrom. The method according to embodiment 538 or 539.

[0552] 541. The method according to any one of embodiments 538 to 540, wherein the first effector moiety comprises MQ1 or a functional variant or fragment thereof.

[0553] 542. The method according to any one of embodiments 538 to 541, wherein the first effector portion comprises the sequence of SEQ ID NO: 19 or 87, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0554] 543. The method according to any one of embodiments 538 to 542, wherein the first effector portion comprises the sequence of SEQ ID NO: 129, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0555] 544. The method according to any one of embodiments 538 to 543, wherein the RNA comprises a nucleotide sequence encoding the first targeting portion, and the nucleotide sequence encoding the first targeting portion comprises the sequence according to SEQ ID NO: 131 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0556] 545. The method according to any one of embodiments 538 to 544, wherein the RNA comprises a nucleotide sequence encoding the first effector portion, and the nucleotide sequence encoding the first effector portion comprises the sequence according to SEQ ID NO: 132, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0557] 546. The method according to any one of embodiments 538 to 545, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 130, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0558] 547. The RNA further encodes a second expression repressor, and the second expression repressor a second targeting moiety that binds to a second genomic locus, and a second effector moiety The method according to any one of embodiments 538 to 546, comprising.

[0559] 548. The method according to 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.

[0560] 549. The method according to embodiment 547 or 548, wherein the second targeting moiety comprises a zinc finger domain.

[0561] 550. The method according to any one of embodiments 547 to 549, wherein the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 7, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0562] 551. The method according to any one of embodiments 547 to 550, wherein the second effector moiety comprises KRAB or a functional variant or fragment thereof.

[0563] 552. The method according to any one of embodiments 547 to 551, wherein the second effector portion comprises the amino acid sequence according to SEQ ID NO: 18, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0564] 553. The method according to any one of embodiments 547 to 552, wherein the second expression repressor comprises the amino acid sequence according to SEQ ID NO: 24, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or a sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0565] 554. The method according to any one of embodiments 547 to 553, wherein the RNA comprises the nucleotide sequence according to SEQ ID NO: 113.

[0566] 555. The method according to any one of embodiments 5209 to 554, wherein the nucleic acid is formulated into lipid nanoparticles (LNP), and optionally the nucleic acid is encapsulated in the LNP.

[0567] 556. The method according to any one of embodiments 520 to 555, wherein the nucleic acid encoding the expression repressor (for example, RNA, for example, mRNA) is administered as an intravenous injection, for example, over 80 to 120 minutes, for example, every two weeks.

[0568] 557. The method according to any one of embodiments 520 to 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, for example, every 1, 2, 3, 4, 5, 6, or 7 weeks.

[0569] The method according to any one of embodiments 520 to 557, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor and the compound having the general structure of formula (I) are administered on different days.

[0570] The method according to any one of embodiments 520 to 558, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor and the compound having the general structure of formula (I) are administered on the same day.

[0571] The method according to embodiment 559, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor is administered before the administration of the compound having the general structure of formula (I) or formula (II).

[0572] The method according to embodiment 559 or 560, wherein the compound having the general structure of formula (I) or formula (II) is administered within 1 to 24 hours after the completion of the administration of the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor.

[0573] Definitions As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0574] Agent: As used herein, the term "agent" may be used to refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations or complexes thereof. As will be apparent to those skilled in the art from the context, in some embodiments, the term may be used to refer to a cell or organism, or a fraction, extract, or component thereof, or an entity containing the same. Alternatively, or in addition, in some embodiments, as will be understood by those skilled in the art in light of the context, the term may be used to refer to a natural product in the sense that it is found in nature and / or is obtainable from nature. In some embodiments, also as will be understood by those skilled in the art in light of the context, the term may be used to refer to one or more entities that are artificial in the sense that they are designed, engineered, and / or fabricated by human artifice and / or are not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, a collection or library of potential agents may be provided, for example, which may be screened to identify or characterize agents that are active therein. In some embodiments, the term "agent" may refer to a compound or entity that is a polymer or contains a polymer; in some embodiments, the term may refer to a compound or entity that contains one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and / or does not substantially contain any polymers and / or does not contain one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or substantially does not contain any polymer moieties.

[0575] Anchor sequence: As used herein, the term "anchor sequence" refers to a nucleic acid sequence recognized by a nucleation agent 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 either an enhancer or a promoter. In some embodiments, the anchor sequence is at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least 1 kb away from any transcription start site. In some embodiments, the anchor sequence is located within a region not associated with genomic imprinting, allelic expression, and / or allelic epigenetic marks. In some embodiments, the anchor sequence has one or more functions selected from binding to an endogenous nucleation polypeptide (e.g., CTCF), formation of anchor sequence-mediated conjugation by interaction with a second anchor sequence, or shielding of an enhancer outside of the anchor sequence-mediated conjugation. In some embodiments of the present disclosure, there is provided a technique capable of specifically targeting a particular one or more anchor sequences without targeting other anchor sequences (e.g., sequences that may contain nucleation agent (e.g., CTCF) binding motifs in another context); such targeted anchor sequences may also be referred to as "target anchor sequences". In some embodiments, the sequence and / or activity of the target anchor sequence is regulated, but the sequence and / or activity of one or more other anchor sequences that may be present within the same system as the targeted anchor sequence (e.g., within the same cell and / or in some embodiments, within the same nucleic acid molecule, e.g., on the same chromosome) is not regulated.In some embodiments, the anchor sequence comprises, or is, a nucleation polypeptide binding motif. In some embodiments, the anchor sequence is adjacent to a nucleation polypeptide binding motif.

[0576] Anchor sequence-mediated junction: As used herein, the term "anchor sequence-mediated junction" refers to a DNA structure, and in some cases a complex, that occurs and / or is maintained by physical interaction or binding of at least two anchor sequences in DNA by one or more polypeptides such as a nucleation polypeptide, or one or more proteins and / or nucleic acid entities (e.g., RNA or DNA), which binds to the anchor sequences and enables spatial proximity and functional linkage of the anchor sequences to each other (see, e.g., FIG. 1).

[0577] Associated with: As used herein, two events or entities are "associated with" each other if the presence, level, form, and / or function of one correlates with that of the other. For example, in some embodiments, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or medical condition if its presence, level, form, and / or function correlates with the incidence and / or susceptibility to that disease, disorder, or medical condition. In some embodiments, two or more entities are physically "associated with" each other if they directly or indirectly interact with each other, are physically proximate to each other, and / or remain physically proximate to each other. In some embodiments, two or more entities that are physically associated with each other are covalently bonded to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently bonded to each other but are non-covalently bonded, e.g., by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, a nucleic acid is at least partially present within a target genome or transcription complex, and the DNA sequence is "associated" with the target genome or transcription complex if the expression of the gene in the DNA sequence is affected by the formation or breakdown of the target genome or transcription complex.

[0578] Biologically active portion of an effector domain: As used herein, a "biologically active portion of an effector domain" is a portion that maintains (e.g., fully, partially, minimally) the function of the effector domain (e.g., a "minimal" or "core" domain).

[0579] Domain: As used herein, the term "domain" refers to a section or portion of an entity. In some embodiments, a "domain" is associated with specific structural and / or functional characteristics of the entity, and when the domain is physically separated from the remainder of its parent entity, it substantially or completely retains the above specific structural and / or functional characteristics. Alternatively, or in addition, in some embodiments, a domain is a part of or can include an entity that, when separated from the (parent) entity and linked to another (recipient) entity, substantially retains and / or confers on the recipient entity one or more structural and / or functional characteristics that characterized it in the parent entity. In some embodiments, a domain is a section or part of or includes a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, polypeptide, etc.). In some embodiments, a domain is a section of or includes a polypeptide. In some such embodiments, a domain is characterized by specific structural elements (e.g., a specific amino acid sequence or sequence motif, an α-helix feature, a β-sheet feature, a coiled-coil feature, a random coil feature, etc.) and / or specific functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signal transduction activity, etc.).

[0580] Effector portion: As used herein, the term "effector portion" refers to a domain that can modify the expression of a target gene when located at an appropriate site within the nucleus of a cell. In some embodiments, the effector portion recruits components of the transcriptional machinery. In some embodiments, the effector portion inhibits the recruitment of components of a transcription factor or a transcriptional repressor. In some embodiments, the effector portion includes an epigenetic modification portion (e.g., epigenetically modifies a target DNA sequence).

[0581] Epigenetic modification moiety: As used herein, the term "epigenetic modification moiety" refers to a domain that, when the epigenetic modification moiety is placed at an appropriate position relative to a nucleic acid (e.g., by a targeting moiety), modifies i) the chromatin structure, e.g., the secondary structure, and / or ii) epigenetic markers (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone SUMOylation, histone phosphorylation, and RNA-related silencing). In some embodiments, the epigenetic modification moiety comprises an enzyme that affects (e.g., increases or decreases the level of) one or more epigenetic markers, or a functional fragment or variant thereof. In some embodiments, the epigenetic modification moiety comprises a DNA methyltransferase, a histone methyltransferase, a CREB-binding protein (CBP), or a functional fragment of any of these.

