CXCL-MODULATING COMPOSITIONS AND METHODS

Abstract

The present disclosure relates to an expression repressor for reducing expression of a target multigene in a cell. In some embodiments, the target multigene comprises CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, and IL-8. In some embodiments, the expression repressor targets the E1 cRE of the CXCL locus. In some embodiments, the expression repressor targets the E2 cRE of the CXCL locus.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 325,524, filed March 30, 2022, U.S. Provisional Patent Application No. 63 / 379,849, filed October 17, 2022, and U.S. Provisional Patent Application No. 63 / 478,855, filed January 6, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on March 28, 2023, is named O2057-7032WO_SL and is 661,150 bytes in size. [Background technology]

[0003] Misregulation of gene expression is an underlying cause of many diseases (e.g., in mammals, e.g., humans). Many diseases and conditions are associated with multiple related genes. There is a need for new tools, systems, and methods for altering, e.g., reducing, the expression of multiple related genes. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides, inter alia, expression repressors or systems comprising expression repressors that can be used to regulate, e.g., reduce, the expression of one or more target genes, e.g., one or more CXCL genes, within a CXCL locus that comprises a cis-acting regulatory element.

[0005] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a first targeting moiety that binds to a target site comprising a cis-acting regulatory element, e.g., an enhancer (e.g., an enhancer for a CXCL gene); a first effector part; The present invention provides an expression repressor comprising:

[0006] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a first targeting moiety that binds to a target site, the target site being within a cis-acting regulatory element of the CXCL locus; optionally a first effector moiety; and an expression repressor comprising Here, the expression repressor is able to reduce the expression of the CXCL gene.

[0007] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a first targeting moiety that binds to a target site comprising the IL-8 promoter; a first effector part; The present invention provides an expression repressor comprising:

[0008] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a first targeting moiety that binds to a target site, the target site being within an E1 cis-acting regulatory element of the CXCL locus or an E2 cis-acting regulatory element of the CXCL locus; optionally a first effector moiety; and an expression repressor comprising Here, the expression repressor is able to reduce the expression of the CXCL gene.

[0009] In some embodiments, the target site is within the genomic coordinates chr4:74591400-74593000 or chr4:74982639-74983600 (based on the hg19 human genome reference assembly).

[0010] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a first targeting moiety that binds to a target site within genomic coordinates chr4:74591400-74593000 or chr4:74982639-74983600 (based on the hg19 human genome reference assembly); and optionally a first effector moiety; and an expression repressor comprising Here, the expression repressor is able to reduce the expression of the CXCL gene.

[0011] In some embodiments, the target site is selected from the following: a)GRCh37:chr4:74591777-74591797; b)GRCh37:chr4:74591834-74591854; c)GRCh37:chr4:74591896-74591916; d)GRCh37:chr4:74592082-74592102; e)GRCh37:chr4:74592107-74592127; f)GRCh37:chr4:74592156-74592176; g)GRCh37:chr4:74592210-74592230; h)GRCh37:chr4:74592057-74592077; i)GRCh37:chr4:74591977-74591997; j)GRCh37:chr4:74591856-74591876; k)GRCh37:chr4:74591768-74591790; l)GRCh37:chr4:74591844-74591866; m)GRCh37:chr4:74591892-74591914; n)GRCh37:chr4:74592088-74592110; o)GRCh37:chr4:74982748-74982770; p)GRCh37:chr4:74982841-74982863; k)GRCh37:chr4:74982882-74982904; r)GRCh37:chr4:74982960-74982982; s)GRCh37:chr4:74983108-74983130; and t)GRCh37:chr4:74983181-74983203.

[0012] In certain embodiments, the first targeting moiety binds within 500, 300, 200, 100, or 50 nucleotides upstream or downstream of a target site selected from the following: a)GRCh37:chr4:74591777-74591797; b)GRCh37:chr4:74591834-74591854; c)GRCh37:chr4:74591896-74591916; d)GRCh37:chr4:74592082-74592102; e)GRCh37:chr4:74592107-74592127; f)GRCh37:chr4:74592156-74592176; g)GRCh37:chr4:74592210-74592230; h)GRCh37:chr4:74592057-74592077; i)GRCh37:chr4:74591977-74591997; j)GRCh37:chr4:74591856-74591876; k)GRCh37:chr4:74591768-74591790; l)GRCh37:chr4:74591844-74591866; m)GRCh37:chr4:74591892-74591914; n)GRCh37:chr4:74592088-74592110; o)GRCh37:chr4:74982748-74982770; p)GRCh37:chr4:74982841-74982863; q)GRCh37:chr4:74982882-74982904; r)GRCh37:chr4:74982960-74982982; s)GRCh37:chr4:74983108-74983130; and t)GRCh37:chr4:74983181-74983203.

[0013] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a first targeting moiety that binds to a target site comprising at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides of any one of SEQ ID NOs: 163 or 164, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or differing therefrom in no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position; optionally a first effector moiety; and an expression repressor comprising Here, the expression repressor is able to reduce the expression of the CXCL gene.

[0014] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a first targeting moiety that binds to a target site, the target site being within the IL-8 promoter; optionally, a first effector moiety; Here, the expression repressor is able to reduce the expression of IL-8.

[0015] In some embodiments, the target site (e.g., a target site within the IL8 promoter) is within genomic coordinates chr4:74606112-74606462 (hg19). In some embodiments, the target site (e.g., a target site within the IL8 promoter) is within genomic coordinates chr:74606112-74606462 (e.g., chr4:74606112-74606662, chr4:74606112-74606862, chr4:74606112-74607062, chr4:74606112-74607262, chr4:74606112-74607462, chr4:74605912-74606462, chr4:74605712-74606 462, chr4:74605512-74606462, chr4:74605312-74606462, chr4:74605112-74606462, chr4:74605912-74606662, chr4:74605912- 74606862, chr4:74605912-74607062, chr4:74605912-74607262, chr4:74605912-74607462, chr4:74605712-74606662, chr4:74605 712-74606862, chr4:74605712-74607062, chr4:74605712-74607262, chr4:74605712-74607462, chr4:74605512-74606662, chr4: 74605512-74606862, chr4:74605512-74607062, chr4:74605512-74607262, chr4:74605512-74607462, chr4:74605312-74606662, c Located within 1 kb of hr4:74605312-74606862, chr4:74605312-74607062, chr4:74605312-74607262, chr4:74605312-74607462, chr4:74605112-74606662, chr4:74605112-74606862, chr4:74605112-74607062, chr4:74605112-74607262, or chr4:74605112-74607462).In certain embodiments, the target site (e.g., a target site within the IL8 promoter) is located 500 bp upstream from the transcription start site. In certain embodiments, the target site (e.g., a target site within the IL8 promoter) is located at chr4:74605723-74606223. In some embodiments, the target sites (e.g., target sites within the IL8 promoter) are chr4:74605723-74606426, chr4:74605723-74606626, chr4:74605723-74606826, chr4:74605723-74607026, chr4:74605723-74607226, chr4:74605523-74606226, chr4:74605323-74606226, chr4:74605123- 74606226, chr4:74604923-74606226, chr4:74604723-74606226, chr4:74605523-74606426, chr4:74605523-74606626, chr4: 74605523-74606826, chr4:74605523-74607026, chr4:74605523-74607226, chr4:74605323-74606426, chr4:74605323-74606 626, chr4:74605323-74606826, chr4:74605323-74607026, chr4:74605323-74607226, chr4:74605123-74606426, chr4:74605 123-74606626, chr4:74605123-74606826, chr4:74605123-74607026, chr4:74605123-74607226, chr4:74604923-74606426, c Located at hr4:74604923-74606626, chr4:74604923-74606826, chr4:74604923-74607026, chr4:74604923-74607226, chr4:74604723-74606426, chr4:74604723-74606626, chr4:74604723-74606826, chr4:74604723-74607026, or chr4:74604723-74607226.In some embodiments, the target site (e.g., a target site within the IL8 promoter) is located 1000 bp upstream from the transcription start site. In certain embodiments, the target site (e.g., a target site within the IL8 promoter) is located at chr4:74605223-74606223. In some embodiments, the target sites (e.g., target sites within the IL8 promoter) are chr4:74605226-74606426, chr4:74605226-74606626, chr4:74605226-74606826, chr4:74605226-74607026, chr4:74605226-74607226, chr4:74605026-74606226, chr4:74604826-74606226, chr4:74604626- 74606226, chr4:74604426-74606226, chr4:74604226-74606226, chr4:74605026-74606426, chr4:74605026-74606626, chr4: 74605026-74606826, chr4:74605026-74607026, chr4:74605026-74607226, chr4:74604826-74606426, chr4:74604826-74606 626, chr4:74604826-74606826, chr4:74604826-74607026, chr4:74604826-74607226, chr4:74604626-74606426, chr4:74604 626-74606626, chr4:74604626-74606826, chr4:74604626-74607026, chr4:74604626-74607226, chr4:74604426-74606426, c Located at hr4:74604426-74606626, chr4:74604426-74606826, chr4:74604426-74607026, chr4:74604426-74607226, chr4:74604226-74606426, chr4:74604226-74606626, chr4:74604226-74606826, chr4:74604226-74607026, or chr4:74604226-74607226.

[0016] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: with genomic coordinates GRCh37:chr4:74606162-74606184, GRCh37:chr4:74605723-74606223, or GRCh37:chr4:74605223-74606223 (based on the hg19 human genome reference assembly); optionally a first effector moiety; Here, the expression repressor is able to reduce the expression of IL-8.

[0017] In some embodiments, the expression repressor is: i)GRCh37:chr4:74605780-74605800; ii)GRCh37:chr4:74605961-74605981; iii)GRCh37:chr4:74606122-74606142; iv)GRCh37:chr4:74605955-74605975; v)GRCh37:chr4:74605842-74605862; vi)GRCh37:chr4:74606145-74606165; vii)GRCh37:chr4:74606039-74606056; viii)GRCh37:chr4:74606113-74606130; ix)GRCh37:chr4:74606137-74606154; x)GRCh37:chr4:74606150-74606167; xi)GRCh37:chr4:74591882-74591899; xii)GRCh37:chr4:74591923-74591940; xiii) GRCh37:chr4:74591897-74591914; and xiv)GRCh37:chr4:74591873-74591890 The target site is selected from the group consisting of:

[0018] In some embodiments, the expression repressor is: i)GRCh37:chr4:74605780-74605800; ii)GRCh37:chr4:74605961-74605981; iii)GRCh37:chr4:74606122-74606142; iv)GRCh37:chr4:74605955-74605975; v)GRCh37:chr4:74605842-74605862; vi)GRCh37:chr4:74606145-74606165; vii)GRCh37:chr4:74606039-74606056; viii)GRCh37:chr4:74606113-74606130; ix)GRCh37:chr4:74606137-74606154; x)GRCh37:chr4:74606150-74606167; xi)GRCh37:chr4:74591882-74591899; xii)GRCh37:chr4:74591923-74591940; xiii) GRCh37:chr4:74591897-74591914; and xiv)GRCh37:chr4:74591873-74591890 The target site is located within 500, 300, 200, 100, or 50 nucleotides upstream or downstream of a target site selected from the group consisting of:

[0019] In some embodiments, the first effector moiety is an effector described herein, e.g., KRAB, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, EZH2, HDAC8, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., G9A), EHMT2 (i.e., G9A), EHMT3 ... i.e., GLP), SUV39H1, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2 or DNMT3, or a functional variant or fragment of any thereof.

[0020] In certain embodiments, the first effector moiety is linked to the targeting moiety via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker can be 2 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 2 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 2 to 20, 5 to 20, 10 to 20, 15 to 20, 2 to 15, 5 to 15, 10 to 15, 2 to 10, 5 to 10, or 2 to 5 amino acids in length, or 2, 5, 10, 15, 20, 25, or 30 amino acids or more (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length).

[0021] In some embodiments, the first effector moiety is C-terminal to the targeting moiety.

[0022] In certain embodiments, the first effector moiety is N-terminal to the targeting moiety.

[0023] In some embodiments, the first effector moiety is encoded by a nucleotide sequence selected from any of SEQ ID NOs: 10, 14, 16, 18, 66, 68, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or differing therefrom at no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0024] In certain embodiments, the first effector moiety comprises an amino acid sequence according to any of SEQ ID NOs: 11, 12, 13, 15, 17, 19, 67, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto or differing therefrom at no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0025] In some embodiments, the first effector moiety is MQ1 or a functional variant or fragment thereof, for example, wherein the first effector moiety comprises the amino acid sequence of SEQ ID NO: 11 or 12, or a sequence having 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 difference therefrom, and optionally, the first effector moiety is C-terminal to the first targeting moiety.

[0026] In certain embodiments, the first effector moiety is KRAB, or a functional variant or fragment thereof, for example, wherein the first effector moiety comprises the amino acid sequence of SEQ ID NO: 1, or a sequence having 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 difference therefrom, and optionally, the first effector moiety is C-terminal to the first targeting moiety.

[0027] In some embodiments, the first effector moiety is DNMT3a / 3L, or a functional variant or fragment thereof, for example, wherein the first effector moiety comprises the amino acid sequence of SEQ ID NO: 15, or a sequence having 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 difference therefrom, and optionally, the first effector moiety is C-terminal to the first targeting moiety.

[0028] In certain embodiments, the first effector moiety is EZH2, or a functional variant or fragment thereof, for example, wherein the first effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom.

[0029] In some embodiments, the first effector moiety is HDAC8, or a functional variant or fragment thereof, for example, wherein the first effector moiety comprises the amino acid sequence of SEQ ID NO: 19, or a sequence having 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 difference therefrom, and optionally, the first effector moiety is C-terminal to the first targeting moiety.

[0030] In certain embodiments, the first effector moiety is G9A, or a functional variant or fragment thereof, for example, wherein the first effector moiety comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having 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 difference therefrom, and optionally, the first effector moiety is N-terminal to the first targeting moiety.

[0031] In some embodiments, the effector moiety comprises a DNA methyltransferase, for example, MQ1, or a fragment or variant thereof.

[0032] In certain embodiments, the effector moiety comprises a transcriptional repressor, for example, KRAB or a fragment or variant thereof.

[0033] In some embodiments, the target site has a length of 15-20, 20-25, 25-30, or 30-35 nucleotides.

[0034] In some embodiments, the first targeting moiety comprises a zinc finger domain.

[0035] In certain embodiments, a zinc finger domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (and optionally up to 11, 10, 9, 8, 7, 6, or 5 zinc fingers).

[0036] In some embodiments, a 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.

[0037] In some embodiments, the zinc finger domain comprises 3, 7, or 9 zinc fingers. In some embodiments, the zinc finger domain targets a site comprising 21 nucleotides.

[0038] In certain embodiments, the first targeting moiety comprises a CRISPR-Cas domain.

[0039] In certain embodiments, the expression repressors described herein are capable of reducing expression of multiple CXCL genes (e.g., 2, 3, 4, 5, 6, 7, or 8 CXCL genes). In certain embodiments, the expression repressors described herein are capable of reducing expression of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8.

[0040] In certain embodiments, the first effector moiety is a long-lasting effector moiety or a transient effector moiety.

[0041] In some embodiments, the first targeting moiety comprises a zinc finger domain and the first effector moiety comprises a transcriptional repressor, for example, KRAB, or a fragment or variant thereof.

[0042] In some embodiments, the first targeting moiety comprises a zinc finger domain and the first effector moiety comprises an epigenetic-modifying moiety, eg, a DNA methyltransferase, eg, MQ1, or a fragment or variant thereof.

[0043] In some embodiments, the expression repressor comprises the amino acid sequence of any one of SEQ ID NOs: 152-161, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 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 difference therefrom.

[0044] In some embodiments, the expression repressors described herein (i) comprise one or more nuclear localization signal sequences (NLS), or (ii) do not comprise an NLS.

[0045] In some embodiments, the expression repressors described herein comprise a first NLS at the N-terminus, for example, the first NLS has the sequence of SEQ ID NO: 63 or 64.

[0046] In some embodiments, an expression repressor described herein comprises an NLS at the C-terminus, e.g., a second NLS, e.g., having the sequence of SEQ ID NO: 63 or 64.

[0047] In some embodiments, the first and second NLSs have the same sequence. In certain embodiments, the first and second NLSs have different sequences.

[0048] In some embodiments, binding of the expression repressor to the target site increases methylation at the site in the CXCL locus, for example, increasing methylation at the E1 cis-acting regulatory element of the CXCL locus or the E2 cis-acting regulatory element of the CXCL locus.

[0049] In one aspect, the present disclosure provides a method for producing a method of manufacturing a semiconductor device comprising: a) a first expression repressor according to any of the above embodiments; and b) a second expression repressor, for example, a second expression repressor that reduces expression of a CXCL gene.

[0050] In some embodiments, the second expression repressor is: a second target site that binds to a second target site within the CXCL locus; and optionally a second effector moiety.

[0051] In certain embodiments, the second expression repressor binds to the E1 cis-acting regulatory element of the CXCL locus, the E2 cis-acting regulatory element of the CXCL locus, or the IL8 promoter.

[0052] In certain embodiments, the second target site is within the coordinates GRCh37:chr4:74606162-74606184, GRCh37:chr4:74605723-74606223, or GRCh37:chr4:74605223-74606223. In some embodiments, the second target site is within the following coordinates: a) chr4:74606112-74606462, chr4:74606112-74606662, chr4:74606112-74606862, chr4:74606112-74607062, chr4:74606 112-74607262, chr4:74606112-74607462, chr4:74605912-74606462, chr4:74605712-74606462, chr4:74605512-74606462 , chr4:74605312-74606462, chr4:74605112-74606462, chr4:74605912-74606662, chr4:74605912-74606862, chr4:746059 12-74607062, chr4:74605912-74607262, chr4:74605912-74607462, chr4:74605712-74606662, chr4:74605712-74606862, c hr4:74605712-74607062, chr4:74605712-74607262, chr4:74605712-74607462, chr4:74605512-74606662, chr4:74605512 -74606862, chr4:74605512-74607062, chr4:74605512-74607262, chr4:74605512-74607462, chr4:74605312-74606662, chr r4:74605312-74606862, chr4:74605312-74607062, chr4:74605312-74607262, chr4:74605312-74607462, chr4:74605112-74606662, chr4:74605112-74606862, chr4:74605112-74607062, chr4:74605112-74607262, or chr4:74605112-74607462; b) chr4:74605723-74606223, chr4:74605723-74606426, chr4:74605723-74606626, chr4:74605723-74606826, chr4:74605 723-74607026, chr4:74605723-74607226, chr4:74605523-74606226, chr4:74605323-74606226, chr4:74605123-74606226, chr4:74604923-74606226, chr4:74604723-74606226, chr4:74605523-74606426, chr4:74605523-74606626, chr4:7460552 3-74606826, chr4:74605523-74607026, chr4:74605523-74607226, chr4:74605323-74606426, chr4:74605323-74606626, chr r4:74605323-74606826, chr4:74605323-74607026, chr4:74605323-74607226, chr4:74605123-74606426, chr4:74605123- 74606626, chr4:74605123-74606826, chr4:74605123-74607026, chr4:74605123-74607226, chr4:74604923-74606426, chr4 :74604923-74606626, chr4:74604923-74606826, chr4:74604923-74607026, chr4:74604923-74607226, chr4:74604723-74606426, chr4:74604723-74606626, chr4:74604723-74606826, chr4:74604723-74607026, or chr4:74604723-74607226; or c) chr4:74605223-74606223, chr4:74605226-74606426, chr4:74605226-74606626, chr4:74605226-74606826, chr4:74605 226-74607026, chr4:74605226-74607226, chr4:74605026-74606226, chr4:74604826-74606226, chr4:74604626-74606226 , chr4:74604426-74606226, chr4:74604226-74606226, chr4:74605026-74606426, chr4:74605026-74606626, chr4:746050 26-74606826, chr4:74605026-74607026, chr4:74605026-74607226, chr4:74604826-74606426, chr4:74604826-74606626, chr4:74604826-74606826, chr4:74604826-74607026, chr4:74604826-74607226, chr4:74604626-74606426, chr4:7460462 6-74606626, chr4:74604626-74606826, chr4:74604626-74607026, chr4:74604626-74607226, chr4:74604426-74606426, c hr4:74604426-74606626, chr4:74604426-74606826, chr4:74604426-74607026, chr4:74604426-74607226, chr4:74604226-74606426, chr4:74604226-74606626, chr4:74604226-74606826, chr4:74604226-74607026, or chr4:74604226-74607226 It's inside.