[0582] Expression control sequence: As used herein, the term "expression control sequence" refers to a nucleic acid sequence that increases or decreases the transcription of a gene, and includes (but is not limited to) promoters and enhancers. An "enhancer sequence" refers to a subtype of expression control sequence that increases the likelihood of gene transcription. A "silencing or repressor sequence" refers to a subtype of expression control sequence that decreases the likelihood of gene transcription.

[0583] Expression repressor: As used herein, the term "expression repressor" refers to an agent or entity having one or more functions 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 the target gene, or a transcriptional control element operably linked to the target gene). An expression repressor comprises at least one targeting moiety and, optionally, one effector moiety.

[0584] Expression suppression system: As used herein, the term "expression suppression system" refers to a plurality of expression repressors that reduce the expression of a target gene in a cell. In some embodiments, the expression suppression system includes a first expression repressor and a second expression repressor, where 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, mixture, or pharmaceutical composition. In some embodiments, the expression suppression system includes a first expression repressor and a second expression repressor, where 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, after the first expression repressor is present in the cell for a first period, the second expression repressor may be present in the cell for a second period, where the first and second periods may be either overlapping or non-overlapping.

[0585] Fusion molecule: As used herein, the term "fusion molecule" refers to a compound comprising two or more moieties covalently linked, such as a targeting moiety and an effector moiety. The fusion molecule and its moieties can include any combination of polypeptides, nucleic acids, glycans, small molecules, or other components described herein (e.g., the targeting moiety may include a nucleic acid and the effector moiety may include a polypeptide). In some embodiments, the fusion molecule is a fusion protein comprising, for example, one or more polypeptide domains covalently linked by peptide bonds. In some embodiments, the fusion molecule is a conjugate molecule comprising a targeting moiety and an effector moiety linked by a covalent bond other 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 other than a peptide bond or a phosphodiester bond). In some embodiments, the expression repressor is a fusion molecule or comprises a fusion molecule.

[0586] Genomic complex: As used herein, the term "genomic complex" is a complex that brings together two genomic sequence elements spaced apart from each other on one or more chromosomes by the interaction of multiple proteins and / or other components (possibly including 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 include an anchor sequence-mediated junction. In some embodiments, the genomic sequence element may be or include a CTCF binding motif, a promoter, and / or an enhancer. In some embodiments, the genomic sequence element includes 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 the genomic sequence element and / or by the binding of the genomic sequence element to one or more protein components. As will be understood by those skilled in the art, in some embodiments, DNA topology in or near the genomic sequence element (e.g., between sequences in some embodiments) is altered by the co-localization (e.g., conjugation) of genomic sites due to the formation of the complex. In some embodiments, the genomic complex includes anchor sequence-mediated conjugation, which includes one or more loops. In some embodiments, the genomic complex described herein is nucleated by a nucleating polypeptide such as, for example, CTCF and / or cohesin. In some embodiments, the genomic complex described herein may include one or more of, for example: CTCF, cohesin, non-coding RNA (e.g., eRNA, transcription machinery proteins (e.g., RNA polymerase, one or more transcription factors selected from the group consisting of, for example, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc.), transcriptional regulatory factors (e.g., Mediator, P300, enhancer binding proteins, repressor binding proteins, histone modifying factors, etc.).In some embodiments, the genomic complexes described herein comprise one or more polypeptide components and / or one or more nucleic acid components (e.g., one or more RNA components), which, in some embodiments, 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 confine a segment of genomic DNA within a topological conformation (e.g., a loop) that would not be adopted in the absence of complex formation.

[0587] 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.

[0588] Modulator: As used herein, the term "modulator" refers to an agent that comprises one or more targeting moieties and one or more effector moieties that can modify (e.g., increase or decrease) the expression of a target gene, e.g., MYC.

[0589] MYC: As used herein, the term "MYC locus" refers to a portion of the human genome that forms an ASMC containing a MYC polypeptide (e.g., the polypeptide disclosed in NCBI accession number NP002458.2, or a mutant thereof), a promoter operably linked to MYC ("MYC promoter"), and an anchor sequence encoding the MYC gene. In some embodiments, the MYC locus encodes a nucleic acid having NCBI accession number NM-002467. In some embodiments, the MYC gene is an oncogene, and in some embodiments the MYC gene is a cancer gene. In certain examples, the MYC gene is found at 8q24.21 on chromosome 8. In certain examples, the MYC gene starts at 128,816,862 bp from pter and ends at 128,822,856 bp from pter. In certain examples, the MYC gene is about 6 kb. In certain examples, the MYC gene encodes at least eight distinct mRNA sequences - mutants that undergo five alternative splicings and mutants that do not undergo three splicings.

[0590] Nucleic acid: As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and / or substance that is incorporated into, or can be incorporated into, an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into, or can be incorporated into, an oligonucleotide chain via a phosphodiester bond. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain containing individual nucleic acid residues. In some embodiments, "nucleic acid" is RNA or includes RNA; in some embodiments, "nucleic acid" is DNA or includes 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 comprises or consists of one or more "peptide nucleic acids", which are known in the art, have peptide bonds instead of phosphodiester bonds in the backbone, and are considered to be within the scope of the present disclosure. Alternatively, or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, a 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, pyrrolo-pyrimidine, 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 natural 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) by polymerization based on a complementary template, replication in a recombinant cell or recombinant 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 completely single-stranded; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence encoding a polypeptide or comprising at least one element that is a complement of a sequence encoding a polypeptide.In some embodiments, the nucleic acid has enzymatic activity.

[0591] Nucleation polypeptide: As used herein, the term "nucleation polypeptide" or "junction nucleation polypeptide" refers to a protein that associates directly or indirectly with an anchor sequence and interacts with one or more junction nucleation polypeptides (which can interact with an anchor sequence or other nucleic acid) to form a dimer (or higher-order structure) that may or may not be identical to each other. When junction nucleation polypeptides associated with different anchor sequences associate with each other such that they keep their different anchor sequences physically close to each other, the resulting structure is an anchor sequence-mediated junction. That is, the interaction of a nucleation polypeptide-anchor sequence with another nucleation polypeptide-anchor sequence in close physical proximity at a short distance results in an anchor sequence-mediated junction having the anchor sequence at the beginning and end (e.g., in some cases, a DNA loop). As will be readily understood by those skilled in the art upon reading this specification, terms such as "nucleation polypeptide", "nucleation molecule", "nucleation protein", "junction nucleation protein" may sometimes be used to refer to a junction nucleation polypeptide. Similarly, as will be readily understood by those skilled in the art upon reading this specification, a collection of two or more junction nucleation polypeptides (which in some embodiments may include multiple copies of the same agent and / or in some embodiments may include one or more of each of a plurality of different agents) may be referred to as a "complex", "dimer", "multimer", etc.

[0592] Operably linked: As used herein, the phrase "operably linked" refers to an arrangement in which the components described are in a relationship that enables them to function in their intended manner. A functional element, such as a gene, that is "operably linked" to a transcriptional control element is joined such that the expression and / or activity of the functional element, such as a gene, is achieved under conditions compatible with the transcriptional control element. In some embodiments, an "operably linked" transcriptional control element is contiguous with (e.g., shares) the coding element of interest, such as a gene; in some embodiments, an operably linked transcriptional control element acts in trans with respect to the functional element of interest, such as a gene, or at a locus that is otherwise at a defined distance from it. In some embodiments, operably linked means that two nucleic acid sequences are included on the same nucleic acid molecule. In another embodiment, operably linked may further mean that two nucleic acid sequences are in proximity to each other on the same nucleic acid molecule, e.g., within 1000, 500, 100, 50, or 10 base pairs of each other, or adjacent to each other.

[0593] Peptide, polypeptide, protein: As used herein, the terms "peptide", "polypeptide", and "protein" refer to compounds comprising 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 to the maximum number of amino acids that can comprise the sequence of a protein or peptide. A polypeptide includes 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 term refers to both short chains, such as are generally referred to in the art as peptides, oligopeptides, and oligomers, and long chains, such as are generally referred to in the art as proteins (of which there are many types).

[0594] Pharmaceutical Composition: As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a modulator, e.g., a disruptor) formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit doses in an amount appropriate for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition is adapted for: oral administration, e.g., a drink (aqueous or non-aqueous solution or suspension), a tablet, e.g., buccal, sublingual, and those targeted for systemic absorption, a bolus, a powder, a granule, a paste applied to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or a sustained release formulation; topical application, e.g., as a cream, an ointment, or a release control patch or spray applied to the skin, the lung, or the oral cavity; intravaginally or rectally, e.g., as a pessary, a cream, or a foam; sublingually; intravitreally; transdermally; or intranasally, including those for administration to the lung and / or other mucosal surfaces, and may be specially formulated for administration in solid or liquid form.

[0595] Proximity: As used herein, "proximity" refers to the closeness of two sites, e.g., nucleic acid sites, such that binding of a transcriptional repressor at a first site and / or modification of the first site by a transcriptional repressor would produce the same or substantially the same effect as binding and / or modification of another site. For example, a targeting moiety may bind to a first site that is in proximity to an enhancer (a second site), and if an effector moiety associated with the targeting moiety epigenetically modifies the first site, it may be substantially the same as if the second site (enhancer sequence) had been bound and / or modified, and the effect of the enhancer on the expression of the target gene may be modified. In some embodiments, a site that is in proximity to a target gene (e.g., an exon, intron, or splice site within the target gene), in proximity to a transcriptional control element operably linked to the target gene, or in proximity 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 (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 the target gene), transcriptional control element, or anchor sequence.

[0596] Specific: As used herein, the term "specific" when referring to an agent having activity is understood by one of ordinary skill in the art to mean that the agent discriminates between a potential target entity or condition. For example, in some embodiments, an agent is said to "specifically" bind to a target if it preferentially binds to that target in the presence of one or more competing other targets. In some embodiments, a specific interaction depends on the presence of specific structural features (e.g., epitope, cleft, binding site) of the target entity. It should be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to the specificity of the binder for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binder. In some embodiments, specificity is evaluated relative to the specificity of a reference non-specific binding agent. In some embodiments, an agent or entity shows no detectable binding to a competing other target under conditions where it binds to its target entity. In some embodiments, a binder binds to its target entity with a fast on-rate, a slow off-rate, a high affinity, a low dissociation, and / or a high stability when compared to one or more competing other targets.

[0597] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in the context in which the binding occurs. In some embodiments, a binder that interacts with one 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 evaluated by detecting or determining the degree of association between the binder and its partner; in some embodiments, specific binding is evaluated by detecting or determining the degree of dissociation of the binder-partner complex. In some embodiments, specific binding is evaluated by detecting or determining the ability of a binder to compete with alternative interactions between its partner and another entity. In some embodiments, specific binding is evaluated by performing such detection or determination at various concentrations.

[0598] Substantially: As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all or the extent of the targeted feature or characteristic. One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, reach completion, and / or proceed to completeness, and / or achieve or avoid absolute results. Thus, the term "substantially" may be used in some embodiments herein to account for the possibility of lack of completeness inherent in many biological and chemical phenomena.

[0599] Symptom alleviation: As used herein, the phrase "symptom alleviation" 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 decreases. In some embodiments, a delay in the onset of a particular symptom is considered one form of decreasing the frequency of that symptom.

[0600] Target: A drug or entity is considered to "target" another drug or entity according to the present disclosure if they specifically bind to a targeting drug or entity under conditions where they contact each other. In some embodiments, for example, an antibody (or an antigen-binding fragment thereof) targets its cognate epitope or antigen. In some embodiments, a nucleic acid having a particular sequence targets a nucleic acid having a substantially complementary sequence.

[0601] Target gene: As used herein, the term "target gene" means a gene that is targeted for regulation, e.g., regulation of expression. In some embodiments, the target gene is part of a targeted genomic complex (e.g., a gene having at least a portion of its genomic sequence as part of the target genomic complex within an anchor sequence-mediated conjugation), and this genomic complex is targeted by one or more regulatory agents described herein. In some embodiments, the regulation includes inhibition of 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 suppression system described herein, e.g., an expression repressor. In some embodiments, the target gene is abnormally expressed (e.g., overexpressed) in a cell, e.g., a cell of a subject (e.g., a patient).

[0602] 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 an anchor sequence). In some embodiments, the genomic sequence element is proximal to and / or operably linked to a target gene (e.g., MYC).

[0603] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used for the alleviation, amelioration, reduction, inhibition, prevention, delay in onset, decrease in severity, and / or decrease in incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent includes an expression suppression system described herein, e.g., an expression repressor. In some embodiments, a therapeutic agent includes a nucleic acid encoding an expression suppression system described herein, e.g., an expression repressor. In some embodiments, a therapeutic agent includes a pharmaceutical composition described herein.

[0604] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, induces a desired biological response. In some embodiments, a therapeutically effective amount of a substance, when administered to a subject having or susceptible to a disease, disorder, and / or medical condition, is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or medical condition. As will be appreciated by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, in some embodiments, the effective amount of a compound in a formulation for treating a disease, disorder, and / or medical condition is an amount that improves, alleviates, reduces, inhibits, prevents, delays the onset of, reduces the severity of, and / or decreases the incidence of one or more symptoms or characteristics of the disease, disorder, and / or medical condition. In some embodiments, the therapeutically effective amount is administered as a single dose; in some embodiments, multiple doses are required to deliver the therapeutically effective amount.

[0605] Halo: As used herein, the term "halo" represents a halogen selected from bromine, chlorine, iodine, and fluorine.

[0606] Alkyl: As used herein, the term "alkyl" refers to a saturated or unsaturated (i.e., having one or more double and / or triple bonds), straight-chain or branched-chain hydrocarbon radical having from 1 to 24 carbon atoms (C1-C 24 alkyl), having from 1 to 12 carbon atoms (C1-C 12a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, having 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), and being 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 (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, propenyl, butenyl, pentenyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless specifically specified otherwise in this specification, the alkyl group is optionally substituted.

[0607] Alkylene: As used herein, the term "alkylene" or "alkylene chain" is saturated or unsaturated (i.e., having one or more double (alkenylene) and / or triple bonds (alkynylene)), and, for example, having 1 to 24 carbon atoms (C1-C 24 alkylene), 1 to 15 carbon atoms (C1-C 15 alkylene), 1 to 12 carbon atoms (C1-C 12 alkylene), 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), a straight-chain or branched divalent hydrocarbon chain consisting only of carbon and hydrogen, connecting the rest of the molecule to a radical group, for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain may be attached to the rest of the molecule via a single bond or a double bond, and may also be attached to the radical via a single bond or a double bond. The point at which the alkylene chain is attached to the rest of the molecule and the radical may be via one carbon or any two carbons within the chain. Unless specifically specified otherwise in this specification, the alkylene chain may be optionally substituted.

[0608] Cycloalkyl: As used herein, the term "cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, which may include fused or bridged ring systems, and is attached by a single bond to the remainder of the molecule and is saturated or unsaturated. Examples of monocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic groups include adamantyl, norbomyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise herein, the cycloalkyl group is optionally substituted.

[0609] Cycloalkylene: As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. Unless specifically stated otherwise herein, the cycloalkylene group may be optionally substituted.

[0610] Cycloalkenylene: As used herein, the term "cycloalkenylene", unless otherwise specified, has 3 to 10 carbons (e.g., C3-C 10"Cycloalkenylene" refers to a divalent carbocyclic non-aromatic group. Non-limiting examples of cycloalkenylene 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; cyclopent-3-ene-1,1-diyl; cyclopent-1,3-diene-1,2-diyl; cyclopent-1,3-diene-1,3-diyl; cyclopent-1,3-diene-1,4-diyl; cyclopent-1,3-diene-1,5-diyl; cyclopent-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. Cycloalkenylene may be unsubstituted or substituted as described for cycloalkyl (e.g., optionally substituted cycloalkenylene).

[0611] Heterocyclyl: As used herein, the term "heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 18-membered non-aromatic ring group consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless specifically stated otherwise herein, a heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system that can include a fused or bridged ring system; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl group can optionally be oxidized; the nitrogen atom can optionally be quaternized; and the heterocyclyl group can be partially saturated or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl [l,3] dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise herein, a heterocyclyl group can optionally be substituted.

[0612] Substituted: As used herein, the term "substituted" means that at least one hydrogen atom is replaced by, 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 , wherein R a is C1-C 12 alkyl or cycloalkyl); a carboxyl group (-OC(=O)R a or -C(=O)OR a , wherein R a is H, C1-C 12 alkyl or cycloalkyl); an amine group (-NR a Rb , wherein R a and R b are each independently H, C1-C 12 alkyl or cycloalkyl); C1-C 12 alkyl group; and a cycloalkyl group, etc., any of the above groups replaced by a bond to a non-hydrogen atom (e.g., alkyl, cycloalkyl or heterocyclyl). In some embodiments, the substituent is a C1-C 12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group such as fluoro. 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.

[0613] Optionally: As used herein, the term "optionally" or "optionally (e.g., optionally substituted)" means that the subsequent described event or situation may or may not occur, and the description includes both the case where the event or situation occurs and the case where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both the substituted alkyl group and the unsubstituted alkyl group.

[0614] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to both acid addition salts and base addition salts.

[0615] Pharmaceutically acceptable acid addition salts: For example, the term "pharmaceutically acceptable acid addition salts" refers to salts that are not biologically or otherwise undesirable and include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as 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, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic 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, glycerophosphoric acid, 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., which retain the biological effectiveness and properties of the free base.

[0616] Pharmaceutically acceptable base addition salts: As used herein, the term "pharmaceutically acceptable base addition salts" refers to salts that retain the biological effectiveness and properties of the free acid and are not biologically or otherwise undesirable. Such salts are prepared by the addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts include ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, for example, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0617] Stereoisomers: As used herein, the term "stereoisomers" refers to compounds that are composed of the same atoms bonded by the same bonds but have non-superimposable different three-dimensional structures. The present invention contemplates various stereoisomers and mixtures thereof, including "enantiomers", which refer to two stereoisomers having molecules that are mirror images of each other and cannot be superimposed on each other.

[0618] Tautomers: As used herein, the term "tautomers" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the above compounds.

[0619] Cationic lipids: As used herein, the term "cationic lipids" refers to lipids that can be positively charged. Exemplary cationic lipids include one or more amine groups that carry a positive charge. Exemplary cationic lipids are ionizable and can exist in a positively charged form or a neutral form depending on the pH. Ionization of cationic lipids can 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, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) and the ability to form an endosomolytic non-bilayer structure for intracellular delivery of nucleic acids (Hafez, I.M., et al., Gene Ther 8:1188-1196 (2001)).