[0053] In certain embodiments, the second target site is within GRCh37:chr4:74606162-74606184.

[0054] In some embodiments, the second target moiety is: i)GRCh37:chr4:74605780-74605800; ii)GRCh37:chr4:74605961-74605981; iii)GRCh37:chr4:74606122-74606142; iv)GRCh37:chr4:74605955-74605975; v)GRCh37:chr4:74605842-74605862; vi)GRCh37:chr4:74606145-74606165; vii)GRCh37:chr4:74606039-74606056; viii)GRCh37:chr4:74606113-74606130; ix)GRCh37:chr4:74606137-74606154; x)GRCh37:chr4:74606150-74606167; xi)GRCh37:chr4:74591882-74591899; xii)GRCh37:chr4:74591923-74591940; xiii) GRCh37:chr4:74591897-74591914; and xiv)GRCh37:chr4:74591873-74591890 is selected from.

[0055] In certain embodiments, the second target moiety is: i)GRCh37:chr4:74605780-74605800; ii)GRCh37:chr4:74605961-74605981; iii)GRCh37:chr4:74606122-74606142; iv)GRCh37:chr4:74605955-74605975; v)GRCh37:chr4:74605842-74605862; vi)GRCh37:chr4:74606145-74606165; vii)GRCh37:chr4:74606039-74606056; viii)GRCh37:chr4:74606113-74606130; ix)GRCh37:chr4:74606137-74606154; x)GRCh37:chr4:74606150-74606167; xi)GRCh37:chr4:74591882-74591899; xii)GRCh37:chr4:74591923-74591940; xiii) GRCh37:chr4:74591897-74591914; and xiv)GRCh37:chr4:74591873-74591890 The target site may be selected from the group consisting of:

[0056] In certain embodiments, the second targeting moiety is a clustered regularly interspaced short palindromic repeats (CRISPR) Cas domain.

[0057] In one aspect, the disclosure provides a nucleic acid encoding an expression repressor described herein.

[0058] In one aspect, the present disclosure provides a system comprising: any of the first expression repressors described herein; and a second expression repressor, e.g., a second expression repressor that reduces expression of a CXCL gene, e.g., an expression repressor of any of the aspects of the above embodiments. Nucleic acids encoding the

[0059] In one aspect, the present disclosure provides a method for manufacturing a method for manufacturing a semiconductor device, comprising: a) a first nucleic acid encoding a first expression repressor described herein; b) a second nucleic acid encoding a second expression repressor, e.g., a second expression repressor that reduces expression of a CXCL gene, e.g., an expression repressor of the system of any of the aspects of the above embodiments; The present invention provides a nucleic acid system comprising:

[0060] In some embodiments, the nucleic acid or nucleic acid system comprises a region encoding a first targeting moiety, wherein the region encoding the first targeting moiety comprises the nucleotide sequence of any one of SEQ ID NOs: 122-131, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0061] In some embodiments, the nucleic acid or nucleic acid system comprises a region encoding a first targeting moiety, wherein the region encoding the first targeting moiety comprises the nucleotide sequence of any one of SEQ ID NOs: 194-199, 248-253, or 276-291, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0062] In some embodiments, the nucleic acid or nucleic acid system comprises a region encoding a first effector moiety, wherein the region encoding the first effector moiety comprises the nucleotide sequence of any one of SEQ ID NOs: 10, 14, 16, 18, 66, 68, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0063] In some embodiments, the nucleic acid or nucleic acid system further comprises a region encoding an NLS. In certain embodiments, the region encoding the NLS comprises the nucleotide sequence of SEQ ID NO: 63 or 64, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0064] In certain embodiments, the nucleic acid or nucleic acid system comprises DNA or RNA (eg, mRNA).

[0065] In one aspect, the present disclosure provides a vector comprising the nucleic acid or nucleic acid system of any one of the preceding aspects or embodiments.

[0066] In one aspect, the disclosure provides a pharmaceutical composition comprising the expression repressor, nucleic acid, or nucleic acid system of any one of the preceding aspects or embodiments.

[0067] In some embodiments, the pharmaceutical composition comprises an LNP, for example, where the nucleic acid or nucleic acid system is formulated as an LNP.

[0068] In one aspect, the disclosure provides a human cell comprising: an expression repressor described herein, a nucleic acid or nucleic acid system described herein, or a vector described herein.

[0069] In one aspect, the present disclosure provides a human cell having reduced expression of a CXCL gene, wherein the cell has been produced by a method comprising contacting the cell with an expression repressor of any of the preceding aspects or embodiments, a nucleic acid or nucleic acid system of any of the preceding aspects or embodiments, or a vector of any of the preceding aspects or embodiments.

[0070] In some embodiments, the human cell has reduced expression of the first and second CXCL genes. In certain embodiments, the human cell has reduced expression of the third CXCL gene. In certain embodiments, the human cell has reduced expression of the fourth CXCL gene. In some embodiments, the human cell has reduced expression of the fifth CXCL gene. In certain embodiments, the human cell has reduced expression of the sixth CXCL gene. In some embodiments, the human cell has reduced expression of the seventh CXCL gene. In some embodiments, the human cell has reduced expression of the eighth CXCL gene. In some embodiments, the human cell has reduced expression of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8. In some embodiments, the human cell has reduced expression of one or more of CXCL1, CXCL2, CXCL3, and IL-8.

[0071] In one aspect, the present disclosure provides a method of reducing expression of one or more CXCL genes in a cell, comprising contacting the cell with an expression repressor, system, nucleic acid or nucleic acid system, or vector of any one of the preceding aspects or embodiments.

[0072] In one aspect, the disclosure provides a method of reducing expression of IL-8 in a cell, comprising contacting the cell with an expression repressor, system, nucleic acid, or nucleic acid system described herein.

[0073] In one aspect, the disclosure provides a method of reducing expression of one or more CXCL genes in a cell, comprising contacting the cell with an expression repressor or a nucleic acid comprising a sequence encoding an expression repressor, wherein the expression repressor is a first targeting moiety that binds to a target site, the target site being within an E1 cis-acting regulatory element of the CXCL locus or an E2 cis-acting regulatory element of the CXCL locus; optionally a first effector moiety; and thereby reducing the expression of the CXCL gene.

[0074] In some embodiments, the target site is within the genomic coordinates chr4:74591400-74593000 or chr4:74982639-74983600 (based on the hg19 human genome reference assembly).

[0075] In certain embodiments, the expression of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8 is reduced.

[0076] In some embodiments, expression is reduced for at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or for at least 1, 2, 3, 4, or 5 weeks.

[0077] In some embodiments, the cells are from a subject with an inflammatory disease, e.g., an immune-mediated inflammatory disease. In certain embodiments, the inflammatory disease is an autoimmune disease, e.g., rheumatoid arthritis.

[0078] In some embodiments, the inflammatory disease is associated with a pathogenic infection, e.g., a viral infection, e.g., a SARS-CoV2 infection.

[0079] In certain embodiments, the inflammatory disease is associated with a superinfection, e.g., an infection caused by two or more pathogens, e.g., a virus and a bacterium (e.g., SARS-CoV2 and Streptococcus pneumoniae), e.g., a virus and a fungus (e.g., SARS-CoV2 and mucormycosis).

[0080] In some embodiments, the cell is a cell of a subject having: rheumatoid arthritis, inflammation, arthritis, gout, asthma, neutrophilic asthma, neutrophilic dermatosis, paw edema, acute respiratory distress syndrome (ARDS), COVID-19, psoriasis, inflammatory bowel disease, infection (e.g., by a pathogen, e.g., bacterial, viral, or fungal), traumatic injury (e.g., an abrasion or foreign body), effects of radiation or chemical injury, osteoarthritis, osteoarthritic joint pain, arthralgia, inflammatory pain, acute pain, chronic pain, cystitis, bronchitis, dermatitis, cardiovascular disease, neurodegenerative disease, liver disease, lung disease, kidney disease, pain, swelling, stiffness, tenderness, redness, heat, or an elevation in a biomarker associated with a disease state (e.g., a cytokine, chemokine, growth factor, immune receptor, or inflammatory marker).

[0081] In some embodiments, the cells are from a subject with rheumatoid arthritis, psoriasis, or inflammatory bowel disease.

[0082] In certain embodiments, the cells are from a subject with rheumatoid arthritis, gout, neutrophilic asthma, neutrophilic dermatosis, acute respiratory distress syndrome (ARDS), or COVID-19.

[0083] In some embodiments, the cells are cells of a subject with cancer.

[0084] In certain embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), breast cancer, hepatocellular carcinoma (HCC), prostate cancer, colon cancer, skin cancer, cervical cancer, ovarian cancer, endometrioid carcinoma, endometrial carcinoma, mature B-cell lymphoma, bladder cancer, esophagogastric carcinoma, esophageal adenocarcinoma, bone cancer, melanoma, hepatobiliary carcinoma, thyroid carcinoma, mature B-cell neoplasm, glioma, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), sarcoma, or gastric adenocarcinoma.

[0085] In some embodiments, the cells are in a control.

[0086] In certain embodiments, the cells are ex vivo.

[0087] In some embodiments, the cell is a mammalian cell, for example a human cell.

[0088] In certain embodiments, the cell is a somatic cell.

[0089] In some embodiments, the cells are primary cells.

[0090] In some embodiments, the contacting step occurs ex vivo.

[0091] In some embodiments, the method further comprises removing cells (eg, mammalian cells) from the subject prior to the contacting step.

[0092] In some embodiments, the method further comprises, after the contacting step, administering the cell (eg, a mammalian cell) to a subject.

[0093] In certain embodiments, the contacting step comprises administering to the subject a composition comprising the expression repressor.

[0094] In some embodiments, the expression repressor is administered as a monotherapy.

[0095] In certain embodiments, the expression repressor is administered in combination with a second therapeutic agent.

[0096] In one aspect, the disclosure provides a reaction mixture comprising a cell (e.g., a human cell, e.g., a primary human cell) and an expression repressor or system of any of the preceding aspects or embodiments.

[0097] In one aspect, the disclosure provides a method of treating a subject having an inflammatory disorder, comprising: administering to the subject an expression repressor, system, nucleic acid, nucleic acid system, or reaction mixture of any of the preceding aspects or embodiments in an amount sufficient to treat the disorder (e.g., an inflammatory disorder); thereby treating the above disorders (e.g., inflammatory disorders). Includes:

[0098] In some embodiments, the inflammatory disorder is rheumatoid arthritis, psoriasis, or inflammatory bowel disease.

[0099] In some embodiments, the inflammatory disorder is rheumatoid arthritis, gout, neutrophilic asthma, neutrophilic dermatosis, acute respiratory distress syndrome (ARDS), alcoholic hepatitis, chronic obstructive pulmonary disease (COPD), or COVID-19.

[0100] In certain embodiments, the inflammatory disorder is an autoimmune disorder, for example, rheumatoid arthritis.

[0101] In some embodiments, the inflammatory disease is associated with a pathogenic infection, e.g., a viral infection, e.g., a SARS-CoV2 infection.

[0102] In certain embodiments, the inflammatory disease is associated with infection caused by two or more pathogens, e.g., a virus and a bacterium (e.g., SARS-CoV2 and Streptococcus pneumoni), e.g., a virus and a fungus (e.g., SARS-CoV2 and mucormycosis).

[0103] In one aspect, the present disclosure provides a method of treating a subject having cancer, comprising: administering to the subject an expression repressor, system, nucleic acid, nucleic acid system, or reaction mixture of any one of claims 1 to 102 in an amount sufficient to treat cancer; Thereby, the cancer is treated.

[0104] In certain embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), breast cancer, hepatocellular carcinoma (HCC), prostate cancer, colon cancer, skin cancer, cervical cancer, ovarian cancer, endometrioid carcinoma, endometrial carcinoma, mature B-cell lymphoma, bladder cancer, esophagogastric carcinoma, esophageal adenocarcinoma, bone cancer, melanoma, hepatobiliary carcinoma, thyroid carcinoma, mature B-cell neoplasm, glioma, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), sarcoma, or gastric adenocarcinoma.

[0105] In some embodiments, the subject has an E1 cis-acting regulatory element comprising the sequence of SEQ ID NO: 162, or a sequence containing no more than 8, 7, 6, 5, 4, 3, 2, or 1 alteration thereto.

[0106] In certain embodiments, the subject has an E2 cis-acting regulatory element comprising the sequence of SEQ ID NO: 163, or a sequence containing no more than 8, 7, 6, 5, 4, 3, 2, or 1 alteration thereto.

[0107] In some aspects, the present disclosure is directed to nucleic acids encoding a first expression repressor, a second expression repressor, both, or components thereof (e.g., gRNA, mRNA). In some embodiments, the nucleic acid encoding the expression repressor system is a polycistronic sequence. In some embodiments, the polycistronic sequence is a bicistronic sequence.

[0108] In some aspects, the present disclosure provides an expression repressor, wherein the expression repressor comprises a targeting moiety that targets enhancers operably linked to a plurality of genes. In some aspects, the present disclosure provides a method of reducing expression of a plurality of genes, comprising contacting a cell comprising the plurality of genes with an expression repressor, wherein the expression repressor comprises a targeting moiety that targets enhancers operably linked to the plurality of genes. In some embodiments, the plurality of genes comprises CXCL genes. In some embodiments, the expression repressor targets the E1 cRE of the CXCL locus.

[0109] In one aspect, an expression repressor or a system comprising an expression repressor may be used in combination with a site-specific disrupting agent described herein. For example, an expression repressor that targets a cis-acting regulatory element of the CXCL locus may be used in combination with a site-specific disrupting agent that targets an anchor sequence of the CXCL locus. In some embodiments, the site-specific disrupting agent is the site-specific disrupting agent of any one of embodiments B1-B232. In some embodiments, the site-specific disrupting agent is a site-specific disrupting agent described herein. In some embodiments, the site-specific disrupting agent is one described in International Application PCT / US2021 / 052720, which is incorporated herein by reference in its entirety.

[0110] In one aspect, the site-specific disrupting agent comprises a targeting moiety that specifically binds to a first anchor sequence of ASMC or a site adjacent to the first anchor sequence. In some embodiments, binding of the site-specific disrupting agent occurs in an amount sufficient to modulate, e.g., reduce, the expression of multiple target genes, e.g., a first gene and a second gene. In some embodiments, the site-specific disrupting agent further comprises an effector moiety. Generally, modulation of the expression of multiple target genes by a site-specific disrupting agent comprises binding of the site-specific disrupting agent to the first anchor sequence or a site adjacent thereto. In some embodiments, binding of the site-specific disrupting agent to the first anchor sequence disrupts binding of a nucleation polypeptide, e.g., CTCF, to the first anchor sequence, e.g., thereby disrupting the formation and / or maintenance of ASMC, e.g., thereby modulating, e.g., reducing, the expression of multiple genes. In some embodiments, binding of a site-specific disrupting agent to the first anchor sequence or a site adjacent thereto localizes the functionality of the effector moiety to the first anchor sequence and / or ASMC, e.g., thereby disrupting the formation and / or maintenance of ASMC, e.g., thereby modulating, e.g., reducing, the expression of multiple genes. In some embodiments, binding of a site-specific disrupting agent to the first anchor sequence or a site adjacent thereto localizes the functionality of the effector moiety to the first anchor sequence and / or ASMC, e.g., thereby modulating, e.g., reducing, the expression of multiple genes. Without wishing to be bound by theory, it is believed that in some embodiments, targeting multiple genes within the same ASMC may more efficiently modulate, e.g., reduce, the expression of multiple genes and / or more efficiently achieve a therapeutic effect related to the functionality of multiple genes. For example, in some embodiments, the targeted multiple genes may all be pro-inflammatory genes; as taught herein, targeting multiple pro-inflammatory genes for modulation, e.g., reduction, of expression may reduce inflammation more efficiently than targeting individual genes.Targeting multiple genes contained within the same genomic complex, e.g., ASMC (e.g., by targeting ASMC or anchor sequences of ASMC) can have additive or synergistic effects (e.g., in terms of regulating expression or stability / duration) that are greater than the effect of targeting multiple individual genes.

[0111] In some embodiments, the methods described herein include reducing expression of a first gene and a second gene in a cell. In some embodiments, the method includes contacting the cell with a site-specific disrupting agent comprising a targeting moiety that specifically binds to a first anchor sequence or a site proximal to the first anchor sequence, in an amount sufficient to reduce expression of the first and second genes, wherein the first and second genes are within an anchor sequence-mediated junction comprising the first anchor sequence and the second anchor sequence. In some embodiments, the first and second genes are pro-inflammatory genes. In some embodiments, the first and second genes are CXCL genes.

[0112] In some embodiments, the systems described herein include, and the methods described herein include, the use of a DNA-binding moiety, e.g., a targeting moiety that specifically binds to or near a first anchor sequence in a cell. In some embodiments, the first anchor sequence is part of an anchor sequence-mediated junction that further includes a second anchor sequence, a first gene, and a second gene. In some embodiments, the first gene and the second gene are CXCL genes.

[0113] In some embodiments, the systems described herein include, or the methods described herein include the use of, a site-specific disrupting agent comprising a targeting moiety that specifically binds to or in proximity to a first anchor sequence in a cell, wherein the first anchor sequence is part of an anchor sequence-mediated junction further comprising a second anchor sequence, a first gene, and a second gene, and the first gene and the second gene are CXCL genes.

[0114] In some embodiments, the methods described herein include reducing expression of a first gene and a second gene in a cell, the method comprising: contacting the cell with a site-specific disrupting agent comprising a targeting moiety that specifically binds to a first anchor sequence or a site proximal to the first anchor sequence, in an amount sufficient to reduce expression of the first and second genes, wherein the first and second genes are within an anchor sequence-mediated junction comprising the first anchor sequence and the second anchor sequence, and wherein the first and second genes are CXCL genes; thereby reducing expression of the first and second genes.

[0115] In another aspect, the present disclosure is directed to a reaction mixture comprising a cell (e.g., a human cell, e.g., a primary human cell), a system as described herein (e.g., a system comprising an expression repressor described herein, and optionally further comprising a site-specific disrupting agent described herein).

[0116] In another aspect, the present disclosure is directed to a method of treating a subject having an inflammatory disorder, the method comprising administering to the subject a system described herein (e.g., a system comprising an expression repressor described herein, and optionally further comprising a site-specific disrupting agent described herein) in an amount sufficient to treat the inflammatory disorder.

[0117] In another aspect, the present disclosure is directed to a method of treating inflammation, e.g., local inflammation, in a subject having an infection, e.g., a viral infection, e.g., COVID-19, comprising administering to the subject a system described herein (e.g., a system comprising an expression repressor described herein, and optionally further comprising a site-specific disrupting agent described herein) in an amount sufficient to treat the inflammation.

[0118] In another aspect, the present disclosure is directed to a human cell having reduced expression of a first gene and a second gene, wherein the first gene and the second gene are pro-inflammatory genes, and the cell comprises a disrupted (e.g., completely disrupted) anchor sequence-mediated junction comprising the first and second genes. In some embodiments, the human cell has previously been contacted with a system described herein (e.g., a system comprising an expression repressor described herein and, optionally, further comprising a site-specific disrupting agent described herein). In some embodiments, the human cell no longer comprises a system described herein.

[0119] In some embodiments, the human cells described herein comprise a sequence encoding the genomic coordinates chr4:74595464-74595486, chr4:74595457-74595479, chr4:74595460-74595482, chr4:74595472-74595494, chr4:75000088-75000110, chr4:75000091-75000113, chr4:75000085-75000107, chr4:75000157-75000 179, chr4:75000156-75000178, chr4:74595215-74595237, chr4:74595370-74595392, chr4:74595560-74595582, chr4:745 95642-74595664, chr4:74595787-74595809, chr4:74528428-74528450, chr4:74528567-74528589, chr4:74528609-7452863 1, chr4:74789132-74789154, chr4:74789250-74789272, chr4:74789312-74789334, chr4:74964853-74964875, chr4:74964 906-74964928, chr4:74965538-74965560, chr4:74965737-74965759, chr4:75000031-75000053, chr4:75000115-75000137, chr4:75000231-75000253, chr4:74975146-74975168, chr4:74975369-74975391, chr4:74976318-74976340, chr4:74570348-74570370, chr4:74570503-74570525, or chr4:74570526-74570548, or comprising a mutation within 5, 10, 15, 20, 30, 40, or 50 nucleotides of said region.

[0120] Numbered Embodiment B B1. A method for reducing expression of a first gene and a second gene in a cell, comprising: contacting the cell with a site-specific disrupting agent comprising a targeting moiety that specifically binds to the first anchor sequence or a site proximal to the first anchor sequence, in an amount sufficient to reduce expression of the first and second genes; the first and second genes are within an anchor sequence-mediated junction comprising a first anchor sequence and a second anchor sequence; Optionally, the first gene and the second gene are pro-inflammatory genes; thereby reducing expression of the first and second genes.

[0121] B2. DNA binding, e.g., comprising a targeting moiety that binds specifically to or in close proximity to a first anchor sequence within a cell; the first anchor sequence is part of an anchor sequence-mediated junction further comprising a second anchor sequence, a first gene, and a second gene; Optionally, the site-specific interfering agent, wherein the first gene and said second gene are pro-inflammatory genes.

[0122] B3. A site-specific blocking agent according to embodiment B2, wherein the first or second anchor sequence is located between IL-8 and RASSF6; between the IL-8 enhancer and RASSF6; between CXCL1 and CXCL4; between CXCL2 and EPGN; or between the E2 enhancer and EPGN.

[0123] B4. The site-specific blocking agent of embodiment B2 or B3, wherein the site-specific blocking agent further comprises an effector moiety.

[0124] B5. The site-specific disrupting agent of any of embodiments B2-B4, wherein the targeting moiety comprises a TAL effector molecule, a CRISPR / Cas molecule (e.g., a catalytically inactive CRISPR / Cas protein), a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0125] B6. The effector moiety is an effector described herein, e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, EZH2, HDAC8, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, HDA The site-specific disrupting agent of any of embodiments B2-B5, comprising C1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2 or DNMT3, or a functional variant or fragment of any of them.

[0126] B7. The site-specific blocking agent of any of embodiments B2-6, wherein the effector moiety is linked to the targeting moiety via a linker.

[0127] B8. The site-specific blocking agent of any of embodiments B2-B7, wherein the effector moiety is C-terminal to the targeting moiety.

[0128] B9. The site-specific blocking agent of any of embodiments B2-B7, wherein the effector moiety is N-terminal to the targeting moiety.

[0129] B10. The site-specific blocking agent of any of embodiments B2-B9, wherein the effector moiety is encoded by a nucleotide sequence selected from any of SEQ ID NOs: 10, 14, 16, 18, 66, 68, or a sequence having at least 80, 85, 90, 95, 99, or 100% identity thereto, or differing therefrom at no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 position.

[0130] B11. The site-specific blocking agent of any of embodiments B2-B10, wherein the effector moiety comprises an amino acid sequence according to any of SEQ ID NOs: 11, 12, 13, 15, 17, 19, 67, or a sequence having 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 difference therefrom.

[0131] B12. The site-specific blocking agent of any of embodiments B2-B11, wherein the effector moiety is MQ1, or a functional variant or fragment thereof, e.g., the effector moiety comprises the amino acid sequence of SEQ ID NO: 11 or 12, or a sequence having 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 difference therefrom, and optionally, the effector moiety is C-terminal to the targeting moiety.

[0132] B13. The site-specific blocking agent of any of embodiments B2-B11, wherein the effector moiety is KRAB, or a functional variant or fragment thereof, e.g., the effector moiety comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 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 difference thereto, and optionally, the effector moiety is C-terminal to the targeting moiety.

[0133] B14. The site-specific disrupting agent of any of embodiments B2-B11, wherein the effector moiety is DNMT3a / 3L, or a functional variant or fragment thereof, e.g., the effector moiety comprises the amino acid sequence of SEQ ID NO: 15, or a sequence having 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 difference therefrom, and optionally, the effector moiety is C-terminal to the targeting moiety.

[0134] B15. The site-specific blocking agent of any of embodiments B2-B11, wherein the effector moiety is EZH2, or a functional variant or fragment thereof, e.g., the effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom.

[0135] B16. The site-specific blocking agent of any of embodiments B2-B11, wherein the effector moiety is HDAC8, or a functional variant or fragment thereof, e.g., the effector moiety comprises the amino acid sequence of SEQ ID NO: 19, or a sequence having 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 difference therefrom, and optionally, the effector moiety is C-terminal to the targeting moiety.

[0136] B17. The site-specific blocking agent of any one of embodiments B2-B11, wherein the effector moiety is G9A, or a functional variant or fragment thereof, e.g., the effector moiety comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having 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 difference therefrom, and optionally, the effector moiety is N-terminal to the targeting moiety.

[0137] B18. The site-specific blocking agent of any of embodiments B2-B17, further comprising a second effector moiety.

[0138] B19. The site-specific blocking agent of embodiment B18, wherein the targeting moiety is located between the first effector moiety and the second effector moiety.

[0139] B20. The site-specific blocking agent of any of embodiments B2-B19, wherein the effector moiety comprises a polymer, eg, an oligonucleotide; eg, a gRNA.

[0140] B21. The site-specific blocking agent of embodiment B20, wherein the oligonucleotide has a sequence comprising the complement of the anchor sequence or the complement to a sequence adjacent to the anchor sequence.

[0141] B22. The site-specific disrupting agent of any of embodiments B2-B21, wherein the targeting moiety further comprises a gRNA, e.g., a gRNA that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 20-62, e.g., the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 20-62.

[0142] B23. The site-specific disrupting agent of any of embodiments B2-22, wherein the targeting domain comprises a CRISPR / Cas molecule, e.g., a catalytically inactive CRISPR / Cas protein, and a gRNA, e.g., a gRNA that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 20-62, e.g., wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 20-62, and the effector moiety comprises an effector selected from DNMT3a / 3l, MQ1, KRAB, G9A, HDAC8, or EZH2.

[0143] B24. The site-specific disrupting agent of embodiment B23, wherein the targeting domain comprises a CRISPR / Cas molecule, e.g., a catalytically inactive CRISPR / Cas protein, and a gRNA, e.g., a gRNA that binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 20-62, e.g., wherein the gRNA comprises a sequence comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 20-62; the first effector moiety comprises an effector selected from DNMT3a / 3l, MQ1, KRAB, G9A, HDAC8, or EZH2; and the second effector moiety comprises an effector selected from DNMT3a / 3l, MQ1, KRAB, G9A, HDAC8, or EZH2.

[0144] B25. The site-specific disrupting agent of any of embodiments B2-B24, wherein the targeting domain binds to a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of any of SEQ ID NOs: 20-62.

[0145] B26. The site-specific disrupting agent of any of embodiments B2-B25, wherein the targeting domain binds to a genomic locus within 50 nucleotides (eg, upstream or downstream) of any of SEQ ID NOs: 20-62.

[0146] B27. The site-specific disrupting agent of any of embodiments B2-B26, wherein the targeting domain binds to a genomic locus within 100 nucleotides (eg, upstream or downstream) of any of SEQ ID NOs: 20-62.

[0147] B28. The site-specific disrupting agent of any of embodiments B2-B27, wherein the targeting domain binds to a genomic locus within 200 nucleotides (eg, upstream or downstream) of any of SEQ ID NOs: 20-62.

[0148] B29. The site-specific disrupting agent of any one of embodiments B2-B28, wherein the targeting domain binds to a genomic locus within 300 nucleotides (eg, upstream or downstream) of any of SEQ ID NOs: 20-62.

[0149] B30. A site-specific disrupting agent according to any of embodiments B2 to B29, which (i) comprises one or more nuclear localization signal sequences (NLS), or (ii) does not comprise an NLS, optionally wherein the NLS comprises the amino acid sequence of SEQ ID NO: 63 and / or 64.

[0150] B31. The site-specific disrupting agent of any of embodiments B18-B30, wherein the first and / or second effector moiety comprises a DNA methyltransferase, a histone methyltransferase, a histone deacetylase, a histone demethylase, or a recruiter of a histone-modifying complex.

[0151] B32. The site-specific blocking agent of any of embodiments B2-B31, wherein ASMC comprises two loops.

[0152] B33. The site-specific disrupting agent of any of embodiments B2-B32 or the method of embodiment B1, wherein the first gene is within the first loop of ASMC and the second gene is within the second loop of ASMC.

[0153] B34. The site-specific blocking agent or method of embodiment B33, wherein the first anchor sequence is located between the first loop and said second loop.

[0154] B35. A nucleic acid encoding a site-specific blocking agent according to any one of embodiments B2 to B34.

[0155] B36. The site-specific disrupting agent of embodiment B1 or any of embodiments B2-B36, wherein the anchor sequence-mediated junction further comprises a third gene, and optionally, the method results in reduced expression of the third gene.

[0156] B37. The method or site-specific disrupting agent of embodiment B36, wherein the anchor sequence-mediated junction further comprises a fourth gene, and optionally, said method results in reduced expression of the fourth gene.

[0157] B38. The method or site-specific disrupting agent of embodiment B37, wherein the anchor sequence-mediated junction further comprises a fifth gene, and optionally, the method results in reduced expression of the fifth gene.

[0158] B39. The method or site-specific disrupting agent of embodiment B38, wherein the anchor sequence-mediated junction further comprises a sixth gene, and optionally, the method results in reduced expression of the sixth gene.

[0159] B40. The method or site-specific disrupting agent of embodiment B39, wherein the anchor sequence-mediated junction further comprises a seventh gene, and optionally, said method results in reduced expression of the seventh gene.

[0160] B41. The method or site-specific disrupting agent of embodiment B40, wherein the anchor sequence-mediated junction further comprises an eighth gene, and optionally, said method results in reduced expression of the eighth gene.

[0161] B42. A human cell having reduced expression of a first gene and a second gene, the first gene and the second gene are pro-inflammatory genes; A human cell, wherein the cell comprises a disrupted (eg, completely disrupted) anchor sequence-mediated junction comprising the first and second genes.

[0162] B43. The human cell of embodiment B42, wherein CTCF binding to anchor sequences constituted by anchor sequence-mediated junctions is reduced, eg, reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0163] B44. The human cell of any of embodiments B42 or B43, wherein the human cell has reduced expression of a third pro-inflammatory gene.

[0164] B45. The human cell of embodiment B44, wherein the human cell has reduced expression of a fourth pro-inflammatory gene.

[0165] B46. The human cell of embodiment B45, wherein the human cell has reduced expression of a fifth pro-inflammatory gene.

[0166] B47. The human cell of embodiment B46, wherein the human cell has reduced expression of a sixth pro-inflammatory gene.

[0167] B48. The human cell of embodiment B47, wherein the human cell has reduced expression of the seventh pro-inflammatory gene.

[0168] B49. The human cell of embodiment B48, wherein the human cell has reduced expression of an eighth pro-inflammatory gene.

[0169] B50. Human cells were cloned into chr4:74595464-74595486, chr4:74595457-74595479, chr4:74595460-74595482, chr4:74595472-74595494, chr4:75000088-75000110, chr4:75000091-75000113, chr4:75000085-75000107, chr4:75000157-75000179, chr4:75000156-7500 00178, chr4:74595215-74595237, chr4:74595370-74595392, chr4:74595560-74595582, chr4:74595642-74595664, chr4:74 595787-74595809, chr4:74528428-74528450, chr4:74528567-74528589, chr4:74528609-74528631, chr4:74789132-7478915 4, chr4:74789250-74789272, chr4:74789312-74789334, chr4:74964853-74964875, chr4:74964906-74964928, chr4:749655 38-74965560, chr4:74965737-74965759, chr4:75000031-75000053, chr4:75000115-75000137, chr4:75000231-75000253, chr The human cell of any of embodiments B42 to B49, comprising a mutation in chr4:74975146-74975168, chr4:74975369-74975391, chr4:74976318-74976340, chr4:74570348-74570370, chr4:74570503-74570525, or chr4:74570526-74570548, or within 5, 10, 15, 20, 30, 40, or 50 nucleotides of the above regions.

[0170] B51.chr4:74595464-74595486, chr4:74595457-74595479, chr4:74595460-74595482, chr4:74595472-74595494, chr4:7 5000088-75000110, chr4:75000091-75000113, chr4:75000085-75000107, chr4:75000157-75000179, chr4:75000156-75 000178, chr4:74595215-74595237, chr4:74595370-74595392, chr4:74595560-74595582, chr4:74595642-74595664, chr 4:74595787-74595809, chr4:74528428-74528450, chr4:74528567-74528589, chr4:74528609-74528631, chr4:74789132- 74789154, chr4:74789250-74789272, chr4:74789312-74789334, chr4:74964853-74964875, chr4:74964906-74964928, c hr4:74965538-74965560, chr4:74965737-74965759, chr4:75000031-75000053, chr4:75000115-75000137, chr4:7500023 1-75000253, chr4:74975146-74975168, chr4:74975369-74975391, chr4:74976318-74976340, chr4:74570348-74570370, chr4:74570503-74570525, or chr4:74570526-74570548, or a mutation within 5, 10, 15, 20, 30, 40, or 50 nucleotides of the above regions.

[0171] B52. The human cell of any of embodiments B27 or B28, wherein the mutation comprises a deletion, substitution, or insertion (e.g., of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides).

[0172] B53. The human cell of any of embodiments B50-B52, wherein CTCF binding to the mutation is reduced, e.g., reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 100%, compared to a human cell with undisturbed ASMC.

[0173] B54. The human cell of any one of embodiments 42-53, wherein expression of the first and second genes is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a human cell having undisturbed ASMC.

[0174] B55. A first site-specific blocking agent comprising a first targeting moiety and, optionally, a first effector moiety, wherein the first site-specific blocking agent specifically binds to a first anchor sequence of an anchor sequence-mediated junction (ASMC), wherein the ASMC comprises a first gene and a second gene; and A system comprising a second site-specific blocking agent comprising a second targeting moiety and optionally a second effector moiety, wherein the second site-specific blocking agent binds to a second anchor sequence of ASMC.

[0175] B56. The system of embodiment B55, wherein the first anchor sequence is between IL-8 and RASSF6; between the IL-8 enhancer and RASSF6; between CXCL1 and CXCL4; between CXCL2 and EPGN; or between the E2 enhancer and EPGN.

[0176] B57. The system of embodiment B55 or B56, wherein the second anchor sequence is between IL-8 and RASSF6; between the IL-8 enhancer and RASSF6; between CXCL1 and CXCL4; between CXCL2 and EPGN; or between the E2 enhancer and EPGN.

[0177] B58. The system of any of embodiments B55-B57, wherein the first anchor sequence is between the IL-8 enhancer and RASSF6, and the second anchor sequence is between CXCL1 and CXCL4.

[0178] B59. The system of any of embodiments B55-B58, wherein the first anchor sequence is between the IL-8 enhancer and RASSF6, and the second anchor sequence is between the E2 enhancer and EPGN.

[0179] B60. The system of any of embodiments B55-B59, wherein the first anchor sequence is between CXCL1 and CXCL4, and the second anchor sequence is between the E2 enhancer and EPGN.

[0180] B61. The system of any of embodiments B55-B60, wherein the first site-specific blocking agent is a site-specific blocking agent described herein, eg, a site-specific blocking agent described in any of embodiments B2-B9.

[0181] B62. The system of any of embodiments B55-B61, wherein the second site-specific blocking agent is a site-specific blocking agent described herein, eg, a site-specific blocking agent described in any of embodiments B2-B9.

[0182] B63. The system of any of embodiments B55-B62, wherein the first targeting moiety and the second targeting moiety each independently comprise a TAL effector molecule, a CRISPR / Cas molecule, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0183] B64. The system of any of embodiments B55-B63, wherein the first effector and second effector each independently comprise an effector described herein, e.g., MQ1, EZH2, HDAC8, KRAB, G9A, or DNMT3a / 3l, or a functional variant or fragment of any of them.

[0184] B65. The system of any of embodiments B55-B62, wherein the first effector and second effector each independently comprise 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 thereof.

[0185] B66. The system of any of embodiments B55-B65, wherein the first effector moiety and the second effector moiety each independently comprise a protein selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any of them.

[0186] B67. The system of any one of embodiments B55-B43, wherein the first effector and second effector each independently comprise a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a / 3l, or a functional variant or fragment of any thereof.

[0187] B68. The system of any of embodiments B55-B67, wherein the first effector and second effector each independently comprise a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of these.

[0188] B69. The system of any of embodiments B55-B68, wherein the first effector and second effector each independently comprise a polymer, eg, an oligonucleotide.

[0189] B70. The system of any of embodiments B55-B69, wherein the first oligonucleotide and the second oligonucleotide are identical.

[0190] B71. The system of any of embodiments B55-B70, wherein the first oligonucleotide and the second oligonucleotide are different.

[0191] B72. The system of any of embodiments B55-B71, wherein the first oligonucleotide has a sequence comprising the complement of a first anchor sequence or a complement to a sequence adjacent to the first anchor sequence, and the second oligonucleotide has a sequence comprising the complement of a second anchor sequence or a complement to a sequence adjacent to the second anchor sequence.

[0192] B73. The system of any of embodiments B55-B72, wherein the anchor sequence-mediated junction further comprises a third gene.

[0193] B74. The system of any of embodiments B55-B73, wherein the anchor sequence-mediated junction further comprises a fourth gene.

[0194] B75. The system of any of embodiments B55-B74, wherein the anchor sequence-mediated junction further comprises a fifth gene.

[0195] B76. The system of any of embodiments B55-B75, wherein the anchor sequence-mediated junction further comprises a sixth gene.

[0196] B77. The system of any of embodiments B55-B76, wherein the anchor sequence-mediated junction further comprises a seventh gene.

[0197] B78. The system of any of embodiments B55-B77, wherein the anchor sequence-mediated junction further comprises an eighth gene.

[0198] B79. The system of any of embodiments B55-B78, wherein the ASMC comprises two loops.

[0199] B80. A nucleic acid composition encoding a system according to any one of embodiments B55 to B79.

[0200] B81. The nucleic acid of embodiment B80, wherein a single nucleic acid encodes both the first site-specific blocking agent and the second site-specific blocking agent.

[0201] B82. The nucleic acid of embodiment B81, wherein the first nucleic acid encodes a first site-specific interfering agent and the second nucleic acid encodes a second site-specific interfering agent.

[0202] B83. A method for reducing expression of a first gene and a second gene in a cell, comprising contacting said cell with a system described in any of embodiments B55-B79 of a nucleic acid composition described in any of embodiments B80-82.

[0203] B84. The method of embodiment B83, wherein said cells are contacted with the first site-specific blocking agent and the second site-specific blocking agent simultaneously.

[0204] B85. The method of embodiment 83, wherein the cells are contacted sequentially with a first site-specific blocking agent and a second site-specific blocking agent.

[0205] B86. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL1 and the second gene is CXCL2.

[0206] B87. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL1 and the second gene is CXCL3.

[0207] B88. The method, human cell, site-specific blocking agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL1 and the second gene is IL-8.

[0208] B89. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL1 and the second gene is CXCLX4.

[0209] B90. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL1 and the second gene is CXCL5.

[0210] B91. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL1 and the second gene is CXCL6.

[0211] B92. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL1 and the second gene is CXCL7.

[0212] B93. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL2 and the second gene is CXCL3.

[0213] B94. The method, human cell, site-specific blocking agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL2 and the second gene is IL-8.

[0214] B95. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL2 and the second gene is CXCL4.

[0215] B96. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL2 and the second gene is CXCL4.

[0216] B97. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL2 and the second gene is CXCL5.

[0217] B98. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL2 and the second gene is CXCL6.

[0218] B99. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL2 and the second gene is CXCL7.

[0219] B100. The method, human cell, site-specific blocking agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL3 and the second gene is IL-8.

[0220] B101. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL3 and the second gene is CXCL4.

[0221] B102. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL3 and the second gene is CXCL5.

[0222] B103. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL3 and the second gene is CXCL6.

[0223] B104. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL3 and the second gene is CXCL7.

[0224] B105. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL4 and the second gene is CXCL5.

[0225] B106. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL4 and the second gene is CXCL6.

[0226] B107. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL4 and the second gene is CXCL7.

[0227] B108. The method, human cell, site-specific blocking agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL4 and the second gene is IL-8.

[0228] B109. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL5 and the second gene is CXCL6.

[0229] B110. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL5 and the second gene is CXCL7.