[0620] Neutral lipids: As used herein, the term "neutral lipid" refers to any of several lipid species that exist in either a non-charged or neutral zwitterionic form at the 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), phosphotidylethanolan1ines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and derivatives thereof. Neutral lipids may be synthetic or of natural origin.

[0621] Charged lipids: As used herein, the term "charged lipid" refers to any of several lipid species that exist in either a positively charged or negatively charged form regardless of the pH within the useful physiological range, e.g., pH 3 to pH 9. Charged lipids may be synthetic or of natural origin. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, hemisuccinate sterols, dialkyltrimethylammonium propanes (e.g., DOTAP, DOTMA), dialkyldimethylaminopropanes, ethylphosphocholines, dimethylaminoethane carbamoyl sterols (e.g., DCChol).

[0622] Lipid Nanoparticles: As used herein, the term "lipid nanoparticle" refers to particles comprising a specified lipid such that at least one dimension is in the nanometer range (e.g., 1 - 1,000 nm). Lipid nanoparticles may also include nucleic acids (e.g., mRNA). In some embodiments, lipid nanoparticles are included in formulations 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, lipid nanoparticles contain a nucleic acid. Such lipid nanoparticles typically include 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, may be encapsulated in an aqueous space enclosed by the lipid portion of the lipid nanoparticle or a part or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects caused by the host organism or cell machinery, such as a harmful immune response.

[0623] This patent or application file contains at least one drawing executed 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.

[0624] The following detailed description of embodiments of the disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the disclosure, embodiments exemplified herein are shown in the drawings. However, it should be understood that the disclosure is not limited to the specific arrangements and means shown in the embodiments of the drawings.

Brief Description of the Drawings

[0625]

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Mode for Carrying Out the Invention

[0626] The present disclosure provides, for example, techniques for regulating, for example, reducing the expression of a target gene, such as MYC, in the cells of a subject or patient using the expression repressors or systems described herein.

[0627] Many different diseases and syndromes can be caused by dysregulation of gene expression, including cancer, autoimmunity, cardiovascular disease, and obesity. In particular, it has long been known that overexpression of transcription factors contributes to tumorigenesis, and recent studies have pointed out that overexpression of oncogenic transcription factors can change the central self-regulatory circuits of cells.

[0628] The transcription factor and cell master regulator MYC is frequently dysregulated in more than 50% of human cancers and plays a central role in almost all aspects of the tumorigenic process. Except for early response genes, MYC typically upregulates gene expression. MYC is the most frequently amplified oncogene, and increased expression of its gene product is associated with tumor aggression and poor clinical outcome. Elevated levels of c-MYC can promote tumorigenesis in a wide range of tissues. Most tumor cells are transcription factor c-MYC dependent for their growth and proliferation. MYC overexpression is also associated with chronic liver diseases, such as virus-related and alcohol-related liver diseases. The levels of MYC overexpression vary in specific cancer subtypes. Without wishing to be bound by theory, it is believed that modulating, e.g., reducing, the level of MYC in a subject suffering from MYC dysregulation disorder, e.g., in a whole or in a particular one or more target tissues, may alleviate or eliminate the symptoms of the MYC dysregulation disorder.

[0629] Expression repressor As described herein, the disclosure provides, in part, expression repressors for modulating, e.g., reducing, the expression of a target gene, e.g., MYC. In some embodiments, the expression repressor can include a targeting moiety that binds to a target gene promoter, e.g., the MYC promoter, and optionally an effector moiety. In some embodiments, the targeting moiety specifically binds to a target DNA sequence, e.g., the MYC DNA sequence, thereby localizing the function of the expression repressor to the DNA sequence. In some embodiments, the expression repressor includes a targeting moiety and one effector moiety. In some embodiments, the expression repressor includes a targeting moiety and multiple effector moieties, as described, for example, in International Publication No. WO 2022 / 132195, which is incorporated herein by reference in its entirety.

[0630] In some embodiments, the expression repressor includes one or more targeting moieties as described, for example, in WO 2022 / 132195 (which is incorporated herein by reference in its entirety).

[0631] In some embodiments, the expression repressor includes, for example, a targeting moiety and an effector moiety covalently linked by a peptide bond. In some embodiments, the targeting moiety and the effector moiety are located on the same polypeptide chain and are connected, for example, by one or more peptide bonds and / or linkers. In some embodiments, the expression repressor is or includes a fusion molecule that includes a targeting moiety and an effector moiety linked by, for example, a peptide bond and / or a linker. In some embodiments, the expression repressor includes a targeting moiety disposed on the N-terminal side of the effector moiety on the same polypeptide chain. In some embodiments, the expression repressor includes a targeting moiety disposed on the C-terminal side of the effector moiety on the same polypeptide chain. In some embodiments, the expression repressor includes 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 includes a targeting moiety and a plurality of effector moieties, where the targeting moiety and the plurality of effector moieties are covalently linked, for example, by a peptide bond (e.g., 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 all other effector moieties).

[0632] In other embodiments, the expression repressor comprises a targeting portion and an effector portion that are not covalently linked, as described, for example, in WO 2022 / 132195, which is incorporated herein by reference in its entirety.

[0633] Generally, an expression repressor as described herein binds to a genomic sequence element proximal to and / or operably linked to a target gene, such as MYC (e.g., via the targeting portion). In some embodiments, when the expression repressor binds to the genomic sequence element, the expression of the target gene, such as MYC, is regulated (e.g., decreased). For example, when an expression repressor comprising an effector portion that recruits or inhibits the recruitment of components of the transcriptional machinery binds to the genomic sequence element, the expression of the target gene, such as MYC, can be regulated (e.g., decreased). As a further example, when an expression repressor comprising an effector portion with enzymatic activity (e.g., an epigenetic modification portion) binds, the expression of the target gene, such as MYC, can be regulated (e.g., decreased) through the localization of the enzymatic activity of the effector portion. As a further example, both the binding of the expression repressor to the genomic sequence element and the localization of the enzymatic activity of the expression repressor can contribute to the regulation (e.g., decrease) of the expression of the resulting target gene, such as MYC.

[0634] In some embodiments, the expression repressor comprises an effector portion, where the effector portion is selected from the proteins 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., the KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof.

[0635] In some embodiments, the present disclosure provides nucleic acid sequences encoding an expression repressor, an expression repressor system, a targeting portion and / or an effector portion as described herein. Those skilled in the art will recognize that the nucleic acid sequence of RNA is identical to the corresponding DNA sequence, except that typically thymine (T) is replaced by uracil (U). When the nucleotide sequence is represented by a DNA sequence (e.g., including A, T, G, C), the present disclosure also provides the corresponding RNA sequence (e.g., including A, U, G, C) in which "T" is replaced by "U". Conventional notation is used herein to describe polynucleotide sequences: the left end of a single-stranded polynucleotide sequence is the 5' end; the left-handed direction of a double-stranded polynucleotide sequence is called the 5' direction.

[0636] Due to the degeneracy of the genetic code, a number of nucleotide sequences encoding expression repressors containing the targeting portion and / or effector portion described herein can be produced, some of which will be understood by those skilled in the art to have similarity to the nucleic acid sequences disclosed herein, e.g., 90%, 95%, 96%, 97%, 98%, or 99% identity. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at all positions in the nucleic acid molecules of the invention where arginine is specified by a codon, the codon can be modified to any of the foregoing corresponding codons without modifying the encoded polypeptide.

[0637] In some embodiments, the nucleic acid agglomerates encoding expression repressors containing the targeting portion and / or effector portion can be part or all of a codon-optimized coding region optimized according to the codon usage frequency in mammals, e.g., humans. In some embodiments, the nucleic acid agglomerates encoding the targeting portion and / or effector portion are codons 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 compared to the level of the protein when encoded by a non-codon-optimized nucleic acid sequence.

[0638] Expression repression system The expression suppression system of the present disclosure may include two or more expression repressors. In some embodiments, the expression suppression system includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more (and optionally, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less) expression repressors. In some embodiments, the expression suppression system targets two or more different sequences (e.g., the first and second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and / or more DNA sequences, and optionally the 20th, 19th, 18th, 17th, 16th, 15th, 14th, 13th, 12th, 11th, 10th, 9th, 8th, 6th, 5th, 4th, 3rd, or 2nd or less sequences). In some embodiments, the expression suppression system includes a plurality of expression repressors, wherein each member of the plurality of expression repressors does not bind detectably to another member of the plurality of expression repressors, e.g., does not bind. In some embodiments, the expression suppression system includes a first expression repressor and a second expression repressor, wherein the first expression repressor does not bind detectably to the second expression repressor, e.g., does not bind.

[0639] In some embodiments, the expression suppression system of the present disclosure includes two or more expression repressors, wherein the expression repressors are present together in a composition, pharmaceutical composition, or mixture. In some embodiments, the expression suppression system of the present disclosure includes two or more expression repressors, wherein one or more of the expression repressors are not mixed with at least one other expression repressor. For example, the expression suppression system may include a first expression repressor and a second expression repressor, wherein the presence of the first expression repressor in the nucleus of a cell does not overlap with the presence of the second expression repressor in the nucleus of the same cell, wherein the expression suppression system achieves a decrease in the expression of the MYC gene by the non-overlapping presence of the first and second expression repressors. In some embodiments, the expression suppression system achieves a greater decrease in the expression of the MYC gene compared to the decrease in the expression of the MYC gene achieved by the first or second expression repressor alone.