[0230] B111. The method, human cell, site-specific blocking agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL5 and the second gene is IL-8.

[0231] B112. The method, human cell, site-specific interfering agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL6 and the second gene is CXCL7.

[0232] B113. The method, human cell, site-specific blocking agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL6 and the second gene is IL-8.

[0233] B114. The method, human cell, site-specific blocking agent, or system of any of embodiments B1-B85, wherein the first gene is CXCL7 and the second gene is IL-8.

[0234] B115. The method, human cell, site-specific interfering agent, or system of any of embodiments B36-B85, wherein the first gene is CXCL1, the second gene is CXCL2, and the third gene is CXCL3.

[0235] B116. The method, human cell, site-specific blocking agent, or system of any of embodiments B36-B85, wherein the first, second, and third genes are selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8.

[0236] B117. The method, human cell, site-specific blocking agent, or system of any of embodiments B36-B85, wherein the first, second, third, and fourth genes are selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8.

[0237] B118. The method, human cell, site-specific blocking agent, or system of any of embodiments B36-B85, wherein the first, second, third, fourth, and fifth genes are selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8.

[0238] B119. The method, human cell, site-specific blocking agent, or system of any of embodiments B36-B85, wherein the first, second, third, fourth, fifth, and sixth genes are selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8.

[0239] B120. The method, human cell, site-specific blocking agent, or system of any of embodiments B36-B85, wherein the first, second, third, fourth, fifth, sixth, and seventh genes are selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8.

[0240] B121. The method, human cell, site-specific blocking agent, or system of any of embodiments B36-B85, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth genes are selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8.

[0241] B122. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the first gene is a cytokine.

[0242] B123. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the second gene is a cytokine.

[0243] B124. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the third gene is a cytokine.

[0244] B125. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the fourth gene is a cytokine.

[0245] B126. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the fifth gene is a cytokine.

[0246] B127. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the sixth gene is a cytokine.

[0247] B128. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the seventh gene is a cytokine.

[0248] B129. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the eighth gene is a cytokine.

[0249] B130. The method, human cell, site-specific blocking agent, or system of any of the preceding embodiments, wherein the anchor sequence-mediated junction comprises three, four, or five pro-inflammatory genes.

[0250] B131. The method, human cell, site-specific interfering agent, or system of any preceding embodiment, wherein the site-specific interfering agent comprises a nucleic acid (e.g., DNA or RNA) comprising a nucleotide sequence selected from SEQ ID NOs: 20-62, or a sequence having at least 90%, 95%, 98%, or 99% identity thereto, or differing therefrom at no more than 1, 2, 3, 4, or 5 positions.

[0251] B132. The method of any preceding embodiment, wherein the site-specific blocking agent comprises a nucleic acid (e.g., DNA or RNA) comprising a nucleotide sequence selected from SEQ ID NOs: 21, 22, 24, 40, or a sequence having at least 90%, 95%, 98%, or 99% identity thereto, or differing therefrom at no more than 1, 2, 3, 4, or 5 positions.

[0252] B133. The method or site-specific disrupting agent of any of the preceding embodiments, wherein the site-specific disrupting agent binds to a sequence that at least partially overlaps with a region having genomic coordinates selected from Table 4, 5, 6, 7, or a sequence within 5, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides of said region.

[0253] B134. The method of any of the preceding embodiments, which results in a reduction in the level of a cytokine, e.g., a chemokine, upon stimulation of the cells, e.g., with TNF-alpha (e.g., measured as described in Examples 2-11).

[0254] B135. The method or human cell of any preceding embodiment, which, upon stimulation of the cells, e.g., with TNF-alpha, results in a reduced level of a cytokine, e.g., a chemokine (e.g., measured as described in Examples 2-11).

[0255] B136. The method or human cell of any of the preceding embodiments, wherein, e.g., upon stimulation of the cell with TNF-alpha, the transcription levels of one or more (e.g., two, three, or all) of CXCL1, CXCL2, CXCL3, and IL8 are reduced (e.g., measured as described in Examples 2 or 4-11).

[0256] B137. The method or human cell of any of the preceding embodiments, wherein the transcription levels of one or more (e.g., two, three, or all) of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, and IL8 are reduced upon stimulation of the cell, e.g., with TNF-alpha.

[0257] B138. The method or human cell of any of the preceding embodiments, wherein the reduction is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction compared to pre-treatment levels or to human cells with undisturbed ASMC.

[0258] B139. The method or human cell of any preceding embodiment, wherein, e.g., upon stimulation of the cell with TNF-alpha, protein levels (e.g., secreted protein levels) of one or more (e.g., two, three, or all) of CXCL1, CXCL2, CXCL3, and IL8 are reduced (e.g., measured as described in Example 3).

[0259] B140. The method or human cell of any of the preceding embodiments, wherein, upon stimulation of the cell with, e.g., TNF-alpha, protein levels (e.g., secreted protein levels) of one or more (e.g., two, three, or all) of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, and IL8 are reduced.

[0260] B141. The method or human cell of any of the preceding embodiments, wherein the reduction is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction compared to pre-treatment levels or human cells with undisturbed ASMC.

[0261] B142. The method of any of the preceding embodiments, resulting in a reduction in binding of CTCF to the first anchor sequence, e.g., a complete loss of binding, or at least a 20, 30, 40, 50, 60, 70, 80, 90, or 100% loss compared to human cells with undisturbed ASMC, e.g., as measured by ChIP and quantitative PCR.

[0262] B143. The method of any preceding embodiment, resulting in disruption of an anchor sequence-mediated junction.

[0263] B144. The method of any of the preceding embodiments, wherein a population of cells is contacted with a site-specific disrupting agent and the first anchor sequence is edited in at least 50%, 60%, 70%, 80%, 90%, or 95% of cells in said population.

[0264] B145. The method of any of the preceding embodiments, wherein the effect (e.g., reduction in cytokine levels) is additive or synergistic compared to the effect of inhibiting the first gene or the second gene individually.

[0265] B146. The method of any of the preceding embodiments, wherein expression is reduced for at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks.

[0266] B147. The method, human cell, site-specific interfering agent, or system of any of the preceding embodiments, wherein the cell is a cell of a subject having an inflammatory disease, eg, an immune-mediated inflammatory disease.

[0267] B148. The method, human cell, site-specific interfering agent, or system of any of the preceding embodiments, wherein the inflammatory disease is an autoimmune disorder, for example, rheumatoid arthritis.

[0268] B149. The method, human cell, site-specific interfering agent, or system of any of the preceding embodiments, wherein the inflammatory disease is associated with a pathogenic infection, e.g., a viral infection, e.g., a SARS-CoV2 infection.

[0269] B150. The method, human cell, site-specific interfering agent, or system of any of the preceding embodiments, wherein the inflammatory disease is associated with a co-infection, e.g., an infection caused by two or more pathogens, e.g., a virus and a bacterium (e.g., SARS-CoV2 and Streptococcus pneumoniae), e.g., a virus and a fungus (e.g., SARS-CoV2 and mucormycosis).

[0270] B151. The method, human cell, site-specific interfering agent, or system of any of the preceding embodiments, wherein the cell is a cell of a subject having rheumatoid arthritis, inflammation, arthritis, gout, asthma, neutrophilic asthma, neutrophilic dermatosis, paw edema, acute respiratory distress syndrome (ARDS), COVID-19, psoriasis, inflammatory bowel disease, infection (e.g., by a pathogen, e.g., bacterial, viral, or fungal), external injury (e.g., an abrasion or foreign body), effects of radiation or chemical injury, osteoarthritis, osteoarthritic joint pain, arthralgia, inflammatory pain, acute pain, chronic pain, cystitis, bronchitis, dermatitis, dermatosis, cardiovascular disease, neurodegenerative disease, liver disease, lung disease, kidney disease, pain, swelling, stiffness, tenderness, redness, fever, or elevated biomarkers associated with a disease state (e.g., cytokines, chemokines, growth factors, immune receptors, infection markers, or inflammation markers).

[0271] B152. The method, human cell, site-specific interfering agent, or system of any of the preceding embodiments, wherein said cell is a cell of a subject with rheumatoid arthritis, psoriasis, or inflammatory bowel disease.

[0272] B153. The method, human cell, site-specific disrupting agent, or system of any preceding embodiment, wherein the cell is a cell of a subject with rheumatoid arthritis, gout, neutrophilic asthma, neutrophilic dermatosis, acute respiratory distress syndrome (ARDS), or COVID-19.

[0273] B154. The method, site-specific blocking agent, or system of any of the preceding embodiments, wherein the anchor sequence-mediated junction comprises an internal enhancing sequence.

[0274] B155. The method, site-specific blocking agent, or system of any of the preceding embodiments, wherein the second gene (and optionally said third, fourth, fifth, sixth, seventh, or eighth gene) is transcribed in the same direction as the first gene.

[0275] B156. The method, site-specific blocking agent, or system of any of the preceding embodiments, wherein the first anchor sequence comprises a binding motif selected from a CTCF binding motif, a USF1 binding motif, a YY1 binding motif, a TAF3 binding motif, or a ZNF143 binding motif.

[0276] B157. The method, site-specific blocking agent, or system of any of the preceding embodiments, wherein the first anchor sequence comprises a CTCF binding motif.

[0277] B158. The method, site-specific blocking agent, or system of any preceding embodiment, wherein the site-specific blocking agent specifically binds to or in close proximity to the first anchor sequence with sufficient affinity to compete with binding of an endogenous nucleation polypeptide (e.g., CTCF, USF1, YY1, TAF3, or ZNF143) in said cell.

[0278] B159. The method, site-specific blocking agent, or system of any of the preceding embodiments, wherein the site-specific blocking agent adds, deletes, or substitutes one or more nucleotides within or adjacent to the first anchor sequence.

[0279] B160. The method or site-specific disruption agent of any preceding embodiment, wherein the site-specific disruption agent comprises a targeting moiety or effector moiety having a first CRISPR / Cas molecule comprising a first CRISPR / Cas protein and a first guide RNA.

[0280] B161. The method or system of any preceding embodiment, wherein the first site-specific disruption agent comprises a first targeting moiety or a first effector moiety having a first CRISPR / Cas molecule comprising a first CRISPR / Cas protein and a first guide RNA, and the second site-specific disruption agent comprises a second targeting moiety or a second effector moiety having a second CRISPR / Cas molecule comprising a second CRISPR / Cas protein and a second guide RNA.

[0281] B162. The method or site-specific disruption agent of any of the previous embodiments, wherein the site-specific disruption agent comprises a targeting or effector moiety comprising a TAL effector molecule, a CRISPR / Cas molecule, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0282] B163. The method or system of any of the preceding embodiments, wherein the first site-specific disruption agent comprises a first targeting moiety or first effector moiety having a TAL effector molecule, a CRISPR / Cas molecule, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide, and the second site-specific disruption agent comprises a second targeting moiety or second effector moiety having a TAL effector molecule, a CRISPR / Cas molecule, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

[0283] B164. The method or site-specific disruption agent of any of the previous embodiments, wherein the site-specific disruption agent comprises an effector moiety having histone-modifying functionality, eg, histone methyltransferase, histone demethylase, or histone deacetylase activity.

[0284] B165. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific disrupting agent comprises an effector moiety having histone-modifying functionality, e.g., histone methyltransferase, histone demethylase, or histone deacetylase activity.

[0285] B166. The method, site-specific blocking agent, or system of embodiment B164 or B165, wherein the effector moiety 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 thereof.

[0286] B167. The method, site-specific blocking agent, or system of embodiment B164 or B165, wherein the effector moiety comprises a protein selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof.

[0287] B168. The method, site-specific interfering agent, or system of embodiment B164 or B165, wherein the effector moiety comprises EZH2 or a functional variant or fragment of either thereof.

[0288] B169. The method, site-specific blocking agent, or system of embodiment B164 or B165, wherein the effector moiety comprises HDAC8 or a functional variant or fragment of any thereof.

[0289] B170. The method or site-specific disruption agent of any of the previous embodiments, wherein the site-specific disruption agent comprises an effector moiety that comprises a DNA-modifying functionality, e.g., a DNA methyltransferase.

[0290] B171. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific disrupting agent comprises an effector moiety that comprises a DNA-modifying functionality, eg, a DNA methyltransferase.

[0291] B172. The method, site-specific blocking agent, or system of embodiment B170 or B171, wherein the effector moiety comprises a protein selected from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a / 3l, or a functional variant or fragment of any thereof.

[0292] B173. The method, site-specific blocking agent, or system of embodiment B170 or B171, wherein the effector moiety comprises MQ1 or a functional variant or fragment of any thereof.

[0293] B174. The method, site-specific blocking agent, or system of embodiment 170 or 171, wherein the effector moiety comprises DNMT3 (e.g., DNMT3a, DNMT3L, DNMT3a / 3l, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, or DNMT3B6) or a functional variant or fragment of any thereof.

[0294] B175. The method or site-specific disruption agent of any preceding embodiment, wherein the site-specific disruption agent comprises an effector moiety that comprises a transcriptional repressor.

[0295] B176. The method or site-specific disruption agent of any preceding embodiment, wherein the first and / or second site-specific disruption agent comprises an effector moiety that comprises a transcriptional repressor.

[0296] B177. The method, site-specific blocking agent, or system of embodiment B175 or B176, wherein the effector moiety comprises a protein selected from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any of them.

[0297] B178. The method, site-specific blocking agent, or system of embodiment B177, wherein the effector moiety comprises KRAB or a functional variant or fragment of any thereof.

[0298] B179. The method or site-specific blocking agent of any of the previous embodiments, wherein the site-specific blocking agent comprises a polymer.

[0299] B180. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific blocking agent comprises a polymer.

[0300] B181. The method, site-specific blocking agent, or system of embodiment B179 or B180, wherein the polymer comprises a polyamide.

[0301] B182. The method, site-specific blocking agent, or system of embodiment B179 or B180, wherein the polymer is an oligonucleotide.

[0302] B183. The method, site-specific blocking agent, or system of embodiment B182, wherein the oligonucleotide has a sequence comprising the complement of the first anchor sequence or the complement to a sequence adjacent to the first anchor sequence.

[0303] B184. The method, site-specific blocking agent, or system of embodiment B182, wherein the oligonucleotide has a sequence comprising the complement of the second anchor sequence or the complement to a sequence adjacent to the second anchor sequence.

[0304] B185. The method, site-specific blocking agent, or system of any of embodiments B182-B184, wherein the oligonucleotide comprises a chemical modification.

[0305] B186. The method, site-specific blocking agent, or system of embodiment B179 or B180, wherein the polymer is a peptide nucleic acid.

[0306] B187. The method, site-specific blocking agent, or system of any of the preceding embodiments, wherein the site-specific blocking agent comprises a peptide-nucleic acid mixmer.

[0307] B188. The method, site-specific disruption agent, or system of any of the previous embodiments, wherein the site-specific disruption agent (eg, the targeting moiety or effector moiety of the site-specific disruption agent) comprises a peptide or polypeptide.

[0308] B189. The method, site-specific blocking agent, or system of embodiment B188, wherein the polypeptide is a zinc finger polypeptide.

[0309] B190. The method, site-specific disruption agent, or system of embodiment 188, wherein the polypeptide is or comprises a Transcription Activator-Like Effector Nuclease (TALEN) polypeptide.

[0310] B191. The method or site-specific interfering agent of any of the preceding embodiments, wherein the site-specific interfering agent comprises a small molecule.

[0311] B192. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific blocking agent comprises a small molecule.

[0312] B193. The method or site-specific disruption agent of any of the preceding embodiments, wherein the site-specific disruption agent further comprises an effector moiety, e.g., an epigenetic modifier, e.g., a DNA methyltransferase, histone deacetylase, or histone methyltransferase.

[0313] B194. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific disrupting agent further comprises an effector moiety, e.g., an epigenetic modifier, e.g., a DNA methyltransferase, histone deacetylase, or histone methyltransferase.

[0314] B195. The method or site-specific blocking agent of any of the preceding embodiments, wherein the site-specific blocking agent comprises a fusion molecule.

[0315] B196. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific blocking agent comprises a fusion molecule.

[0316] B197. The method or site-specific disruption agent of any preceding embodiment, wherein the site-specific disruption agent comprises a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety comprising a transcriptional repressor, e.g., as a fusion molecule.

[0317] B198. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific disrupting agent comprises, e.g., as a fusion molecule, a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety comprising a transcriptional repressor.

[0318] B199. The method or site-specific blocking agent of embodiment B198, wherein the targeting moiety comprises dCas9 and the effector moiety KRAB or a functional variant or portion thereof.

[0319] B200. The method or system of any preceding embodiment, wherein the first and / or second targeting moiety comprises dCas9 and an effector moiety KRAB or a functional variant or portion thereof.

[0320] B201. The method or site-specific disruption agent of any of the previous embodiments, wherein the site-specific disruption agent comprises a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety comprising a histone methyltransferase, e.g., as a fusion molecule.

[0321] B201. The method or site-specific disruption agent of any of embodiments B1-B177, wherein the first and / or second site-specific disruption agent comprises a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety comprising a histone methyltransferase, e.g., as a fusion molecule.

[0322] B202. The method or system of any of the preceding embodiments, wherein the first and / or second site-specific disrupting agent comprises, e.g., as a fusion molecule, a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety comprising a histone methyltransferase.

[0323] B203. The method, site-specific disruption agent, or system of embodiment B201, wherein the targeting moiety comprises dCas9 and the effector moiety comprises EZH2 or a functional variant or portion thereof.

[0324] B204. The method, site-specific disruption agent, or system of any of embodiments B1-B196, wherein the site-specific disruption agent comprises, e.g., as a fusion molecule, a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety comprising a DNA methyltransferase.

[0325] B205. The method, site-specific disruption agent, or system of embodiment B204, wherein the targeting moiety comprises dCas9 and the effector moiety comprises MQ1 or a functional variant or portion thereof.

[0326] B206. The method, site-specific disruption agent, or system of embodiment B203, wherein the targeting moiety comprises dCas9 and the effector moiety comprises DNMT3, e.g., DNMT3a / 3l, or a functional variant or portion thereof.

[0327] B207. The method, site-specific disruption, or system of any preceding embodiment, wherein the site-specific disruption comprises, e.g., as a fusion molecule, a targeting moiety comprising a CRISPR / Cas molecule, a first effector moiety comprising a histone methyltransferase, and a second effector moiety comprising a transcriptional repressor.

[0328] B208. The method, site-specific blocking agent, or system of embodiment B207, wherein the targeting moiety comprises dCas9, the first effector moiety comprises EZH2 or a functional variant or portion thereof, and the second effector moiety comprises KRAB or a functional variant or portion thereof.

[0329] B209. The method, site-specific disruption agent, or system of any of the previous embodiments, wherein the site-specific disruption agent comprises a targeting moiety comprising a CRISPR / Cas molecule and an effector moiety comprising a histone deacetylase, e.g., as a fusion molecule.

[0330] B210. The method, site-specific disruption agent, or system of embodiment B209, wherein the targeting moiety comprises dCas9 and the effector moiety comprises HDAC8 or a functional variant or portion thereof.

[0331] B211. The method, site-specific disruption agent, or system of any of the previous embodiments, wherein the site-specific disruption agent comprises, e.g., as a fusion molecule, a targeting moiety comprising a CRISPR / Cas molecule, a first effector moiety comprising a histone methyltransferase, and a second effector moiety comprising a histone deacetylase.

[0332] B212. The method, site-specific disruption agent, or system of embodiment B211, wherein the targeting moiety comprises dCas9, the first effector moiety comprises EZH2 or a functional variant or portion thereof, and the second effector moiety comprises HDAC8 or a functional variant or portion thereof.

[0333] B213. The method, site-specific blocking agent, or system of any of embodiments B195-B212, wherein the site-specific blocking agent comprises an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NOs: 69, 71, 85, 201, 202, 204, 205, 207, 209, 211, 213, 215, 217, or 219-242, or a complementary or reverse complementary sequence of any of them, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.

[0334] B214. The method, site-specific blocking agent, or system of any of embodiments B195-B213, wherein the site-specific blocking agent comprises an amino acid sequence selected from any one of SEQ ID NOs: 70, 72, 82, 84, 86, 203, 206, 208, 210, 212, 214, 216, or 218, or encoded by a sequence selected from any one of SEQ ID NOs: 219-242, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.