[0640] In some embodiments, the expression repressors of the expression repressor system each include a different targeting moiety (e.g., the first, second, third, or more expression repressors each include a targeting moiety that is different from each other). For example, the expression repression system may include a first expression repressor and a second expression repressor, where the first expression repressor includes a first targeting moiety (e.g., a Cas9 domain, a TAL effector domain, or a Zn finger domain), and the second expression repressor includes 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 means including individual types of targeting moieties. For example, the first targeting moiety may include a Cas9 domain, and the second DNA targeting moiety may include a Zn finger domain. In other embodiments, different means including individual variants of the same type of targeting moiety. For example, the first targeting moiety may include a first Cas9 domain (e.g., from a first species), and the second targeting moiety may include a second Cas9 domain (e.g., from a second species). In certain embodiments, when the expression repressor system includes two or more targeting moieties of the same type, e.g., two or more Cas9 or ZF domains, those targeting moieties specifically bind to two or more different sequences. For example, in an expression repressor system including two or more Cas9 domains, the two or more Cas9 domains are selected (e.g., and modified so as not to bind to a gRNA corresponding to the target of another Cas9 domain) so as to bind only as much as they can recognize their target sequence. In a further example, in an expression repressor system including two or more effector moieties, the two or more effector moieties are selected (e.g., and modified so as not to bind to the target sequence of another effector moiety) so as to bind only as much as they can recognize their target sequence.

[0641] In some embodiments, the expression repressors of the expression repressor system each bind to a different DNA sequence (e.g., the first, second, third, or more expression repressors each bind to a DNA sequence that is different from each other). For example, the expression repression system may include a first expression repressor and a second expression repressor, where 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 means that there is at least one position that is not identical between the DNA sequence to which one expression repressor binds and the DNA sequence to which the other expression repressor binds, or there is at least one position that exists in the DNA sequence to which one expression repressor binds and does not exist in the DNA sequence to which the other expression repressor binds.

[0642] In some embodiments, the first DNA sequence may be located on the first genomic DNA strand, and the second DNA sequence may be located on the 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.

[0643] In some embodiments, the expression repression system includes two or more (e.g., two) expression repressors, and a plurality (e.g., two) of those expression repressors include targeting moieties that bind to different DNA sequences. In such embodiments, the first targeting moiety may bind to a first DNA sequence, and the second DNA targeting moiety may bind to a second DNA sequence, where the first and second DNA sequences are different and non-overlapping.

[0644] In some embodiments, the expression repressors of the expression repressor system each comprise a different effector moiety (e.g., the first, second, third, or more expression repressors each comprise a different effector moiety from each other). For example, the expression repression system may comprise a first expression repressor and a second expression repressor, wherein the first expression repressor comprises a first effector moiety (e.g., one comprising a DNA methyltransferase or a functional fragment thereof), and the second expression repressor comprises a second effector moiety different from the first effector moiety (e.g., one comprising a transcriptional repressor (e.g., KRAB) or a functional fragment thereof). In some embodiments, different may mean comprising individual types of effector moieties. In other embodiments, different may mean comprising individual variants of the same type of effector moiety, e.g., the first effector moiety comprises a first DNA methyltransferase (e.g., one having a first site specificity or amino acid sequence), and the second effector moiety comprises a second DNA methyltransferase (e.g., one having a second site specificity or amino acid sequence).

[0645] In some embodiments, the expression repressor system includes a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety, wherein the first effector moiety comprises a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof, and the second effector moiety comprises a different protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment thereof.

[0646] In some embodiments, the first or second effector portion comprises DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof), the other effector portion comprises transcriptional repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or any functional variant or fragment thereof), the first or second effector portion comprises histone methyltransferase activity, and the other effector portion comprises 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 any functional variant or fragment thereof). In some embodiments, the first or second effector portion comprises histone methyltransferase activity and the other effector portion comprises DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or any functional variant or fragment thereof). In some embodiments, the first or second effector portion comprises DNA methyltransferase activity and the other effector portion comprises transcriptional repressor activity. In some embodiments, the first or second effector portion comprises histone methyltransferase activity and the other effector portion comprises transcriptional repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or any functional variant or fragment thereof).

[0647] In some embodiments, two or more (e.g., all) of the expression repressors of the 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 of the expression repressors of the expression repressor system are covalently associated with each other. In certain embodiments, the expression repression system includes, for example, a first expression repressor and a second expression repressor disposed on the same polypeptide, connected by, for example, a peptide bond and optionally a linker, e.g., as a fusion molecule. In some embodiments, the peptide is a self-cleaving peptide, e.g., a T2A self-cleaving peptide. In certain embodiments, the expression repression system includes a first expression repressor and a second expression repressor connected by a non-peptide bond, e.g., conjugated to each other.

[0648] Linker An expression repressor or expression repressor system as disclosed herein may include one or more linkers. The linker may connect a targeting moiety to an effector moiety, connect an effector moiety to another effector moiety, or connect a targeting moiety to another targeting moiety. The linker may be a chemical bond, e.g., one or more covalent or non-covalent bonds. In some embodiments, the linker is covalent. In some embodiments, the linker is non-covalent. In some embodiments, the linker is a peptide linker. Such linker may be 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 2, 5, 10, 15, 20, 25, or 30 amino acids or longer (and optionally 50, 40, 30, 25, 20, 15, 10, or 5 amino acids or shorter). In some embodiments, a linker can be used to separate a first domain or portion from a second domain or portion, e.g., separating a DNA targeting moiety from an effector moiety. In some embodiments, e.g., the linker can be positioned between a DNA targeting moiety and an effector moiety to provide, e.g., molecular flexibility of secondary and tertiary structures. The linker may include flexible, rigid, and / or cleavable linkers as described herein. In some embodiments, the linker provides flexibility by including at least one glycine, alanine, and serine amino acid. In some embodiments, the linker is a hydrophobic linker, such as including a negatively charged sulfonic acid group, a polyethylene glycol (PEG) group, or a pyrophosphate diester group. In some embodiments, the linker is cleavable and selectively releases a moiety (e.g., a polypeptide) from a modulator, but is stable enough to prevent premature cleavage.

[0649] In some embodiments, one or more portions and / or domains of the expression repressors described herein are linked to one or more linkers. In some embodiments, expression repression may include a linker located between a targeting portion and an effector portion. In some embodiments, the linker may have the sequence of ASGSGGGSGGARD (SEQ ID NO: 137), or ASGSGGGSGG (SEQ ID NO: 138). In some embodiments, a system comprising a first and a second repressor may include a first linker located between a first targeting portion and a first effector portion, and a second linker located between a second targeting portion and a second effector portion. 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 the amino acid sequence according to SEQ ID NO: 137 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, and the second linker may comprise the amino acid sequence according to SEQ ID NO: 138 or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0650] As will be known to those skilled in the art, commonly used flexible linkers generally have a sequence consisting mainly of a series of Gly and Ser residues ("GS" linkers). Flexible linkers may be useful for joining domains / portions that require some degree of movement or interaction, and may include small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Incorporating Ser or Thr can also maintain the stability of the linker in an aqueous solution by forming hydrogen bonds with water molecules, and thus reduce unfavorable interactions between the linker and the portion / domain. In some embodiments, the linker is a GS linker or a variant thereof, such as G4S (SEQ ID NO: 207).

[0651] Rigid linkers are useful for keeping domains / portions at a fixed distance and maintaining their independent functions. Rigid linkers can also be useful when it is critically important that domains be spatially separated to ensure the stability or biological activity of one or more components within the fusion. Rigid linkers can have an α-helix structure or a Pro-rich sequence, (XP) n which may have (wherein X means any amino acid, preferably Ala, Lys, or Glu).

[0652] Cleavable linkers can release free functional domains in vivo. In some embodiments, the linker may be cleaved under special conditions, such as the presence of a reducing reagent or protease. In vivo cleavable linkers can utilize the reversible nature of disulfide bonds. An example is a thrombin-sensitive sequence (e.g., PRS) between two Cys residues. Treatment of CPRSC residues with thrombin in vitro results in cleavage of the thrombin-sensitive sequence while the reversible disulfide bond remains intact. Such linkers are known and are described, for example, in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369. In vivo cleavage of the linker within the fusion can also be effected by proteases that are expressed in specific cells or tissues or are confined within certain intracellular compartments under certain conditions in vivo. The specificity of many proteases results in a delay in cleavage of the linker within the confined compartment. In some embodiments, the cleavable linker can be a self-cleaving linker, such as a T2A peptide linker. In some embodiments, the linker can include a "ribosome skipping" sequence, such as a tPT2A linker.

[0653] Examples of molecules suitable for use in the linkers 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 variants thereof, or other N-containing variants; carbonless linkers; carbohydrate linkers; phosphodiester linkers, or other molecules capable of covalently linking two or more components of the expression repressor. Also included are non-covalent linkers such as hydrophobic lipid globules to which a polypeptide is linked through a hydrophobic region of the polypeptide or a hydrophobic extension of the polypeptide, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues. The components of the expression repressor may be linked using charge-based chemistry such that the positively charged component of the expression repressor is linked to the negative charge of another component.