[0335] B215. The method, site-specific interfering agent, or system of any of the preceding embodiments, wherein said cell is in a subject.

[0336] B216. The method of any of embodiments B1-B215, wherein said cells are ex vivo, the site-specific blocking agent.

[0337] B217. The method or site-specific interfering agent, or system according to any of the preceding embodiments, wherein said cell is a mammalian cell, for example a human cell.

[0338] B218. The method, site-specific interfering agent, or system of any of the preceding embodiments, wherein said cell is a somatic cell.

[0339] B219. The method, site-specific interfering agent, or system of any of the preceding embodiments, wherein said cells are primary cells.

[0340] B220. The method of any of the preceding embodiments, wherein the contacting step is performed ex vivo.

[0341] B221. The method of embodiment B220, further comprising removing said cells (eg, mammalian cells) from the subject prior to the contacting step.

[0342] B222. The method of any of embodiments B220 or B221, further comprising, after the contacting step, (b) administering said cells (eg, mammalian cells) to a subject.

[0343] B223. The method of any of embodiments B1-222, wherein the contacting step comprises administering to the subject a composition comprising the site-specific blocking agent.

[0344] B224. The method of embodiment B223, wherein the site-specific blocking agent is administered as monotherapy.

[0345] B225. The method of embodiment B223, wherein the site-specific blocking agent is administered in combination with a second therapeutic agent.

[0346] B226. A reaction mixture comprising cells (eg, human cells, eg, primary human cells) and a site-specific interfering agent or system according to any of the preceding embodiments.

[0347] B227. A method of treating a subject with an inflammatory disorder, comprising: administering to a subject a site-specific disruption agent, system, or reaction mixture of any of the preceding embodiments in an amount sufficient to treat an inflammatory disorder; thereby treating said inflammatory disorder. A method comprising:

[0348] B228. The method of embodiment B227, wherein the inflammatory disorder is rheumatoid arthritis, psoriasis, or inflammatory bowel disease.

[0349] B229. The method of embodiment B227 or B228, wherein the inflammatory disorder is rheumatoid arthritis, gout, neutrophilic asthma, neutrophilic dermatosis, acute respiratory distress syndrome (ARDS), or COVID-19.

[0350] B230. The method of any of embodiments B227-B229, wherein the inflammatory disorder is an autoimmune disorder, eg, rheumatoid arthritis.

[0351] B231. The method of any of embodiments B227-229, wherein the inflammatory disease is associated with a pathogenic infection, e.g., a viral infection, e.g., a SARS-CoV2 infection.

[0352] B232. The method of any of embodiments B227-B229, wherein the inflammatory disease is associated with a superinfection, e.g., an infection caused by two or more pathogens, e.g., a virus and a bacterium (e.g., SARS-CoV2 and Streptococcus pneumoni), e.g., a virus and a fungus (e.g., SARS-CoV2 and mucormycosis).

[0353] B232. A method of treating a subject with cancer, comprising: administering to a subject an expression repressor, system, nucleic acid, nucleic acid system, or reaction mixture of any of the preceding embodiments in an amount sufficient to treat cancer; Thereby, treating cancer A method comprising:

[0354] B233. The method of claim B232, wherein the cancer is lung cancer (e.g., non-small cell lung cancer), breast cancer, hepatocellular carcinoma (HCC), prostate cancer, colon cancer, skin cancer, cervical cancer, ovarian cancer, uterine endometrioid carcinoma, endometrial carcinoma, mature B-cell lymphoma, bladder cancer, esophagogastric carcinoma, esophageal adenocarcinoma, bone cancer, melanoma, hepatobiliary carcinoma, thyroid carcinoma, mature B-cell neoplasm, glioma, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), sarcoma, or gastric adenocarcinoma.

[0355] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein.

[0356] All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) cited herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced herein, e.g., in any Table herein, are incorporated by reference. Unless otherwise indicated, sequence accession numbers listed herein, including in all Tables herein, refer to database entries current as of March 30, 2022. When a gene or protein has multiple sequence accession numbers, all sequence variants are encompassed.

[0357] definition A, an, the: As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly requires otherwise.

[0358] Anchor sequence: As used herein, the term "anchor sequence" refers to a nucleic acid sequence recognized by a nucleation agent and sufficiently bound to form an anchor sequence-mediated junction, e.g., a complex. In some embodiments, the anchor sequence comprises one or more CTCF binding motifs. In some embodiments, the anchor sequence is not located within a gene coding region. In some embodiments, the anchor sequence is located within an intergenic region. In some embodiments, the anchor sequence is not located within an enhancer or promoter. In some embodiments, the anchor sequence is located at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least 1 kb away from any transcription start site. In some embodiments, the anchor sequence is located in a region not associated with genomic imprinting, monoallelic expression, and / or monoallelic epigenetic marks. In some embodiments, the anchor sequence has one or more functions selected from binding to an endogenous nucleation polypeptide (e.g., CTCF), interacting with a second anchor sequence to form an anchor sequence-mediated junction, or isolating from an enhancer outside the anchor sequence-mediated junction. Some embodiments of the present disclosure provide techniques that can specifically target a particular anchor sequence or sequences without targeting other anchor sequences (e.g., sequences that may contain nucleation agent (e.g., CTCF) binding motifs in other contexts); such targeting anchor sequences are sometimes referred to as "target anchor sequences." In some embodiments, the sequence and / or activity of the target anchor sequence is modulated, but the sequence and / or activity of one or more other anchor sequences that may be present in the same system as the targeting anchor sequence (e.g., within the same cell and / or in some embodiments, on the same nucleic acid molecule, e.g., the same chromosome) are not modulated. In some embodiments, the anchor sequence includes or is a nucleation polypeptide-binding motif.In some embodiments, the anchor sequence is adjacent to the nucleation polypeptide binding motif.

[0359] Anchor sequence-mediated junction: As used herein, the term "anchor sequence-mediated junction" refers to a DNA structure, in some cases a complex, that arises from and / or is maintained by the physical interaction or binding of at least two anchor sequences within the DNA by one or more polypeptides, such as nucleation polypeptides, or one or more protein and / or nucleic acid entities (e.g., RNA or DNA), which bind to the anchor sequences, allowing for spatial proximity and functional linkage of the anchor sequences.

[0360] Associated with: As used herein, two events or entities are "associated" with one another when the presence, level, morphology, 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 condition when its presence, level, morphology, and / or function correlates with the incidence of and / or susceptibility to the disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with one another if they directly or indirectly interact to bring and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently bound by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, a DNA sequence is "associated" with a target genome complex or transcription complex when the nucleic acid is at least partially within the target genome complex or transcription complex and expression of genes in the DNA sequence is affected by the formation or disruption of the target genome complex or transcription complex.

[0361] CXCL locus: As used herein, the term "CXCL locus" refers to the portion of the human genome, or a homologous region of the genome in a non-human animal, that encodes CXCL1-7 and IL-8, enhancers E1 and E2, and anchor sequences that form ASMCs containing CXCL1-7 and IL-8. In some embodiments, the CXCL locus is located on human chromosome 4.

[0362] CXCL gene: As used herein, the term "CXCL gene" refers to human CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, or IL-8, or a homologous non-human gene. Human IL-8 is also sometimes referred to as CXCL8.

[0363] Site-specific disrupting agent: As used herein, the term "site-specific disrupting agent" refers to an agent or entity that specifically inhibits, dissociates, disassembles, and / or modifies one or more components of a genome complex, e.g., ASMC, thereby regulating, e.g., reducing, the expression of the target multigenes described herein. In some embodiments, the site-specific disrupting agent interacts with one or more components of a genome complex. In some embodiments, the site-specific disrupting agent binds (e.g., directly, or in some embodiments, indirectly) to one or more genome complex components. In some embodiments, the site-specific disrupting agent binds to an anchor sequence, e.g., the first and / or second anchor sequence, that may be part of the ASMC that contains the target multigene. In some embodiments, the site-specific disrupting agent binds to a site adjacent to an anchor sequence, e.g., the first and / or second anchor sequence, that may be part of the ASMC that contains the target multigene. In some embodiments, the site-specific disrupting agent modifies one or more genome complex components. In some embodiments, the site-specific disrupting agent comprises an oligonucleotide. In some embodiments, the site-specific disrupting agent comprises a polypeptide. In some embodiments, the site-specific disruption agent comprises an antibody (e.g., a monospecific or multispecific antibody construct) or antibody fragment. In some embodiments, the site-specific disruption agent is directed to a specific genomic location and / or genomic complex by a targeting moiety, as described herein. In some embodiments, the site-specific disruption agent comprises a genomic complex component or a variant thereof. In some embodiments, the site-specific disruption agent comprises a targeting moiety. In some embodiments, the site-specific disruption agent comprises an effector moiety. In some embodiments, the site-specific disruption agent comprises multiple effector moieties. In some embodiments, the site-specific disruption agent comprises a targeting moiety and one or more effector moieties. In some embodiments, the site-specific disruption agent specifically binds to a first site in the genome with higher affinity than a second site in the genome (e.g., compared to any other site in the genome). In some embodiments, the site-specific disruption agent preferentially inhibits, dissociates, disassembles, and / or modifies one or more components of a first genomic complex compared to a second genomic complex (e.g., compared to any other genomic complex).In some embodiments, the site-specific interfering agent may be an expression repressor, for example, the site-specific interfering agent may inhibit ASMC, thereby reducing expression of a gene in ASMC.

[0364] Domain: As used herein, the term "domain" refers to a section or portion of an entity. In some embodiments, a "domain" is associated with a particular structural and / or functional characteristic of the entity, and when the domain is physically separated from the rest of its parent entity, it substantially or completely retains said particular structural and / or functional characteristic. Alternatively, or in addition, in some embodiments, a domain may be or comprise a portion of 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 or comprises a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, polypeptide, etc.). In some embodiments, a domain is or comprises a section of a polypeptide. In some such embodiments, the domain is characterized by particular structural elements (e.g., particular amino acid sequences or sequence motifs, α-helical characteristics, β-sheet characteristics, coiled-coil characteristics, random coil characteristics, etc.) and / or particular functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0365] E1 cis-acting regulatory element (E1 cRE): The terms "E1 cRE" and "E1 cis-acting regulatory element" as used herein refer to a nucleic acid sequence that is located adjacent to (e.g., approximately 14 kb upstream of) IL8 in the human genome (see Figure 16B) and that is recognized by a trans-acting factor (e.g., a transcription factor such as p65) that binds sufficiently to upregulate expression of one or more CXCL genes.

[0366] E2 cis-acting regulatory element (E2 cRE): The terms "E2 cRE" and "E2 cis-acting regulatory element," as used herein, refer to a nucleic acid sequence that is located adjacent to CXCL2 in the human genome (see Figure 16B) and is recognized by a trans-acting factor (e.g., a transcription factor, e.g., p65) that binds sufficiently to upregulate expression of one or more CXCL genes.

[0367] Effector moiety: As used herein, the term "effector moiety" refers to a domain having one or more functionalities that, when properly localized in the nucleus of a cell, modulates, e.g., reduces, expression of a target multigene in a cell. In some embodiments, the effector moiety comprises a polypeptide. In some embodiments, the effector moiety comprises a polypeptide and a nucleic acid. The functionality associated with the effector moiety may directly affect the expression of the target multigene, e.g., block the recruitment of transcription factors that can stimulate expression of the genes. The functionality associated with the effector moiety may indirectly affect the expression of the target multigene, e.g., introduce epigenetic modifications or recruit other factors that introduce epigenetic modifications that induce changes in chromosomal topology that inhibit expression of the target multigene.

[0368] Expression repressor: As used herein, the term "expression repressor" refers to an agent or entity that reduces expression of a target gene in a cell and specifically binds to a DNA sequence (e.g., a target gene or a DNA sequence associated with a transcriptional control element operably linked to a target gene) and has one or more functionalities. An expression repressor comprises at least one targeting moiety and optionally an effector moiety. In some embodiments, an expression repressor binds to a site adjacent to an enhancer sequence that may be operably linked to multiple targeted genes. In some embodiments, an expression repressor comprises an oligonucleotide. In some embodiments, an expression repressor comprises a polypeptide. In some embodiments, an expression repressor comprises multiple effector moieties. In some embodiments, an expression repressor comprises a targeting moiety and one or more effector moieties. In some embodiments, an expression repressor specifically binds to a first site in a genome with higher affinity than a second site in the genome (e.g., relative to any other site in the genome).

[0369] Genome complex: As used herein, the term "genome complex" refers to a complex that brings together two genome sequence elements spaced apart from one another on one or more chromosomes through the interaction of multiple proteins and / or other components (perhaps including genome sequence elements). In some embodiments, the genome sequence element is an anchor sequence to which one or more protein components of the complex bind. In some embodiments, the genome complex may comprise an anchor sequence-mediated junction. In some embodiments, the genome sequence element may be or comprise a CTCF binding motif, a promoter, and / or an enhancer. In some embodiments, the genome sequence element comprises at least one or both of a promoter and / or a regulatory site (e.g., an enhancer). In some embodiments, complex formation is nucleated at the genome sequence element and / or by binding of one or more protein components to the genome sequence element. As will be appreciated by one of skill in the art, in some embodiments, colocalization (e.g., coupling) of genome sites through complex formation alters DNA topology at or near (e.g., in some embodiments, between) the genome sequence elements. In some embodiments, the genome complex comprises an anchor sequence-mediated junction comprising one or more loops. In some embodiments, the genome complex described herein is nucleated by a nucleation polypeptide, such as, for example, CTCF and / or cohesin. In some embodiments, the genome complex described herein may comprise, for example, one or more of the following: 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 regulators (e.g., Mediator, P300, enhancer-binding proteins, repressor-binding proteins, histone modifiers, etc.).In some embodiments, the genome 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, can interact with each other and / or with one or more genome sequence elements (e.g., anchor sequences, promoter sequences, regulatory sequences (e.g., enhancer sequences) to trap a stretch of genomic DNA in a topological configuration (e.g., a loop) that it would not adopt absent complex formation.

[0370] Nucleic Acid: As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); 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, a nucleic acid is, comprises, or consists of one or more naturally occurring 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, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds rather than phosphodiester linkages in the backbone, and are considered 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 linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring 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, intervening 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 naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or a 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 by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is partially or 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 includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide.In some embodiments, the nucleic acid has enzymatic activity. In some embodiments, the nucleic acid is an mRNA nucleic acid, which may be monocistronic or polycistronic (e.g., bicistronic, tricistronic, etc.).

[0371] Operably linked: As used herein, the phrase "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A genomic regulatory element (e.g., a transcriptional control element) "operably linked" to a functional element, e.g., a gene, is joined such that expression and / or activity of the functional element, e.g., the gene, is achieved under conditions compatible with the genomic regulatory element (e.g., transcriptional control element). In some embodiments, an "operably linked" genomic regulatory element (e.g., a transcriptional control element) is contiguous with (e.g., shared by) a coding element of interest, e.g., a gene; in some embodiments, an operably linked genomic regulatory element (e.g., a transcriptional control element) acts in cis with or otherwise at a distance from the functional element, e.g., gene, of interest. In some embodiments, an "operably linked" genomic regulatory element (e.g., a transcriptional control element) is contiguous (e.g., covalently linked) with a coding element, e.g., a gene, of interest; in some embodiments, an operably linked genomic regulatory element (e.g., a transcriptional control element) acts in trans or otherwise at a distance from a functional element, e.g., a gene, of interest. In some embodiments, two operably linked nucleic acid sequences are comprised on the same nucleic acid. In other embodiments, two operably linked nucleic acid sequences are proximate to each other on the same nucleic acid, e.g., within 1000, 500, 100, 50, or 10 base pairs of each other, or adjacent to each other.

[0372] Peptide, Polypeptide, Protein: As used herein, the terms "peptide," "polypeptide," and "protein" refer to compounds composed of amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. Polypeptides include any peptide or protein containing 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, e.g., also commonly referred to in the art as peptides or oligopeptides, and longer chains, which are commonly referred to in the art as proteins, of which there are many varieties.

[0373] Proximity: As used herein, "proximity" refers to the proximity of two sites, e.g., nucleic acid sites, such that binding of an expression repressor or site-specific disrupting agent at the first site and / or modification of the first site by the expression repressor or site-specific disrupting agent produces the same, or substantially the same, effect as binding and / or modification of the other site. For example, a DNA targeting moiety can bind to a first site that is proximal to an anchor sequence (second site), and an effector moiety bound to the DNA targeting moiety can epigenetically modify the first site such that binding of the anchor sequence to an endogenous nucleation polypeptide for expression of the target gene is modified in substantially the same manner as if the second site (anchor sequence) had been bound and / or modified. In some embodiments, sites that are adjacent to each other are less than 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, or 5 base pairs from each other.

[0374] Sequence-targeting polypeptide: As used herein, the term "sequence-targeting polypeptide" refers to a protein that recognizes or specifically binds to a target nucleic acid sequence, e.g., a protein comprising a CRISPR / Cas domain, a TAL effector domain, or a Zn finger domain. In some embodiments, the sequence-targeting polypeptide is a catalytically inactive protein that lacks endonuclease activity, such as dCas9, a TAL effector molecule, or a Zn finger domain.

[0375] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in an environment in which binding occurs. In some embodiments, a binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0376] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms, etc.) and plants. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0377] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the art will understand that biological and chemical phenomena rarely, if ever, reach completion and / or proceed to perfection, or achieve or avoid absolute results. Thus, the term "substantially" may be used in some embodiments herein to capture the possible lack of perfection inherent in many biological and chemical phenomena.

[0378] Symptoms are reduced: As used herein, the phrase "symptoms are reduced" may be used when the magnitude (e.g., intensity, severity, etc.) and / or frequency of one or more symptoms of a particular disease, disorder, or condition is reduced. In some embodiments, delaying the onset of a particular symptom is considered a form of reducing the frequency of that symptom.

[0379] Target: An agent or entity is considered to "target" another agent or entity in accordance with the present disclosure if it specifically binds to the targeting agent or entity under conditions in which they contact each other. In some embodiments, for example, an antibody (or antigen-binding fragment thereof) targets its cognate epitope or antigen. In some embodiments, a nucleic acid having a particular sequence targets a nucleic acid of a substantially complementary sequence. In some embodiments, a targeting moiety that specifically binds to an anchor sequence targets the anchor sequence, ASMC containing the anchor sequence, and / or multiple genes within the ASMC.

[0380] Targeted multigene: As used herein, the term "targeted multigene" refers to a group of multiple genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more genes) that are targeted for regulation, e.g., modulation of expression. In some embodiments, the targeted multigene is part of a targeted genome complex. In some embodiments, each gene of the targeted multigene is operably linked to an enhancer, e.g., an E1 enhancer, and the enhancer is targeted by an expression repressor described herein. In some embodiments, modulation comprises inhibition of expression of the targeted multigene. In some embodiments, the targeted multigene is regulated by contacting the targeted multigene, or a genomic regulatory element (e.g., a transcriptional control element) operably linked to one or more of the targeted multigenes, with an expression repressor described herein. In some embodiments, one or more of the targeted multigenes are aberrantly expressed (e.g., overexpressed) in a cell, e.g., a cell of a subject (e.g., a patient). In some embodiments, the targeted multigene has associated functionality. For example, all of the genes in the target multigene may have a pro-inflammatory effect when expressed; such genes in the target multigene may be referred to herein as pro-inflammatory genes or target pro-inflammatory genes. In some embodiments, the genes in the target multigene encode proteins. In some embodiments, the genes in the target multigene encode functional RNA.