[0654] Targeting moiety The present disclosure provides an expression repressor comprising a targeting moiety that specifically targets, e.g., binds to, a genomic sequence element (e.g., a promoter, TSS, or anchor sequence) that is at, proximal to, and / or operably linked to a target gene. The targeting moiety can specifically bind to a DNA sequence, e.g., 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 the targeting moiety.

[0655] In some embodiments, the targeting moiety targets, e.g., binds to, a component of a genomic complex (e.g., an ASMC). In some embodiments, the targeting moiety targets, e.g., binds to, an expression control sequence (e.g., a promoter or enhancer) operably linked to a target gene. In some embodiments, the targeting moiety targets, e.g., binds to, a target gene, or a portion of a target gene. The target of the targeting moiety may be referred to as its targeting component. The targeting component may be any genomic sequence element operably linked to a target gene, or the target gene itself, including, but not limited to, a promoter, enhancer, anchor sequence, exon, intron, UTR coding sequence, splice site, or transcription start site. In some embodiments, the targeting moiety specifically binds to one or more target anchor sequences (e.g., intracellular) and does not bind to non-target anchor sequences (e.g., within the same cell).

[0656] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 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.

[0657] In some embodiments, the present disclosure provides an expression repressor comprising a targeting moiety that binds to a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2 or 77, 82, or 83; and wherein the expression repressor is capable of reducing the expression of a target gene, e.g., MYC. In some embodiments, the expression repressor comprises an effector moiety.

[0658] In some embodiments, the present disclosure provides an expression repressor comprising a targeting portion that binds to a genomic locus within 1400 nt upstream or downstream of SEQ ID NO: 4.

[0659] In some embodiments, the present disclosure provides an expression repressor comprising a targeting portion that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, or 83.

[0660] In some embodiments, the present disclosure provides an expression repressor comprising a targeting portion that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, 96, or 108.

[0661] In some embodiments, the targeting moiety can be or include a CRISPR / Cas domain, a TAL effector domain, a Zn finger domain, a peptide nucleic acid (PNA), or a nucleic acid molecule. In some embodiments, the expression repressor includes one effector moiety. In some embodiments, the expression repressor includes a plurality of targeting moieties, where each targeting moiety does not bind detectably to another targeting moiety, e.g., does not bind. In some embodiments, the expression repression system includes a plurality of expression repressors, where each member of the plurality of expression repressors includes a targeting moiety, where each targeting moiety does not bind detectably to another targeting moiety, e.g., does not bind. In some embodiments, the expression repression system includes a first expression repressor that includes a first targeting moiety and a second expression repressor that includes a second targeting moiety, where the first targeting moiety does not bind detectably to the second targeting moiety, e.g., does not bind. In some embodiments, the expression repression system includes a first expression repressor that includes a first targeting moiety and a second expression repressor that includes a second targeting moiety, where the first targeting moiety does not bind detectably to another first targeting moiety, e.g., does not bind, and where the second targeting moiety does not bind detectably to another second targeting moiety, e.g., does not bind. In some embodiments, the targeting moieties used in the compositions and methods described herein are monomers, e.g., are functional in a non-dimerized state (e.g., bind to a DNA sequence).

[0662] In some embodiments, the targeting moiety can be or include a CRISPR / Cas domain, a TAL effector domain, a Zn finger domain, or a nucleic acid molecule, or can include the same. In some embodiments, the targeting moiety is or includes a DNA binding domain from a nuclease. Exemplary nucleic acid targeting moieties are described, for example, in International Publication No. WO 2022 / 132195, which is incorporated herein by reference in its entirety.

[0663] A system comprising an expression repressor or repressors as disclosed herein may comprise nucleic acids, e.g., one or more nucleic acids. The term "nucleic acid" refers to an oligonucleotide chain or any compound that can be incorporated therein. In some embodiments, the nucleic acid is an oligonucleotide chain or a compound and / or substance that can be incorporated therein via a phosphodiester bond. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, the nucleic acid is or comprises more than 50% ribonucleotides and is referred to herein as ribonucleic acid (RNA). In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone and are considered to be within the scope of the present invention. Alternatively or in addition, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of 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, pyrrolo-pyrimidine, 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, intercalating 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 present in natural 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) by polymerization based on a complementary template, propagation in a recombinant cell or system, and chemical synthesis. As used herein, "recombinant" when used to describe a nucleic acid refers to any nucleic acid that does not occur naturally. 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 residues in length or greater.In some embodiments, the nucleic acid can have a length of about 2 to about 5000 nt, about 10 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to 250 nt, about 200 to about 300 nt, about 250 to about 350 nt, about 300 to about 500 nt, about 10 to about 1000 nt, about 50 to about 1000 nt, about 100 to about 1000 nt, about 1000 to about 2000 nt, about 2000 to about 3000 nt, about 3000 to about 4000 nt, about 4000 to about 5000 nt, or any range therebetween. In some embodiments, the nucleic acid is partially or completely single-stranded; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a polypeptide or includes at least one element that is a complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0664] CRISPR / Cas domain In some embodiments, for example, as described in International Publication No. WO 2022 / 132195, which is incorporated herein by reference in its entirety, the targeting moiety is or includes a CRISPR / Cas domain. The CRISPR / Cas protein can include a CRISPR / Cas effector and optionally one or more other domains. The CRISPR / Cas domain typically has structural and / or functional similarity to a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, such as a Cas protein. The CRISPR / Cas domain optionally includes a guide RNA, such as a single guide RNA (sgRNA). In some embodiments, the gRNA included by the CRISPR / Cas domain is non-covalently bound by the CRISPR / Cas domain.

[0665] In some embodiments, the Cas protein is modified such that the nuclease is inactivated, e.g., nuclease-deficient Cas. In some embodiments, the Cas protein is a Cas9 protein. Wild-type Cas9 generates a double-strand break (DSB) in a specific DNA sequence targeted by the gRNA, whereas several CRISPR endonucleases with modified functionality are available, e.g., the "nickase" version of Cas9 generates only single-strand breaks; catalytically inactive Cas9 ("dCas9") does not cleave the target DNA. In some embodiments, the binding of the DNA sequence to dCas interferes with the transcription of the site due to steric hindrance. In some embodiments, the DNA targeting moiety is a catalytically inactive Cas, e.g., dCas, or includes it. Many catalytically inactive Cas proteins are known in the art. In some embodiments, dCas9 includes mutations, e.g., D10A and H840A mutations, in each endonuclease domain of the Cas protein.

[0666] TAL effector domain In some embodiments, for example, as described in International Publication No. WO 2022 / 132195, which is incorporated herein by reference in its entirety, the DNA targeting moiety is a TAL effector domain or includes it. A TAL effector domain, e.g., a TAL effector domain that specifically binds to a DNA sequence, includes a plurality of TAL effector repeats or fragments thereof, and optionally, one or more additional portions of a naturally occurring TAL effector repeat (e.g., on the N-terminal side and / or C-terminal side of a plurality of TAL effector domains), where each TAL effector repeat recognizes a nucleotide. A TAL effector protein can include a TAL effector domain and optionally one or more other domains. Many TAL effector domains are known to those skilled in the art and are commercially available, e.g., from Thermo Fisher Scientific.

[0667] Zn finger domain In some embodiments, the DNA targeting moiety is or comprises a Zn finger domain. The Zn finger domain comprises Zn fingers, e.g., naturally occurring Zn fingers or engineered Zn fingers, or fragments thereof. Many Zn fingers are known to those skilled in the art and are commercially available, e.g., from Sigma-Aldrich. Generally, the Zn finger domain comprises multiple Zn fingers, where each Zn finger recognizes three nucleotides. A Zn finger protein can 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 WO 2022 / 132195, which is hereby incorporated by reference in its entirety.

[0668] Furthermore, as disclosed in the above and other references, zinc fingers and / or multi-finger zinc finger domains may be linked to each other using any suitable linker sequence, such as a linker having a length of 5 amino acids or more. For exemplary linker sequences having a length of 6 amino acids or more, see U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949. The proteins described herein may comprise any combination of suitable linkers between the individual zinc fingers of the protein. In addition, for enhancement of binding specificity to a zinc finger binding domain, see, e.g., co-owned WO 02 / 077227.

[0669] In certain embodiments, the DNA targeting moiety comprises a Zn finger domain that includes engineered zinc fingers that (sequence specifically) bind to a target DNA sequence. In some embodiments, the Zn finger domain comprises one zinc finger or a fragment thereof. In some embodiments, the Zn finger domain comprises multiple zinc fingers (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more zinc fingers (and optionally, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer fingers). In some embodiments, the Zn finger domain comprises at least 3 zinc fingers. In some embodiments, the Zn finger domain comprises 4, 5, or 6 zinc fingers. In some embodiments, the Zn finger domain comprises 8, 9, 10, 11, or 12 zinc fingers. In some embodiments, a Zn finger domain comprising 3 zinc fingers recognizes a target DNA sequence that comprises 9 or 10 nucleotides. In some embodiments, a Zn finger domain comprising 4 zinc fingers recognizes a target DNA sequence that comprises 12-14 nucleotides. In some embodiments, a Zn finger domain comprising 6 zinc fingers recognizes a target DNA sequence that comprises 18-21 nucleotides.