[0381] Targeting moiety: As used herein, the term "targeting moiety" refers to a component or set of components, e.g., an agent or entity that specifically interacts with (e.g., targets) DNA. In some embodiments, a component or multiple components participate in a genome complex described herein (e.g., anchor sequence-mediated junction). In some embodiments, a targeting moiety in accordance with the present disclosure targets one or more target components of a genome complex described herein. In some embodiments, a targeting moiety targets a genome regulatory element (e.g., an E1 enhancer). In some embodiments, a targeting moiety targets an anchor sequence. In some embodiments, a targeting moiety targets a genome complex component other than a genome regulatory element. In some embodiments, a targeting moiety targets a plurality or combination of genome complex components, and in some embodiments, a plurality may include a genome sequence element. In some aspects, effective inhibition, dissociation, disassembly, and / or modification of one or more genome complexes described herein can be achieved by targeting a complex component that includes a genome sequence element. In some embodiments, the present disclosure provides improved (e.g., with respect to the degree of specificity for a particular genome complex relative to other genome complexes that may form or exist in a given system; efficacy of inhibition, dissociation, degradation, or modification [e.g., in terms of the effect on the number of complexes detected in a population]) inhibition, dissociation, degradation, or modification can be achieved by targeting one or more complex components that are not genome sequence elements, and may optionally include targeting genome sequence elements, where the improved inhibition, dissociation, degradation, or modification is compared to that typically achieved by targeting genome sequence elements alone. In some embodiments, the site-specific disrupting agents described herein promote the inhibition, dissociation, degradation, or modification of a target genome complex.For example, by way of non-limiting example, in some embodiments, the site-specific disrupting agents described herein inhibit, dissociate, degrade (e.g., degrade its components), and / or modify (e.g., modify its components) the anchor sequence-mediated junction by targeting at least one component of a given genomic complex (e.g., including the anchor sequence-mediated junction). In some embodiments, the site-specific disrupting agents described herein inhibit, dissociate, degrade (e.g., degrade its components), and / or alter (e.g., modify its components) a specific genomic complex (i.e., a target genomic complex) without inhibiting, dissociating, degrading (e.g., degrade its components), and / or modify (e.g., modify its components) at least one other specific genomic complex (i.e., a non-target genomic complex) that may be present in other cells (e.g., non-target cells) and / or may be present at a different site within the same cell (i.e., a target cell). The expression repressors or site-specific disrupting agents described herein may include a targeting moiety. In some embodiments, the targeting moiety also acts as an effector moiety (e.g., a disrupting moiety); in some such embodiments, the expression repressor or site-specific disrupting agent provided may lack any effector moiety (e.g., a disrupting, modifying, or other effector moiety) that is distinct from (or significantly different from) the targeting moiety.

[0382] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be understood by one of skill 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, and the target cell or tissue. For example, in some embodiments, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that ameliorates, alleviates, reduces, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple doses are required to deliver a therapeutically effective amount.

[0383] Genomic regulatory sequence: As used herein, the term "genomic regulatory sequence" refers to a nucleic acid sequence that increases or decreases the transcription of a gene. An "enhancing sequence" increases the likelihood of gene transcription. A "silencing or repressor sequence" decreases the likelihood of gene transcription. Examples of genomic regulatory sequences include promoters and enhancers. In some embodiments, a genomic regulatory sequence is a cis-acting regulatory element. In some embodiments, an ASMC comprises a genomic regulatory sequence. Such a genomic regulatory sequence is referred to as an internal genomic regulatory sequence (e.g., an enhancing sequence contained within an ASMC is referred to as an internal enhancing sequence).

[0384] The following detailed description of the embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings embodiments which are presently illustrated. It should be understood, however, that the present disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0385] [Figure 1] 1 shows a schematic diagram illustrating exemplary placement of gRNA sequences in anchor sequences. FIG. 1 discloses SEQ ID NOs: 244-245, respectively, in order of appearance. [Figure 2] 2 shows a schematic diagram illustrating exemplary placement of gRNA sequences in anchor sequences and restriction site information. FIG. 2 discloses SEQ ID NOs: 246-247, respectively, in order of appearance. [Figure 3] 1 shows a graph of the expression (mRNA) of various chemokines in TNF-treated cells with and without treatment with a site-specific blocking agent comprising a CRISPR / Cas molecule and a first exemplary gRNA. [Figure 4] 1 shows a graph of the expression (mRNA) of various chemokines in TNF-treated cells with and without treatment with a site-specific blocking agent comprising a CRISPR / Cas molecule and a second exemplary gRNA. [Figure 5] 1 is a diagram depicting different types of genomic complexes, e.g., ASMCs, e.g., loops, and a model of how to alter expression of genes contained therein. [Figure 6] Graphs of cytokine expression, as measured by RNA levels of CXCL1, CXCL2, CXCL3, and IL-8, are shown in THP-1 cells treated with a site-specific blocking agent comprising a CRISPR / Cas molecule and an sgRNA targeting an anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC). [Figure 7] Graphs of cytokine secretion (CXCL1 and IL-8) of THP-1 cells treated with site-specific blocking agents containing CRISPR / Cas molecules and different sgRNAs targeting anchor sequences of genomic complexes containing genes encoding cytokines (e.g., ASMC). [Figure 8]Shown (top) is a graph of cytokine expression (CXCL3) as measured by RNA levels in THP-1 cells treated with a site-specific blocking agent containing a CRISPR / Cas molecule and an sgRNA targeting an anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC), and a flowchart (bottom) showing how cells were processed in the experiment. [Figure 9A] Shown (top) is a graph of cytokine expression (CXCL1) as measured by RNA levels in THP-1 cells 3 weeks after treatment with a site-specific blocking agent containing a CRISPR / Cas molecule and an sgRNA targeting an anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC), and a flowchart (bottom) showing how cells were processed in the experiment. [Figure 9B] Graph of cytokine expression (CXCL3) as measured by RNA levels in THP-1 cells 3 weeks after treatment with a site-specific blocking agent containing a CRISPR / Cas molecule and an sgRNA targeting the anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC). [Figure 10] Graph of cytokine expression (CXCL1) as measured by RNA levels in THP-1 cells after treatment with a site-specific blocking agent comprising a catalytically inactive CRISPR / Cas molecule and a transcriptional repressor (KRAB) and an sgRNA targeting an anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC). [Figure 11] Graph of cytokine expression (CXCL1) as measured by RNA levels in THP-1 cells after treatment with a site-specific disruptor comprising a catalytically inactive CRISPR / Cas molecule and a histone methyltransferase (EZH2) and an sgRNA targeting an anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC). [Figure 12]Graph of cytokine expression (CXCL1) as measured by RNA levels in THP-1 cells after treatment with a site-specific blocking agent comprising a catalytically inactive CRISPR / Cas molecule and a DNA methyltransferase (MQ1) and an sgRNA targeting an anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC). [Figure 13] Shown is a graph (top) of cytokine expression (CXCL1) measured by RNA levels in THP-1 cells after treatment with different site-specific blocking agents for 72 hours, 3 weeks, or 4 weeks, and a flowchart (bottom) showing how cells were processed in the experiment. [Figure 14] Shown (top) is a graph of cytokine expression (CXCL3) as measured by RNA levels in THP-1 cells after treatment with different site-specific blocking agents and sgRNA targeting the anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC), and a flowchart (bottom) showing how cells were processed in the experiment. [Figure 15] A graph (top) shows cytokine expression (CXCL1) as measured by RNA levels in THP-1 cells after treatment with different site-specific blocking agents and sgRNA targeting the anchor sequence of a genomic complex containing a gene encoding a cytokine (e.g., ASMC). [Figure 16A] Human CXCL IGD and gene cluster organization. Figure 16A shows a schematic isolated genomic domain (IGD) illustrating two loops within the CXCL1-8 gene cluster. The CXCL8, CXCL6, and CXCL1 genes reside in the left loop of the IGD. The CXCL2-5 and CXCL7 genes reside in the right loop of the IGD. Examination of IGD data from different cell lines suggests that middle CTCF is present only in cells that secrete CXCLs (e.g., not in lymphocytes). Figure 16B shows that guides were designed against four different CTCF targets: left CTCF-2, left CTCF, middle CTCF, and right CTCF. [Figure 16B] Same as above [Figure 17] We show that CXCL1-8 genes were downregulated when dCas9-EZH2 guide 30183 targeted the middle CTCF motif located within the CXCL1-8 cluster in TNF-alpha-treated human A549 lung cancer epithelial cells. Cells stimulated with TNF-alpha served as a control. [Figure 18] We show that CXCL1, 2, 3, and 8 genes were downregulated when dCas9-EZH2 guide 30183 targeted the middle CTCF motif located within the CXCL1-8 cluster in TNF-alpha-treated human IMR-90 normal lung fibroblasts. Cells stimulated with TNF-alpha served as a control. [Figure 19] Figure 1 shows that when controller A targeted the left CTCF motif located within the CXCL1-8 cluster in TNF-alpha-treated human monocytes, CXCL1, 2, 3, and 8 genes were downregulated. Cells stimulated with TNF-alpha served as a control. [Figure 20A-1] Figure 20 shows the mouse CXCL IGD and gene cluster organization. Figure 20A shows a schematic isolated genomic domain (IGD) illustrating two loops within the CXCL gene cluster. Figure 20B illustrates two loops within the CXCL1-5, 7, and 15 gene clusters. The CXCL4, CXCL5, and CXCL7 genes reside in the left loop of the IGD. The CXCL1-3 and CXCL15 genes reside in the right loop of the IGD guides designed against four different CTCF targets: left (L), middle 1 (M1), middle 2 (M2), and right (R) CTCF. [Figure 20A-2] Same as above [Figure 20B] Same as above [Figure 21A] Shown is an IGD guide designed for four different CTCF targets: central 1 (M1), central 2 (M2), and right (R) CTCF. [Figure 21B]Figure 1 shows in vitro downregulation of mouse CXCL IGD in Hep1.6 cells using dCas9-MQ1. dCas9-MQ1 transfected with guides targeting either the right or one of the two central CTCF motifs of the CXCL gene cluster showed no downregulation of any of the seven CXCL genes after TNF-alpha stimulation (orange). Transfecting dCas9-MQ1 with a combination guide targeting both the central and right CTCF downregulated the entire gene cluster (blue). [Figure 22] (Figure 22A) Schematic experimental design for determining the effect of dCas9-MQ1 on reducing leukocyte filtration in inflamed lungs. Each mouse was treated with LNP alone or 3 mg / kg of dCas9-MQ1 targeting the two central and right CTCFs at -2 h. Mice were simulated with 5 mg / kg LPS at 0 h, followed by a second administration of 3 mg / kg of LNP alone or dCas9-MQ1 targeting the two central and right CTCFs at +8 h. Dexamethasone was administered intraperitoneally at a dose of 10 mg / kg at 0, 24, and 48 h. Animals were sacrificed at 72 h, and bronchial lavage fluid was collected from the lungs for flow cytometry. (Figure 22B) Systemic administration of dCas9-MQ1 reduced leukocyte infiltration in inflamed lungs. The total leukocyte counts / mL in bronchial lavage fluid obtained from dCas9-MQ1-treated mice showed a significant difference compared to LPS+ diseased animals. [Figure 23] (Figure 23A) The composition of infiltrating cells found in bronchial lavage fluid obtained from the lungs of inflamed mice. The leukocyte cell type that constitutes the majority of infiltrating cells is neutrophils, followed by B cells, T cells, macrophages, and other types of hematopoietic cells. (Figure 23B) dCas9-MQ1 significantly reduces the number of neutrophils infiltrating the lungs compared to the +LPS disease group. [Figure 24]This graph shows that the reduction in white blood cells in BALF is lung-specific and not due to a reduction in white blood cells in peripheral blood. This graph demonstrates that the effect of dCas9-MQ1 treatment on the reduction of white blood cell counts in BALF is lung-specific and not due to a reduction in white blood cell populations in the mice themselves. Hematopoietic cell populations in peripheral blood were similar in all groups. [Figure 25A-B] The results show that CXCL1-5, CXCL7, and CXCL15 gene expression was reduced in lung tissue. After treating animals with LNP alone or with dCas9-MQ1, lung tissue was processed and CXCL gene expression was checked by qPCR. Treatment with dCas9-MQ1 showed downregulation of all CXCL genes. CXCL2 expression was the most downregulated. [Figure 25C-E] Same as above [Figure 25F-G] Same as above [Figure 26A-D] These results indicate that reducing CXCL expression and recruiting cells to the inflammatory site had beneficial downstream effects of reducing the presence of other cytokines. Chemokine protein levels secreted in BALF showed a decrease in CXCL1 and 2 protein levels. Reducing CXCL expression and recruiting cells to the inflammatory site had beneficial downstream effects of reducing the presence of GM-CSF (Figure 26C) and IL6 (Figure 26D). [Figure 27]

[0023] Figure 1 is a bar graph showing the % downregulation of the CXCL gene (relative to cells + IL-1A) using expression repressors targeting different sites in the E1 cRE. Overall, these graphs demonstrate that multiple effectors targeting two different sites in the E1 cRE can achieve downregulation of multiple genes near the E1 cRE. [Figure 28] Same as above [Figure 29] 1 is a bar graph showing % downregulation of the CXCL gene (relative to cells + IL-1A) using expression repressors targeting a single site in the E2 cRE. [Figure 30]31 is a bar graph showing how dCas9-KRAB (FIG. 30) and dCas9-MQ1 (FIG. 31), which target a single site in the E1 cRE, can achieve downregulation of multiple genes near the E1 cRE. *p<0.05, ***p<0.001, ****p<0.0001 [Figure 31] Same as above [Figure 32] 32A-33C are bar graphs showing how dCas9-KRAB (FIG. 32) and dCas9-MQ1 (FIG. 33), which target a single site in the E1 cRE, can achieve downregulation of multiple genes near the E1 cRE. *p<0.05, ***p<0.001, ****p<0.0001 [Figure 33] Same as above [Figure 34] 1 is a bar graph showing how an expression repressor (dCas9-KRAB) targeting the IL8 promoter successfully downregulates IL8 expression. [Figure 35] This bar graph shows how two expression repressors containing zinc finger domain targeting sites to different sites in the E1 cRE can achieve downregulation of multiple genes near the E1 cRE. Additionally, the graph shows that the dCas9-KRAB expression repressor targeting the IL8 promoter reduced IL8 expression by over 90%. [Figure 36] 1 is a bar graph showing that an E1 cRE-targeted expression repressor (zinc finger-KRAB), an IL8 promoter-targeted expression repressor (dCas9-KRAB), and a combination thereof do not interfere with each other, and that the combination of expression repressors has a greater effect on IL8 compared to either expression repressor alone. [Figure 37] 1 is a bar graph showing the reduction of IL8 expression using expression repressors targeting a site in the E1 cRE or the IL8 promoter, as measured by IL8 mRNA 1 hour after IL1A stimulation. [Figure 38]38A and 38B are bar graphs showing reduction of IL8 expression using expression repressors targeting the E1 cRE or a site in the IL8 promoter, where IL8 protein levels are measured by ELISA 6 hours (FIG. 38) and 24 hours (FIG. 39) after IL1A stimulation. [Figure 39] Same as above [Figure 40] 1 is a bar graph depicting downregulation of CXCL1-3 and IL8 mRNA levels (percent downregulation calculated using normalization to IL1A-treated controls) by two expression repressors directed against two sites in the E1 cRE. [Figure 41] 1 is a bar graph showing the ability of two expression repressors (MR32105 and MR32104, which contain zinc finger targeting sites and KRAB effector domains) directed against two sites in the E1 cRE to increase H3Kme3 as measured by ChIP qPCR. [Figure 42] Figure 1 shows bar graphs depicting downregulation of CXCL1-3 and IL-8 3-7 days after transfection with an expression repressor (MR32105) targeting the E1 cRE. Percent downregulation of CXCL1-3 and IL-8 genes was calculated using normalization to IL-1A-treated controls. Downregulation of CXCL1, CXCL2, CXCL3, and IL-8 is shown from left to right for groups on days 3-7. [Figure 43] 1 is a bar graph showing the downregulation of IL8 using expression repressors targeted to different sites in the IL8 promoter. Overall, this graph shows that multiple effectors targeted to different sites in the IL8 promoter can achieve downregulation of IL8. [Figure 44A]The enrichment of the MR-32105-induced E1-targeted expression repressor at the E1 site (top panel) results in an increase in on-target DNA histone methylation (H3K9me3) (middle panel) and a decrease in on-target histone acetylation (H3K27ac) (bottom panel) (Figure 44A). Figure 44B shows the deletion of the P65 transcription factor at the E1 locus resulting from the MR-32105-induced expression repressor. [Figure 44B] Same as above [Figure 45] 1 is a bar graph showing downregulation of CXCL1-3 and IL8 in response to IL1A stimulation 1 hour after introduction of an expression repressor (MR-32104 or MR-32105) targeting the E1 cRE. [Figure 46A] Box-and-whisker blot showing downregulation of the CXCL gene after introduction of expression repressors (MR-32104 and MR-32105) targeting the E1 cRE. [Figure 46B] Same as above [Figure 47] HA-ChIP Seq shows enrichment of MR-32712-derived IL8 target expression repressors in target IL8 (top panel), resulting in increased on-target DNA histone methylation (H3K9me3) (middle panel) and decreased on-target P65 binding (bottom panel). [Figure 48] 1 is a bar graph showing CXCL gene expression in IMR-90 cells after administration of an IL8-targeting expression repressor (MR-32712). [Figure 49]

[0033] Figure 11 is a boxplot showing RNA levels of CXCL gene expression after introduction of an IL8-targeting expression repressor (MR-32172). Overall, the boxplot shows a significant decrease in IL8 RNA. [Figure 50] Shows enrichment of E1-targeted expression repressors at 24 hours, but no detectable signal at 24 hours by HA-ChIP Seq. [Figure 51]1 is a bar graph showing downregulation of CXCL genes and proteins in small airway epithelial cells (COPD) after introduction of an expression repressor targeting IL8 (MR-32172) and a bicistronic expression repressor targeting E1 cRE and IL8 (MR-32905). [Figure 52] 1 is a bar graph showing downregulation of CXCL gene and protein in bronchial smooth muscle cells (asthma) after introduction of an expression repressor targeting IL8 (MR-32172) and a bicistronic expression repressor targeting E1 cRE and IL8 (MR-32905). [Figure 53] 1 is a bar graph showing downregulation of CXCL genes and proteins in primary lung fibroblasts after introduction of an expression repressor targeting IL8 (MR-32172) and a bicistronic expression repressor targeting E1 cRE and IL8 (MR-32905). [Figure 54] Graph showing downregulation of CXCL1-3 and IL8 over 13 days following introduction of an expression repressor targeting IL8 (MR-32172) and a bicistronic expression repressor targeting E1 cRE and IL8 (MR-32905). [Figure 55] FIG. 10 is a graph showing the reduction in neutrophil migration after introduction of an expression repressor targeting the E1 cRE (MR-32105) and / or an expression repressor targeting IL8 (MR-32712). [Figure 56A] Graph showing reduction in neutrophil migration after introduction of an expression repressor targeting IL8 (MR-32712) and / or a bicistronic expression repressor targeting E1 cRE and IL8 (MR-32905). [Figure 56B] Same as above [Figure 57] Figure 4 depicts the loci of functional enhancers in the mouse CXCL cluster, with the three candidate E1 positions tested in Example 41 indicated by arrows. [Figure 58]1 is a bar graph showing downregulation of CXCL1 and CXCL2 after directing expression repressors with guides targeting mouse P1 and P6, homologs to human E1 and E2, respectively. [Figure 59] 1 is a bar graph showing CXCL2 RNA qPCR results after directing an expression repressor with a guide targeting the mouse homologue to human CXCL. [Figure 60] 1 is a bar graph showing CXCL1 RNA qPCR results after directing expression repressors with guides targeting the mouse homologue to human CXCL. [Figure 61] 1 is a bar graph showing the results of CXCL1 protein expression after introduction of an expression repressor with a guide targeting the mouse homologue to human CXCL. [Figure 62] 1 is a bar graph showing downregulation of CXCL1 and CXCL2 in mouse homologues to human CXCL. [Figure 63] 1 is a bar graph showing the results of CXCL1 protein expression after introduction of an expression repressor targeting the mouse homologue to human CXCL1. [Figure 64] 1 is a bar graph showing the results of IL-8 mRNA levels in multiple cancer cell lines after introduction of the E1 cRE and a bicistronic expression repressor targeting IL8 (MR-32905). IL-8 mRNA levels are normalized to IL-8 mRNA in TNFα-stimulated cells. [Figure 65] 1 is a bar graph showing the results of IL-8 protein expression levels in multiple cancer cell lines after introduction of the E1 cRE and a bicistronic expression repressor targeting IL8 (MR-32905). IL-8 mRNA levels are normalized to IL-8 mRNA in TNFα-stimulated cells. [Figure 66]1 is a bar graph showing the results of CXCL1 mRNA levels in multiple cancer cell lines after introduction of a bicistronic expression repressor (MR-32905) targeting E1 cRE and IL8. CXCL1 mRNA levels are normalized to CXCL1 mRNA in TNFα-stimulated cells. [Figure 67] 1 is a bar graph showing the results of endogenous IL-8 mRNA levels in breast cancer cell lines after introduction of the E1 cRE and a bicistronic expression repressor targeting IL8 (MR-32905). IL-8 mRNA levels are normalized to IL-8 mRNA in TNFα-stimulated cells. [Figure 68] 1 is a graph showing tumor volume (mm 3 ) in an A549 NSCLC xenograft model after introduction of a bicistronic expression repressor (MR-32905) targeting E1 cRE and IL8. [Figure 69] 1 is a graph showing the mean weight change (percentage) in A549 NSCLC xenograft model mice groups. Error bars represent the standard error of the mean (SEM). The experiment was performed as described in Example 47. [Figure 70] 1 is a bar graph showing the area under the curve (AUC) of percent body weight change in A549 NSCLC xenograft model mice groups. The percent body weight change AUC was calculated for each animal throughout the study up to day 04. The calculation was performed using a trapezoidal rule transformation. Error bars represent the SEM for each group. This experiment was performed as described in Example 47. [Figure 71] 1 is a graph showing the mean tumor volume (mm3) in an A549 NSCLC xenograft model after introduction of a bicistronic expression repressor (MR-32905) or a GFP control. The mean tumor volume was calculated from length and width measurements. The mean value for each group was calculated, and the error bars represent the SEM for each group. This experiment was performed as described in Example 47. [Figure 72]1 is a bar graph showing the area under the curve (AUC) of percent weight change in A549 NSCLC xenograft model mice groups. AUC was calculated using trapezoidal rule transformation for the tumor volume measured for each animal in this study. Group means were calculated and shown with error bars representing the SEM for each group. Groups were compared using ANOVA test. This experiment was performed as described in Example 47. [Figure 73] Figure 73 is a graph showing the mean tumor volume (percentage) in the A549 NSCLC xenogeneic model after transfection with a bicistronic expression repressor (MR-32905) or a GFP control. Mean tumor volume was calculated from length and width measurements. Group means were calculated and shown with error bars representing the SEM for each group. This experiment was performed as described in Example 47. [Figure 74] 1 is a bar graph showing the area under the curve (AUC) of weight change (percentage) in A549 NSCLC xenograft model mouse groups. AUC was calculated for the tumor volume measured for each animal during the study. This calculation was performed using trapezoidal rule transformation. Group means were calculated and shown with error bars representing the SEM for each group. Groups were compared using ANOVA test. This experiment was performed as described in Example 47. [Figure 75]

[0033] Figure 1 shows a schematic experimental design for determining the efficacy of expression repressors for use in acute respiratory distress syndrome (ARDS). The experiment was carried out as described in Example 48. [Figure 76] 1 is a graph showing percent change in body weight (BW) from baseline in LPS-induced C57BL / 6 mice. The experiment was performed as described in Example 48. [Figure 77] 1 is a bar graph showing BALF cell concentration in C57BL / 6 mice. The experiment was performed as described in Example 48. [Figure 78A]Figure 78A is a bar graph showing BALF immune cell concentrations in LPS-induced C57BL / 6 mice. Figure 78A is a bar graph showing BALF mouse leukocyte concentrations (cells / mL). Figure 78B is a bar graph showing BALF mouse alveolar macrophage concentrations (cells / mL). Figure 78C is a bar graph showing BALF mouse neutrophil concentrations (cells / mL). Figure 78D is a bar graph showing BALF mouse T cell concentrations (cells / mL). Figure 78E is a bar graph showing BALF mouse B cell concentrations (cells / mL). This experiment was performed as described in Example 48. [Figure 78B] Same as above [Figure 78C] Same as above [Figure 78D] Same as above [Figure 78E] Same as above [Figure 79A] 1 is a bar graph showing BALF immune cell frequencies in LPS-induced C57BL / 6 mice. The experiment was performed as described in Example 48. [Figure 79B] Same as above [Figure 79C] Same as above [Figure 79D] Same as above [Figure 80A] 1 is a bar graph showing blood immune cell concentrations in LPS-induced C57BL / 6 mice. The experiment was performed as described in Example 48. [Figure 80B] Same as above [Figure 80C] Same as above [Figure 80D] Same as above [Figure 80E] Same as above [Figure 81A] 1 is a bar graph showing blood immune cell frequencies in LPS-induced C57BL / 6 mice. The experiment was performed as described in Example 48. [Figure 81B] Same as above [Figure 81C] Same as above [Figure 81D] Same as above [Figure 82A] 1 is a bar graph showing histology scores and evaluation in LPS-induced C57BL / 6 mice. The experiment was performed as described in Example 48. [Figure 82B] Same as above [Figure 82C] Same as above [Figure 82D] Same as above [Figure 82E] Same as above [Figure 82F] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0386] The present disclosure provides techniques for reducing the expression of multiple target CXCL genes in a cell, e.g., a subject or patient, by using, for example, an expression repressor, a system comprising two or more expression repressors, or a system comprising an expression repressor and a site-specific blocking agent. In some embodiments, the expression repressor comprises a targeting moiety. In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety. Without wishing to be bound by theory, some diseases and conditions are associated with groups of genes with related functionality involving a common enhancer. Enhancer inhibition may be an improved approach for reducing the expression of multiple target genes (e.g., in terms of improved efficiency, potency, and / or stability of the modification) relative to regulating individual target genes. Optionally, an expression repressor may be used in combination with a site-specific blocking agent, e.g., a site-specific blocking agent that blocks anchor sequence-mediated junctions. Site-specific blocking agents may also suppress the expression of multiple genes (e.g., the same or overlapping genes as the expression repressor). The improvement can translate into a corresponding improvement in the treatment of diseases and conditions associated with targeting multiple genes. For example, the multiple genes can be CXCL genes, and the expression repressor can target an E1 cRE operably linked to the multiple genes to reduce expression of the multiple genes, thereby achieving an anti-inflammatory effect (e.g., a superior anti-inflammatory effect compared to targeting the multiple genes individually). Examples of expression repressors, site-specific blocking agents, targeting moieties, effector moieties, and targeting multiple genes are provided herein.

[0387] An expression repressor can reduce the expression of target multiple genes through one or more modalities. In some embodiments, an expression repressor for a target site, e.g., the E1 cRE, can physically or sterically compete for binding with a factor that binds to the target site. Without wishing to be bound by theory, physical or steric blocking of an enhancer sequence (e.g., the E1 cRE), such that binding of the factor to the enhancer sequence is inhibited (e.g., prevented), is one mechanism by which an expression repressor can regulate, e.g., reduce, the expression of target multiple genes. An expression repressor can destabilize the interaction between an enhancer sequence and a factor, for example, by altering (e.g., reducing) the affinity and / or avidity with which the factor binds to the enhancer sequence. Blocking or destabilizing the binding of a factor to a target sequence can be achieved by one or more means, including epigenetic modification of the enhancer sequence or a sequence adjacent thereto, genetic modification of the enhancer sequence or a sequence adjacent thereto, or binding of an expression repressor to the enhancer sequence or a sequence adjacent thereto. Inhibition of genomic regulatory elements operably linked to the target multiple genes can regulate, e.g., reduce, the expression of the genes of the target multiple genes. In some embodiments, the expression repressor comprises a targeting moiety, a first effector moiety, and a second effector moiety. In some embodiments, the first effector moiety has a sequence different from that of the second effector moiety. In some embodiments, the first effector moiety has a sequence identical to that of the second effector moiety.

[0388] The expression repressors described herein (e.g., those that target enhancer sequences) can also be used in combination with site-specific disrupting agents (e.g., those that target anchor sequences). In some embodiments, the site-specific disrupting agents comprise a targeting moiety. In some embodiments, the site-specific disrupting agents comprise a targeting moiety and an effector moiety. Without wishing to be bound by theory, many diseases and conditions are associated with a common genomic complex, e.g., a group of genes with related functionality that are associated with ASMC. Modulation, e.g., disruption, of a genomic complex that includes (in whole or in part) a target multigene, e.g., ASMC, can represent an improved approach for altering (e.g., reducing) the expression of a target multigene relative to modulation of individual target genes (e.g., in terms of improved efficiency, efficacy, and / or stability of alteration). Such improvements can lead to corresponding improvements in the treatment of diseases and conditions associated with the target multigene. For example, multiple genes may be associated with a pro-inflammatory effect, and a site-specific disrupting agent can target a genomic complex that includes multiple genes (in whole or in part), e.g., ASMC, to modulate, e.g., reduce, expression of the multiple genes, thereby achieving an anti-inflammatory effect (e.g., a superior anti-inflammatory effect compared to targeting multiple genes individually). Examples of site-specific disrupting agents, targeting moieties, effector moieties, and target multiple genes are provided herein.

[0389] Site-specific disrupting agents can modulate, e.g., reduce, the expression of target multiple genes through one or more modalities. In some embodiments, site-specific disrupting agents bind to target sites, e.g., anchor sequences, and physically or sterically compete for binding with other genome complex components, e.g., nucleation polypeptides. Without wishing to be bound by theory, physical or steric blocking of anchor sequences, e.g., inhibiting (e.g., preventing) binding of genome complex components (e.g., nucleation polypeptides) to the anchor sequences, is one mechanism by which site-specific disrupting agents can modulate, e.g., reduce, the expression of target multiple genes. Site-specific disrupting agents can destabilize the interaction between genome complex components (e.g., nucleation polypeptides) and anchor sequences, for example, by altering (e.g., reducing) the affinity and / or avidity with which the genome complex components bind to the anchor sequences. Blocking or destabilizing the binding of a genome complex component (e.g., a nucleating polypeptide) to an anchor sequence can be achieved by one or more means, including epigenetic modification of the anchor sequence or a sequence adjacent thereto, genetic modification of the anchor sequence or a sequence adjacent thereto, or binding of a site-specific disrupting agent to the anchor sequence or a sequence adjacent thereto. Inhibiting (e.g., preventing) the binding of a genome complex component (e.g., a nucleating polypeptide) to an anchor sequence can inhibit (e.g., disrupt or prevent the formation of) a genome complex, e.g., ASMC. Inhibiting a genome complex, e.g., ASMC, that contains, in whole or in part, a target multigene can modulate, e.g., reduce, the expression of genes in the target multigene. In some embodiments, the site-specific disrupting agent comprises a targeting moiety, a first effector moiety, and a second effector moiety. In some embodiments, the first effector moiety has a sequence different from that of the second effector moiety. In some embodiments, the first effector moiety has a sequence identical to that of the second effector moiety.

[0390] The present disclosure further provides, in part, a system comprising two or more expression repressors, each comprising a targeting moiety and, optionally, an effector moiety. In some embodiments, the targeting moieties target two or more different sequences (e.g., each expression repressor may target a different sequence). In some embodiments, a first expression repressor binds to a first genomic regulatory element (e.g., an enhancer, e.g., E1 cRE) operably linked to target multiple genes, e.g., human CXCL1-8, and a second expression repressor binds to a second genomic regulatory element (e.g., an enhancer, promoter, or transcription start site (TSS)) operably linked to multiple genes, e.g., human CXCL1-8. In some embodiments, the system comprises an expression repressor and a site-specific disrupting agent. In some embodiments, the expression repressor binds to a transcriptional regulatory element (e.g., an enhancer (e.g., E1 cRE)) operably linked to a target gene multiplicity, e.g., human CXCL1-8, and the site-specific disrupting agent binds to an anchor sequence of an anchor sequence-mediated junction (ASMC) that includes the target gene multiplicity, e.g., human CXCL1-8.

[0391] In some embodiments, regulating the expression of a target multigene, e.g., human CXCL1-8, by the system comprises binding of a first expression repressor and a second expression repressor to a first and a second DNA sequence, respectively. In some embodiments, regulating the expression of a target multigene, e.g., human CXCL1-8, by the system comprises binding of an expression repressor and a site-specific blocking agent to a first and a second DNA sequence, respectively. Binding of the first and second DNA sequences localizes the functionality of the first and second effector moieties to those sites. Without wishing to be bound by theory, in some embodiments, the functionality of both the first and second effector moieties is utilized to stably repress expression of a target multigene associated with or comprising the first and / or second DNA sequence, e.g., wherein the first and / or second DNA sequence is or comprises the sequence of the target multigene or one or more operably linked genomic regulatory elements (e.g., transcriptional control elements).

[0392] Expression repressors In some embodiments, the expression repressor comprises a targeting moiety. In some embodiments, the targeting moiety specifically binds to a DNA sequence, e.g., the E1 cRE, thereby modulating, e.g., disrupting, the function of the DNA sequence. In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety. In some embodiments, the targeting moiety specifically binds to a DNA sequence, thereby localizing the functionality of the effector moiety to the DNA sequence or a region adjacent thereto. In some embodiments, the expression repressor comprises one targeting moiety and one effector moiety. In some embodiments, the expression repressor comprises one targeting moiety and multiple effector moieties, e.g., two, three, four, or five effector moieties, each of which may be the same as or different from another of the multiple effector moieties. In some embodiments, the expression repressor may comprise two effector moieties, where the first effector moiety comprises functionality different from the second effector moiety. For example, an expression repressor may comprise two effector moieties, where a first effector moiety comprises DNA methyltransferase functionality (e.g., comprises MQ1, G9A, or EZH2, or a functional fragment or variant thereof), and a second effector moiety comprises transcription repressor functionality (e.g., comprises KRAB, or a functional fragment or variant thereof). In some embodiments, an expression repressor comprises effector moieties whose functionalities are complementary to each other with respect to reducing expression of target multiple genes, where the functionalities together inhibit expression, and optionally, when present individually, do not inhibit expression, or only negligibly inhibit expression. In some embodiments, an expression repressor comprises multiple effector moieties, where each effector moiety complements each of the other effector moieties, and where each effector moiety reduces expression of target multiple genes.

[0393] In some embodiments, the expression repressor comprises a combination of effector moieties whose functionality synergizes with each other with respect to reducing the expression of multiple target genes. Without wishing to be bound by theory, in some embodiments, the epigenetic modifications to a genomic locus are cumulative, in that multiple transcriptionally activating epigenetic markers (e.g., multiple different types of epigenetic markers and / or overproduction of a given type) together inhibit expression more efficiently (e.g., greater and / or longer reduction in expression) than individual modifications alone. In some embodiments, the expression repressor comprises multiple effector moieties, where each effector moiety synergizes with each of the other effector moieties, e.g., each effector moiety reduces the expression of multiple target genes. In some embodiments, the expression repressor (comprising multiple effector moieties that synergize with each other) is more effective at inhibiting the expression of multiple target genes than an expression repressor comprising individual effector moieties. In some embodiments, the expression repressor comprising multiple effector moieties is at least 1.05x (i.e., 1.05-fold), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x more effective at reducing expression of multiple target genes than an expression repressor comprising individual effector moieties.

[0394] In some embodiments, the expression repressor comprises one or more targeting moieties, e.g., a Cas domain, a TAL effector domain, or a zinc finger domain. In one embodiment, when the system comprises two or more targeting moieties of the same type, e.g., two or more Cas domains or two or more zinc finger domains, the targeting moieties specifically bind to two or more different sequences. As a non-limiting example, in an expression repressor system comprising two or more zinc finger domains, the two or more zinc finger domains can be selected or modified to bind predominantly only to their target sequences (e.g., not to bind predominantly to the targets of other zinc finger domains). As another non-limiting example, in an expression repressor system comprising two or more Cas domains, the two or more Cas domains can be selected or modified to bind predominantly only to gRNAs corresponding to their target sequences (e.g., not to bind predominantly to gRNAs corresponding to the targets of other Cas domains).

[0395] In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety covalently linked, e.g., by a peptide bond. In some embodiments, the targeting moiety and the effector moiety are located on the same polypeptide chain and are connected, e.g., by one or more peptide bonds and / or linkers. In some embodiments, the expression repressor comprises a fusion molecule comprising a targeting moiety and an effector moiety linked, e.g., by a peptide bond and / or a linker. In some embodiments, the expression repressor comprises a targeting moiety located N-terminal to the effector moiety on the same polypeptide chain. In some embodiments, the expression repressor comprises a targeting moiety located C-terminal to the effector moiety on the same polypeptide chain. In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety covalently linked by a non-peptide bond. In some embodiments, the targeting moiety is conjugated to the effector moiety by a non-peptide bond. In some embodiments, the expression repressor comprises a targeting moiety and multiple effector moieties, where the targeting moiety and multiple effector moieties are covalently linked, e.g., by peptide bonds (e.g., the targeting moiety and multiple effector moieties are all connected by a series of covalent bonds, although individual moieties may not share covalent bonds with every other moiety).

[0396] In other embodiments, the expression repressor comprises a targeting moiety and an effector moiety that are not covalently linked, e.g., non-covalently bound to each other. In some embodiments, the expression repressor comprises a targeting moiety that is non-covalently bound to an effector moiety, or an effector moiety that is non-covalently bound to a targeting moiety. In some embodiments, the expression repressor comprises a targeting moiety and multiple effector moieties, wherein the targeting moiety and at least one effector moiety are not covalently linked, e.g., non-covalently bound to each other, and the targeting moiety and at least one other multiple effector moiety are covalently linked, e.g., by a peptide bond.

[0397] In some embodiments, the expression repressor comprises a first effector moiety comprising G9A and a second effector moiety comprising KRAB. In some embodiments, the expression repressor comprises a first effector moiety comprising G9A and a second effector moiety comprising EZH2. In some embodiments, the expression repressor comprises a first effector moiety comprising EZH2 and a second effector moiety comprising KRAB.

[0398] In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety, wherein the C-terminus of the effector moiety, e.g., selected from KRAB or MQ1, or a functional variant or fragment thereof, is covalently linked to the N-terminus of the targeting moiety. In some embodiments, the expression repressor comprises a targeting moiety and an effector moiety, wherein the C-terminus of the effector moiety, e.g., selected from HDAC8, MQ1, DNMT3a / 3L, KRAB, or a functional variant or fragment thereof, is covalently linked to the C-terminus of the targeting moiety. In some embodiments, the expression repressor comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the C-terminus of the first effector moiety is covalently linked to the N-terminus of the targeting moiety, and the C-terminus of the targeting moiety is covalently linked to the N-terminus of the second effector moiety. The covalent linkage may be, for example, via a linker sequence.

[0399] In some embodiments, the expression repressor comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is EZH2, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom. the first effector moiety is N-terminal to the targeting moiety; the second effector moiety is KRAB, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0400] In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is EZH2, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom. wherein the first effector moiety is N-terminal to the targeting moiety; and the second effector moiety is HDAC8, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 19, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0401] In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is G9A, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having 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 difference therefrom. the first effector moiety is N-terminal to the targeting moiety; the second effector moiety is KRAB, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0402] In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is G9A, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having 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 difference therefrom. the first effector moiety is N-terminal to the targeting moiety; the second effector moiety is EZH2, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0403] In some embodiments, the first effector moiety comprises histone methyltransferase activity and the second effector moiety comprises a different histone methyltransferase activity. In some embodiments, the first effector moiety comprises histone methyltransferase activity and the second effector moiety comprises the same histone methyltransferase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises histone deacetylase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises DNA methyltransferase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises DNA demethylase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises transcriptional repression activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises a different histone demethylase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises the same histone demethylase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises DNA methyltransferase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises DNA demethylase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises transcriptional repression activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises a different histone deacetylase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises the same histone deacetylase activity. In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises DNA demethylase activity.In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises a different transcriptional repression activity. In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises a different DNA methyltransferase activity. In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises the same DNA methyltransferase activity. In some embodiments, the first effector moiety comprises DNA demethylase activity and the second effector moiety comprises a different DNA demethylase activity. In some embodiments, the first effector moiety comprises DNA demethylase activity and the second effector moiety comprises the same DNA demethylase activity. In some embodiments, the first effector moiety comprises transcriptional repression activity and the second effector moiety comprises a different transcriptional repression activity. In some embodiments, the first effector moiety comprises transcriptional repression activity and the second effector moiety comprises the same transcriptional repression activity.

[0404] In some embodiments, the first effector moiety comprises DNMT3a / 3l, MQ1, KRAB, G9A, HDAC8, or EZH2, and the second effector moiety comprises DNMT3a / 3l, MQ1, KRAB, G9A, HDAC8, or EZH2.