[0670] In some embodiments, the targeting domain comprises a two-handed Zn finger protein. A two-handed Zn finger protein is a protein in which two clusters of zinc fingers are separated by intervening amino acids such that the two zinc finger domains bind to two discontinuous target sites. An example of a two-handed zinc finger binding protein is SIP1, in which case a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three Zn 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 can bind to a unique target sequence, and the spacing between the two target sequences can include many nucleotides.

[0671] In some embodiments, the expression repressor comprises a targeting moiety comprising a zinc finger domain that binds to an anchor sequence-mediated junction (ASMC) anchor sequence, such as a sequence proximal to an anchor sequence, such as a sequence proximal to a target gene (e.g., MYC), which includes an engineered DNA binding domain (DBD), e.g., a promoter or transcription start site (TSS) sequence operably linked to a target sequence, e.g., a target gene (e.g., MYC), e.g., a sequence proximal to a transcriptional regulatory element, e.g., a zinc finger (ZFN) that binds to a sequence proximal to the anchor sequence of the anchor sequence-mediated junction (ASMC) that includes the target gene (e.g., MYC). 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 zinc 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 the exemplary targeting moieties disclosed herein are listed in Table 5. In some embodiments, the expression repressor or system described herein comprises a targeting moiety having a sequence shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, the nucleic acid described herein comprises a sequence shown in Table 5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0672]

Table 4-1

[0673]

Table 4-2

[0674]

Table 5-1

[0675]

Table 5-2

[0676]

Table 5-3

[0677] In some embodiments, expression repression involves a targeting moiety comprising a zinc finger domain that binds to an engineered DNA binding domain (DBD), such as a target sequence in the mouse genome, such as a promoter or transcription start site (TSS) sequence operably linked to a target gene (e.g., MYC), such as a sequence proximal to a transcriptional regulatory element, such as an anchor sequence of an anchor sequence-mediated junction (ASMC) containing a target gene (e.g., MYC), such as a sequence proximal to the anchor sequence, and includes a zinc finger (ZFN) that binds to the 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 zinc 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 the exemplary targeting moieties disclosed herein are listed in Table 15. In some embodiments, the expression repressor or system described herein comprises a targeting moiety having a sequence shown in Table 14, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, the nucleic acid described herein comprises a sequence shown in Table 15, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0678]

Table 14

[0679]

Table 15

[0680] Effector portion In some embodiments, the expression repressor of the present disclosure includes one or more effector portions. In some embodiments, the effector portion reduces the expression of a target gene in a cell when used as part of the expression repressor or expression suppression system described herein.

[0681] In some embodiments, the effector portion has a function unrelated to the binding of the targeting portion. For example, the effector portion can target, e.g., bind to, or recruit a transcription factor to a genomic sequence element targeted by the targeting portion or a genomic complex component proximal to the genomic sequence element. As a further example, the effector portion can include an enzymatic activity, e.g., a gene modification function.

[0682] In some embodiments, the effector portion includes an epigenetic modification portion. In some embodiments, the effector portion includes a DNA modification function, e.g., a DNA methyltransferase. In some embodiments, the effector portion is a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any of these, or includes the same.

[0683] In some embodiments, the effector portion includes a transcriptional repressor. In some embodiments, the transcriptional repressor blocks, for example, the recruitment of factors that stimulate or promote transcription of a target gene. In some embodiments, the transcriptional repressor recruits factors that inhibit transcription of a target gene, for example. In some embodiments, the effector portion, for example, the transcriptional repressor, is a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or any functional variant or fragment thereof, or includes the same.

[0684] In some embodiments, the effector portion directly or indirectly promotes epigenetic modification, for example. For example, the effector portion may indirectly promote epigenetic modification by recruiting endogenous proteins that epigenetically modify chromatin. The effector portion can directly promote epigenetic modification by catalyzing epigenetic modification, where the effector portion includes enzymatic activity and directly places epigenetic marks on chromatin.

[0685] In some embodiments, the effector portion includes histone modification functions, such as histone methyltransferase, histone demethylase, or histone deacetylase activity. In some embodiments, the effector portion is a protein selected from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or any functional variant or fragment thereof, or includes the same. In some embodiments, the effector portion is a protein selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or any functional variant or fragment thereof, or includes the same.

[0686] In some embodiments, the effector portion comprises a protein having the functions described herein. In some embodiments, the effector portion is a protein selected from, or comprises, the following: KRAB (e.g., as described in NP_056209.2 or the protein encoded by NM_015394.5); SET domain (e.g., the following SET domains: SETDB1 (e.g., as described in NP_001353347.1 or the protein encoded by NM_001366418.1); EZH2 (e.g., as described in NP-004447.2 or the protein encoded by NM_004456.5); G9A (e.g., as described in NP_001350618.1 or the protein encoded by NM_001363689.1); or SUV39H1 (e.g., as described in NP_003164.1 or the protein encoded by NM_003173.4)); histone demethylase LSD1 (e.g., as described in NP_055828.2 or the protein encoded by NM_015013.4); FOG1 (e.g., the N-terminal residue of FOG1) (e.g., as described in NP_722520.2 or the protein encoded by NM_153813.3); or KAP1 (e.g., as described in NP_005753.1 or the protein encoded by NM_005762.3)); any functional fragment or variant thereof, or a polypeptide of a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with any of the sequences mentioned above.

[0687] In some embodiments, the effector portion is a protein selected from, or includes, the following: DNMT3A (e.g., human DNMT3A) (e.g., as described in NP_072046.2 or the protein encoded by NM_022552.4); DNMT3B (e.g., as described in NP_008823.1 or the protein encoded by NM_006892.4); DNMT3L (e.g., as described in NP_787063.1 or the protein encoded by NM_175867.3); DNMT3A / 3L complex, bacterial MQ1 (e.g., as described in CAA35058.1 or P15840.3); a functional fragment of any of these, or a polypeptide of a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the sequences mentioned above.

[0688] In another aspect, the present disclosure relates to an expression repressor or polypeptide comprising one or more (e.g., one) targeting portions and one or more effector portions, wherein the one or more effector portions are a Krüppel-associated box (KRAB), e.g., the protein as described in NP_056209.2 or encoded by NM_015394.5 or a functional variant or fragment thereof, or includes the same. In some embodiments, the KRAB is a synthetic KRAB construct. In some embodiments, the KRAB includes the amino acid sequence of SEQ ID NO: 18: DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 18)

[0689] In some embodiments, the KRAB effector portion is encoded by the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the nucleotide sequences described herein are of the sequence of SEQ ID NO: 51 or have at least 80, 85, 90, 95, 99, or 100% identity thereto, or include sequences with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position different therefrom.

[0690]

Chemical formula

[0691] In some embodiments, the KRAB for use in the polypeptides or expression repressors described herein is a variant, e.g., a variant containing one or more mutations compared to the KRAB sequence of SEQ ID NO: 18. In some embodiments, the KRAB variant contains one or more amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 18.

[0692] In some embodiments, the polypeptide or expression repressor is KRAB or a fusion protein comprising an effector portion containing it and a DNA targeting portion.

[0693] In another aspect, the present disclosure relates to an expression repressor or polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the one or more effector moieties are MQ1, such as bacterial MQ1, or a functional variant or fragment thereof, or comprise the same. 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, the effector domain described herein is SEQ ID NO: 19 or 87, or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or comprises a sequence 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.

Chemical formula

Chemical formula

[0694] In some embodiments, MQ1 is encoded by the nucleotide sequence of SEQ ID NO: 52 or 132. In some embodiments, the nucleic acid described herein is the sequence of SEQ ID NO: 52, 132 or has at least 80, 85, 90, 95, 99, or 100% identity thereto, or comprises a sequence 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.

Chemical formula

Chemical formula

[0695] In some embodiments, the MQ1 for use with the polypeptides or expression repressors described herein is a variant, e.g., a variant that contains one or more mutations compared to wild-type MQ1 (e.g., SEQ ID NO: 19). In some embodiments, the MQ1 variant contains one or more amino acid substitutions, deletions, or insertions compared to wild-type MQ1, e.g., SEQ ID NO: 19. In some embodiments, the MQ1 variant contains the K297P substitution. In some embodiments, the MQ1 variant contains the N299C substitution. In some embodiments, the MQ1 variant contains the E301Y substitution. In some embodiments, the MQ1 variant contains the Q147L substitution (e.g., and has reduced DNA methyltransferase activity compared to wild-type MQ1). In some embodiments, the MQ1 variant contains the K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA binding affinity compared to wild-type MQ1). In some embodiments, the MQ1 variant contains the Q147L, K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA methyltransferase activity and DNA binding affinity compared to wild-type MQ1).

[0696] In some embodiments, one or more effector portions are, or include, DNMT1, such as human DNMT1, or a functional variant or fragment thereof. In some embodiments, DNMT1 is, for example, human DNMT1 corresponding to Gene ID 1786, such as corresponding to UniProt ID P26358.2. In some embodiments, DNMT1 includes the amino acid sequence of SEQ ID NO: 20 as described in WO 2022 / 132195 pamphlet (which is incorporated herein by reference in its entirety). In some embodiments, the effector domain described herein includes, for example, the sequence according to SEQ ID NO: 20 as described in WO 2022 / 132195 pamphlet (which is 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 with differences 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, DNMT1 is encoded by, for example, the nucleotide sequence according to SEQ ID NO: 53 as described in WO 2022 / 132195 pamphlet (which is 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 with differences at no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3...

Claims

1. A combination for treating a target cancer that requires cancer treatment, wherein the combination is (1) A nucleic acid encoding an expression repressor, wherein the expression repressor is (a) A first targeting portion, wherein the first targeting portion is: (i) Binds to the MYC promoter, or (ii) A first targeting portion that binds to a genomic locus containing at least 16, 17, 18, 19, or 20 nucleotides of any sequence 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, and (b) Optionally, the first effector section Includes, The expression repressor is a nucleic acid capable of reducing the expression of MYC; and (2) General structure of equation (I): 【Chemistry 27】 (In the formula, R １ is alkyl, cycloalkyl, or aryl; R ２ is a halo, alkyl, or H; R ３ , R ４ , R ５ , R ６ , R ７ Each is independently either H or alkyl; R ８ and R ９ together form an optionally substituted aromatic ring) Compounds having; or its pharmaceutically acceptable salts, tautomers, or stereoisomers A combination that includes this.

2. (a) R １ is alkyl or cycloalkyl; (b) R1 = cyclopropane; (c) R 2 is a halo; (d) R² = Cl; (e) R3, R4, R5, R6, R7 are each H, and / or (f) The combination according to claim 1, wherein the substituted aromatic ring is a substituted phenyl ring.

3. The compound of formula (I) above, 【Chemistry 29】 The combination according to claim 1, or comprising a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

4. The compound of formula (I) (a) characterized by being administered orally; and / or (b) The combination according to claim 1, characterized in that it is administered in a dose of 6 to 10 or 10 to 14 mg per day.

5. The combination according to claim 4, characterized in that the compound of formula (I) is administered in a dose of about 8 or about 12 mg per day.

6. A composition for treating a target cancer requiring cancer treatment, wherein the composition comprises a nucleic acid encoding an expression repressor, (a) A first targeting portion, wherein the first targeting portion is: (i) Binds to the MYC promoter, or (ii) A first targeting portion that binds to a genomic locus containing at least 16, 17, 18, 19, or 20 nucleotides of any sequence 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, and (b) Optionally, the first effector section Includes, The expression repressor can reduce the expression of MYC; The composition has a general structure of formula (I): 【Chemistry 27】 (In the formula, R1 is alkyl, cycloalkyl, or aryl; R2 is a halo, alkyl, or H; R3, R4, R5, R6, and R7 are each independently H or alkyl; R8 and R9 together form an aromatic ring that is optionally substituted. Compounds having; A composition characterized by being administered in combination with a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

7. (a) R1 is alkyl or cycloalkyl; (b) R1 = cyclopropane; (c) R 2 is a halo; (d) R² = Cl; (e) R3, R4, R5, R6, R7 are each H, and / or (f) The composition according to claim 6, wherein the substituted aromatic ring is a substituted phenyl ring.

8. The compound of formula (I) is 【Chemistry 29】 The composition according to claim 6, or comprising a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

9. The compound of formula (I) is (a) characterized by being administered orally; and / or (b) The composition according to claim 6, characterized in that it is administered in a dose of 6 to 10 or 10 to 14 mg per day.

10. The composition according to claim 9, characterized in that the compound of formula (I) is administered in a dose of about 8 or about 12 mg per day.

11. A composition for treating cancer in a person in need of cancer treatment, The composition has a general structure of formula (I): 【Chemistry 27】 (In the formula, R1 is alkyl, cycloalkyl, or aryl; R2 is a halo, alkyl, or H; R3, R4, R5, R6, and R7 are each independently H or alkyl; R8 and R9 together form an aromatic ring that is optionally substituted. Compounds having; or comprising pharmaceutically acceptable salts, tautomers, or stereoisomers thereof; The composition is characterized in that it is administered in combination with a nucleic acid encoding an expression repressor, wherein the expression repressor (a) A first targeting portion, wherein the first targeting portion is: (i) Binds to the MYC promoter, or (ii) A first targeting portion that binds to a genomic locus containing at least 16, 17, 18, 19, or 20 nucleotides of any sequence 83, 2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202, and (b) Optionally, the first effector section Includes, A composition characterized in that the expression repressor can reduce the expression of MYC.

12. (a) R1 is alkyl or cycloalkyl; (b) R1 = cyclopropane; (c) R 2 is a halo; (d) R² = Cl; (e) R3, R4, R5, R6, R7 are each H, and / or (f) The composition according to claim 11, wherein the substituted aromatic ring is a substituted phenyl ring.

13. The compound of formula (I) is 【Chemistry 29】 The composition according to claim 11, or comprising a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

14. The compound of formula (I) is (a) characterized by being administered orally; and / or (b) The composition according to claim 11, characterized in that it is administered in a dose of 6 to 10 or 10 to 14 mg per day.

15. The composition according to claim 14, characterized in that the compound of formula (I) is administered in a dose of about 8 or about 12 mg per day.

16. Whether the aforementioned cancer is lung cancer, for example, NSCLC, or The combination according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 15, wherein the cancer is hepatocellular carcinoma (HCC), fibrous lamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, or secondary liver cancer.

17. The first targeting portion binds to a genomic locus containing at least 16, 17, 18, 19, or 20 nucleotides of the sequence of Sequence ID No. 83, and The expression repressor includes the first effector portion, and the first effector portion includes DNA methyltransferase. A combination according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 15.

18. The first targeting portion includes a zinc finger domain and / or The combination according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 15, wherein the first targeting portion includes an amino acid sequence relating to Sequence ID No. 13 or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto, or a sequence in which the positions of difference therefrom are not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

19. The aforementioned first effector section: (i) MQ1 or its functional variants or fragments; (ii) the sequence of sequence number 19 or 87, or the sequence having at least 80, 85, 90, 95, 99, or 100% identity therewith, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position of difference therewith; and / or (iii) A combination according to any one of claims 1 to 5 or a composition according to any one of claims 6 to 15, comprising the sequence of sequence number 129, or a sequence in which the positions that are at least 80, 85, 90, 95, 99, or 100% identical thereto, or differ therefrom, do not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

20. (i) The nucleic acid comprises RNA, for example, mRNA; (ii) The RNA comprises a nucleotide sequence encoding the first targeting portion, wherein the nucleotide sequence encoding the first targeting portion comprises the sequence relating to Sequence ID No. 131 or a sequence in which at least 80, 85, 90, 95, 99, or 100% identical or different positions thereto are not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; (iii) The RNA comprises a nucleotide sequence encoding the first effector portion, wherein the nucleotide sequence encoding the first effector portion comprises the sequence relating to Sequence ID No. 132, or a sequence in which there are not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position of identity or difference thereto with at least 80, 85, 90, 95, 99, or 100%; and / or (iv) The combination according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 15, wherein the RNA includes the nucleotide sequence relating to Sequence ID No. 130, or a sequence in which the number of positions that are at least 80, 85, 90, 95, 99, or 100% identical thereto, or differ therefrom, is not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

21. The nucleic acid comprises RNA, the RNA further encodes a second expression repressor, the second expression repressor is A second targeting region that binds to a second genomic locus, and Second effect pedal section A combination according to any one of claims 1 to 5, or a composition according to any one of claims 6 to 15, comprising the above.

22. (i) The second targeting portion binds to a second genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of Sequence ID No. 77; (ii) The second targeting portion includes a zinc finger domain; (iii) The second targeting portion includes the amino acid sequence relating to Sequence ID No. 7, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto, or has 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 that differs from it; (iv) The second effector portion includes KRAB or a functional variant or fragment thereof; (v) The second effector portion includes the amino acid sequence relating to Sequence ID No. 18, or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto, or has 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 of difference therefrom; (vi) The second expression repressor includes an amino acid sequence relating to SEQ ID NO: 24 or a sequence that is at least 80, 85, 90, 95, 99, or 100% identical thereto, or a sequence in which the number of differing positions does not exceed 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1; and / or (vii) The combination or composition according to claim 21, wherein the RNA comprises the nucleotide sequence relating to SEQ ID NO: 113, 196, or 208.

23. (i) The nucleic acid is formulated into lipid nanoparticles (LNPs), and optionally the nucleic acid is encapsulated within the LNPs; (ii) The nucleic acid encoding the expression repressor is administered by intravenous infusion, for example, over 80 to 120 minutes, for example, every two weeks; (iii) The nucleic acid is administered in a dose of approximately 0.001 mg / kg to 1.5 mg / kg or approximately 0.002 mg / kg to 1.5 mg / kg, for example, every 1, 2, 3, 4, 5, 6, or 7 weeks; (iv) The nucleic acid encoding the expression repressor and the compound having the general structure of formula (I) are administered on different days; and / or (v) The combination according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 15, characterized in that the nucleic acid encoding the expression repressor and the compound having the general structure of formula (I) are administered on the same day.

24. The nucleic acid encoding the expression repressor is administered before the administration of the compound having the general structure of formula (I); and / or The combination or composition according to claim 23, characterized in that the compound having the general structure of formula (I) is administered within 1 to 24 hours after the completion of administration of the nucleic acid encoding the expression repressor.

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