[0405] Site-specific blocking agents In some embodiments, the site-specific disrupting agent comprises a targeting moiety. In some embodiments, the targeting moiety specifically binds to a DNA sequence, e.g., an anchor sequence, thereby modulating, e.g., disrupting, a genomic complex (e.g., ASMC) comprising the DNA sequence. In some embodiments, the site-specific disrupting agent comprises a targeting moiety and an effector moiety. In some embodiments, the targeting moiety specifically binds to a DNA sequence, thereby localizing the functionality of the effector moiety to the DNA sequence, thereby modulating, e.g., disrupting, a genomic complex (e.g., ASMC) comprising the DNA sequence. In some embodiments, the site-specific disrupting agent comprises one targeting moiety and one effector moiety. In some embodiments, the site-specific disrupting agent comprises one targeting moiety and two or more effector moieties, e.g., two, three, four, or five effector moieties, each of which may be the same as or different from the other of the two or more effector moieties. In some embodiments, a site-specific disruption agent may comprise two effector moieties, wherein a first effector moiety comprises a different functionality than a second effector moiety. For example, a site-specific disruption agent may comprise two effector moieties, wherein a first effector moiety comprises a DNA methyltransferase functionality (e.g., comprises G9A or EZH2, or a functional fragment or variant thereof), and a second effector moiety comprises a transcriptional repressor functionality (e.g., comprises KRAB, or a functional fragment or variant thereof). In some embodiments, a site-specific disruption agent comprises effector moieties whose functionalities are complementary to each other with respect to reducing expression of a target multigene, where the functionalities together inhibit expression and, optionally, do not inhibit, or negligibly inhibit, expression when present individually. In some embodiments, a site-specific disruption agent comprises multiple effector moieties, where each effector moiety complements each of the other effector moieties, and where each effector moiety reduces expression of a target multigene.

[0406] In some embodiments, site-specific disruption agents comprise a combination of effector moieties whose functionality synergizes with each other with respect to reducing expression of multiple target genes. While not wishing to be bound by theory, in some embodiments, epigenetic modifications to a genomic locus are cumulative, in that multiple transcriptionally activated epigenetic markers (e.g., multiple different types of epigenetic markers and / or overproduction of a given type) together inhibit expression more efficiently (e.g., result in greater and / or longer reduction in expression) than individual modifications alone. In some embodiments, site-specific disruption agents comprise multiple effector moieties, where each effector moiety synergizes with each of the other effector moieties, e.g., each effector moiety reduces expression of multiple target genes. In some embodiments, site-specific disruption agents (comprising multiple effector moieties that synergize with each other) are more effective at inhibiting expression of multiple target genes than site-specific disruption agents comprising individual effector moieties. In some embodiments, the site-specific blocking agent comprising multiple effector moieties is at least 1.05x (i.e., 1.05-fold), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x more effective at reducing expression of multiple target genes than a site-specific blocking agent comprising individual effector moieties.

[0407] In some embodiments, the site-specific blocking agent comprises one or more targeting moieties, such as a Cas domain, a TAL effector domain, or a Zn finger domain. In one embodiment, when the system comprises two or more targeting moieties of the same type, e.g., two or more Cas domains, the targeting moieties specifically bind to two or more different sequences. For example, in a site-specific blocking agent system comprising two or more Cas domains, the two or more Cas domains can be selected or engineered to bind significantly only to gRNAs corresponding to their target sequences (e.g., not significantly to gRNAs corresponding to targets of other Cas domains).

[0408] In some embodiments, the site-specific blocking agent comprises a targeting moiety and an effector moiety covalently linked, e.g., by a peptide bond. In some embodiments, the targeting moiety and the effector moiety are located on the same polypeptide chain and are connected, e.g., by one or more peptide bonds and / or linkers. In some embodiments, the site-specific blocking agent comprises a fusion molecule, e.g., comprising a targeting moiety and an effector moiety linked by a peptide bond and / or a linker. In some embodiments, the site-specific blocking agent comprises a targeting moiety located N-terminally of the effector moiety on the same polypeptide chain. In some embodiments, the site-specific blocking agent comprises a targeting moiety located C-terminally of the effector moiety on the same polypeptide chain. In some embodiments, the site-specific blocking agent comprises a targeting moiety and an effector moiety covalently linked by a non-peptide bond. In some embodiments, the targeting moiety is conjugated to the effector moiety by a non-peptide bond. In some embodiments, the site-specific blocking agent comprises a targeting moiety and multiple effector moieties, where the targeting moiety and multiple effector moieties are covalently linked, e.g., by peptide bonds (e.g., the targeting moiety and multiple effector moieties are all connected by a series of covalent bonds, although individual moieties may not share covalent bonds with every other moiety).

[0409] In other embodiments, the site-specific blocking agent comprises a targeting moiety and an effector moiety that are not covalently linked, e.g., non-covalently bound to each other. In some embodiments, the site-specific blocking agent comprises a targeting moiety that is non-covalently bound to an effector moiety, or an effector moiety that is non-covalently bound to a targeting moiety. In some embodiments, the site-specific blocking agent comprises a targeting moiety and multiple effector moieties, wherein the targeting moiety and at least one effector moiety are not covalently linked, e.g., non-covalently bound to each other, and wherein the targeting moiety and at least one other effector moiety are covalently linked, e.g., by a peptide bond.

[0410] In some embodiments, the site-specific blocking agent comprises a first effector moiety having G9A and a second effector moiety having KRAB. In some embodiments, the site-specific blocking agent comprises a first effector moiety having G9A and a second effector moiety having EZH2. In some embodiments, the site-specific blocking agent comprises a first effector moiety having EZH2 and a second effector moiety having KRAB.

[0411] In some embodiments, the site-specific blocking agent comprises a targeting moiety and an effector moiety, wherein the C-terminus of the effector moiety, e.g., selected from EZH2 or G9A, or a functional variant or fragment thereof, is covalently linked to the N-terminus of the targeting moiety. In some embodiments, the site-specific blocking agent comprises a targeting moiety and an effector moiety, wherein the N-terminus of the effector moiety, e.g., selected from HDAC8, MQ1, DNMT3a / 3L, KRAB, or a functional variant or fragment thereof, is covalently linked to the C-terminus of the targeting moiety. In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the C-terminus of the first effector moiety, e.g., an effector moiety selected from EZH2, G9A, or a functional variant or fragment thereof, and the N-terminus of the targeting moiety are covalently linked, and the C-terminus of the targeting moiety and the N-terminus of the second effector moiety, e.g., an effector moiety selected from HDAC8, MQ1, DNMT3a / 3L, KRAB, or a functional variant or fragment thereof, are covalently linked. The covalent linkage may be, for example, via a linker sequence.

[0412] In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is EZH2, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom. the first effector moiety is N-terminal to the targeting moiety; the second effector moiety is KRAB, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0413] In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is EZH2, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom. wherein the first effector moiety is N-terminal to the targeting moiety; and the second effector moiety is HDAC8, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 19, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0414] In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is G9A, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having 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 difference therefrom. the first effector moiety is N-terminal to the targeting moiety; the second effector moiety is KRAB, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0415] In some embodiments, the site-specific blocking agent comprises a targeting moiety, a first effector moiety, and a second effector moiety, wherein the first effector moiety is G9A, or a functional variant or fragment thereof, e.g., the first effector moiety comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having 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 difference therefrom. the first effector moiety is N-terminal to the targeting moiety; the second effector moiety is EZH2, or a functional variant or fragment thereof, e.g., the second effector moiety comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having 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 difference therefrom, and the second effector moiety is C-terminal to the targeting moiety.

[0416] In some embodiments, the first effector moiety comprises histone methyltransferase activity and the second effector moiety comprises a different histone methyltransferase activity. In some embodiments, the first effector moiety comprises histone methyltransferase activity and the second effector moiety comprises the same histone methyltransferase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises histone deacetylase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises DNA methyltransferase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises DNA demethylase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises transcriptional repression activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises a different histone demethylase activity. In some embodiments, the first effector moiety comprises histone demethylase activity and the second effector moiety comprises the same histone demethylase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises DNA methyltransferase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises DNA demethylase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises transcriptional repression activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises a different histone deacetylase activity. In some embodiments, the first effector moiety comprises histone deacetylase activity and the second effector moiety comprises the same histone deacetylase activity. In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises DNA demethylase activity.In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises transcriptional repression activity. In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises a different DNA methyltransferase activity. In some embodiments, the first effector moiety comprises DNA methyltransferase activity and the second effector moiety comprises the same DNA methyltransferase activity. In some embodiments, the first effector moiety comprises DNA demethylase activity and the second effector moiety comprises a different DNA demethylase activity. In some embodiments, the first effector moiety comprises DNA demethylase activity and the second effector moiety comprises the same DNA demethylase activity. In some embodiments, the first effector moiety comprises transcriptional repression activity and the second effector moiety comprises a different transcriptional repression activity. In some embodiments, the first effector moiety comprises a transcriptional repression activity and the second effector moiety comprises the same transcriptional repression activity.

[0417] In some embodiments, the first effector moiety comprises DNMT3a / 3l, MQ1, KRAB, G9A, HDAC8, or EZH2, and the second effector moiety comprises DNMT3a / 3l, MQ1, KRAB, G9A, HDAC8, or EZH2.

[0418] Linker The expression repressor and / or site-specific disrupting agent may comprise one or more linkers. A linker may connect a targeting moiety to an effector moiety, an effector moiety to another effector moiety, or a targeting moiety to another targeting moiety. A linker may be a chemical bond, for example, one or more covalent or non-covalent bonds. In some embodiments, the linker is a covalent bond. In some embodiments, the linker is a non-covalent bond. In some embodiments, the linker is a peptide linker. Such linkers can be 2 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 2 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 2 to 20, 5 to 20, 10 to 20, 15 to 20, 2 to 15, 5 to 15, 10 to 15, 2 to 10, 5 to 10, or 2 to 5 amino acids in length, or 2, 5, 10, 15, 20, 25, or 30 amino acids or more (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length). In some embodiments, a linker can be used to space a first moiety and a second moiety, for example, between a targeting moiety and an effector moiety. In some embodiments, for example, a linker can be placed between the targeting moiety and the effector moiety to provide molecular flexibility, for example, in the secondary and tertiary structure. In some embodiments, the site-specific blocking agent can include a first effector moiety linked to the targeting moiety via a first linker and a second effector moiety linked to the targeting moiety via a second linker. In some embodiments, the first linker has a sequence identical to that of the second linker. In some embodiments, the first linker has a sequence that is not identical to that of the second linker. In some embodiments, the first effector moiety is located at the N-terminus of the targeting moiety. In some embodiments, the first effector moiety is located at the C-terminus of the targeting moiety. In some embodiments, the C-terminus of the first effector moiety is linked to the N-terminus of the targeting moiety via the first linker, and the N-terminus of the second effector moiety is linked to the C-terminus of the targeting moiety via the second linker.

[0419] The linker may comprise a flexible, rigid, and / or cleavable linker as described herein. In some embodiments, to provide flexibility, the linker comprises at least one glycine, alanine, and serine amino acid. In some embodiments, the linker is a hydrophobic linker comprising, for example, a negatively charged sulfonic acid group, a polyethylene glycol (PEG) group, or a pyrophosphate diester group. In some embodiments, the linker is cleavable to selectively release a moiety (e.g., a polypeptide) from the modulating agent, yet is sufficiently stable to prevent premature cleavage.

[0420] In some embodiments, one or more portions of an expression repressor described herein are linked with one or more linkers. In some embodiments, one or more portions of a site-specific blocking agent described herein are linked with one or more linkers.

[0421] As known to those skilled in the art, commonly used flexible linkers have sequences composed primarily of stretches of Gly and Ser residues ("GS" linkers). Flexible linkers may be useful for connecting domains / moieties that require some degree of movement or interaction and may include small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Furthermore, the inclusion of Ser or Thr may maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the moiety / domain.

[0422] Rigid linkers are useful for maintaining a fixed distance between domains / moieties and preserving their independent functions. Rigid linkers may also be useful when spatial separation of domains is critical to preserve the stability or biological activity of one or more components in the fusion. Rigid linkers may be α-helical structures or Pro-rich sequences (XP). n (wherein X refers to any amino acid, preferably Ala, Lys, or Glu).

[0423] A cleavable linker may release a free functional domain / moiety in vivo. In some embodiments, the linker may be cleaved under specific conditions, such as the presence of a reducing agent or a protease. In vivo cleavable linkers may utilize the reversibility of disulfide bonds. An example is a thrombin-sensitive sequence (e.g., PRS) between two Cys residues. In vitro thrombin treatment of CPRSC (SEQ ID NO: 243) results in cleavage of the thrombin-sensitive sequence, while leaving the reversible disulfide bond 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 during fusion may also be performed by a protease expressed in specific cells or tissues, under specific conditions, in vivo, or restricted to a specific cellular compartment. The specificity of many proteases results in slower cleavage of the linker within a restricted compartment.

[0424] Examples of molecules suitable for use in the linkers described herein include hydrophobic linkers, such as negatively charged sulfonic acid groups; lipids, such as poly(--CH2--) hydrocarbon chains, e.g., polyethylene glycol (PEG) groups, unsaturated variants thereof, hydroxylated variants thereof, amidated or other N-containing variants thereof; non-carbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecules capable of covalently linking two or more components of a site-specific blocking agent. Non-covalent linkers are also included, such as hydrophobic lipid globules in which polypeptides are linked through hydrophobic regions of polypeptides or hydrophobic extensions of polypeptides, such as a series of residues rich in leucine, isoleucine, valine, or perhaps even alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues. Components of a site-specific blocking agent may also be linked using charge-based chemistry, such that a positively charged component of a site-specific blocking agent is linked to another negatively charged component.

[0425] nucleic acid In one aspect, the present disclosure provides nucleic acid sequences encoding the expression repressors and / or site-specific disruptors, systems, targeting moieties, and / or effector moieties described herein. Those skilled in the art will recognize that RNA nucleic acid sequences are typically identical to the corresponding DNA sequences, except for thymine (T) substitutions with uracil (U). Where a nucleotide sequence is represented by a DNA sequence (e.g., containing A, T, G, C), it will be understood that the present disclosure also provides the corresponding RNA sequence (e.g., containing A, U, G, C) in which "U" is substituted with "T." Conventional notation is used herein to describe polynucleotide sequences. The left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction.

[0426] Those skilled in the art will understand that the degeneracy of the genetic code may produce numerous nucleotide sequences encoding site-specific disrupting agents comprising the DNA targeting moieties and / or effector moieties described herein, some of which have similarity, e.g., 90%, 95%, 96%, 97%, 98%, or 99% identity, to the nucleic acid sequences disclosed herein. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in a nucleic acid of the present disclosure where arginine is specified by a codon, the codon can be altered to any of the corresponding codons listed above without altering the encoded polypeptide.

[0427] In some embodiments, the nucleic acid sequence encoding the expression repressor, including the targeting moiety and / or one or more effector moieties, can be part or all of a codon-optimized coding region optimized according to codon usage in a mammal, e.g., a human. In some embodiments, the nucleic acid sequence encoding the targeting moiety and / or one or more effector moieties is codon optimized to increase protein expression and / or to increase the duration of protein expression. In some embodiments, the protein produced by the codon-optimized nucleic acid sequence is at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% higher than the level of the protein when encoded by a nucleic acid sequence that is not codon-optimized.

[0428] In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is monocistronic or polycistronic. In some embodiments, the nucleic acid is monocistronic. In certain embodiments, the nucleic acid is polycistronic (e.g., bicistronic, tricistronic, tetracistronic, etc.). In certain embodiments, the nucleic acid is bicistronic. In certain embodiments, the nucleic acid is tricistronic. In certain embodiments, the nucleic acid is tetracistronic.

[0429] Effector part In some embodiments, the systems described herein include, or the methods described herein include the use of, a polypeptide comprising one or more (e.g., one) DNA-targeting moieties and one or more effector moieties, e.g., where the effector moiety is or includes MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof. In some embodiments, the MQ1 is Spiroplasma monobiae MQ1, e.g., MQ1 from strain ATCC33825 and / or corresponding to Uniprot ID P15840. In some embodiments, the MQ1 effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 10, or a sequence having 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 difference therefrom. MQ1 [ka]

[0430] In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the effector domain described herein comprises SEQ ID NO: 11 or 12, or a sequence having 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 difference therefrom. MQ1

[0431] [ka] MQ1 [ka]

[0432] In some embodiments, the MQ1 used in the expression repressors described herein is a mutant relative to wild-type MQ1 (e.g., SEQ ID NO: 11 or SEQ ID NO: 12), e.g., comprising one or more mutations. In some embodiments, the MQ1 mutant comprises one or more amino acid substitutions, deletions, or insertions relative to wild-type MQ1. In some embodiments, the MQ1 mutant comprises a K297P substitution. In some embodiments, the MQ1 mutant comprises an N299C substitution. In some embodiments, the MQ1 mutant comprises an E301Y substitution. In some embodiments, the MQ1 mutant comprises a Q147L substitution (e.g., having reduced DNA methyltransferase activity relative to wild-type MQ1). In some embodiments, the MQ1 mutant comprises K297P, N299C, and E301Y substitutions (e.g., having reduced DNA binding affinity relative to wild-type MQ1). In some embodiments, the MQ1 variant comprises Q147L, K297P, N299C, and E301Y substitutions (eg, has reduced methyltransferase activity and DNA binding affinity relative to wild-type MQ1).

[0433] In some embodiments, the expression repressor comprises one or more linkers described herein, e.g., connecting a portion / domain to another portion / domain. In some embodiments, the expression repressor comprises a targeting portion that is or comprises a CRISPR / Cas molecule, e.g., including a CRISPR / Cas protein, e.g., a dCas9 protein. In some embodiments, the expression repressor is a fusion protein comprising an effector portion that is or comprises MQ1 and a DNA-targeting portion that is or comprises a CRISPR / Cas molecule, e.g., a CRISPR / Cas protein (e.g., a dCas9 protein), e.g., dCas9m4. In some embodiments, the expression repressor comprises additional portions described herein. In some embodiments, the expression repressor reduces expression of a target gene or target genes (e.g., a target gene or target genes described herein). In some embodiments, the expression repressors may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or genomic regulatory element (e.g., a transcriptional control element) described herein. In some embodiments, the system comprises two or more expression repressors.

[0434] In some embodiments, the systems described herein include, or the methods described herein include the use of, expression repressors or polypeptides comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises the Krueppel association box (KRAB) domain of zinc finger protein 10 (e.g., according to NP_056209.2), or the protein encoded by NM_015394.5, or a functional variant or fragment thereof. In some embodiments, the KRAB is a synthetic KRAB construct. In some embodiments, the KRAB used in the expression repressors described herein is a variant compared to wild-type KRAB (e.g., according to the protein encoded by NP_056209.2, or NM_015394.5), e.g., comprising one or more mutations. In some embodiments, the KRAB variant comprises one or more amino acid substitutions, deletions, or insertions compared to wild-type KRAB. In some embodiments, the KRAB mutant comprises a L37P substitution. In some embodiments, the KRAB comprises the amino acid sequence of SEQ ID NO:13. KRAB DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 13)

[0435] In some embodiments, the KRAB effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 14, or a sequence having 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 difference therefrom. KRAB [ka]

[0436] In some embodiments, the KRAB used in the polypeptides or expression repressors described herein is a variant containing, for example, one or more mutations relative to the KRAB sequence of SEQ ID NO: 13. In some embodiments, the KRAB variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 13.

[0437] In some embodiments, the polypeptide or expression repressor is a fusion protein comprising an effector moiety that is or comprises KRAB and a targeting moiety, e.g., a zinc finger domain or Crisper / Cas protein. In some embodiments, the polypeptide or expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or expression repressor reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or expression repressor may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) described herein.

[0438] In some embodiments, the systems described herein include, or the methods described herein include the use of, an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises a DNMT3a / 3L complex, or a functional variant or fragment thereof. In some embodiments, the DNMT3a / 3L complex is a fusion construct. In some embodiments, the DNMT3a / 3L complex comprises DNMT3A, e.g., human DNMT3A (e.g., according to the protein encoded by NP_072046.2 or NM_022552.4) or a protein encoded by NM_022552.4 or a functional variant or fragment thereof, e.g., aa 679-912 of human DNMT3A (e.g., according to the protein encoded by NP_072046.2 or NM_022552.4). In some embodiments, the DNMT3a / 3L complex comprises human DNMT3L or a functional fragment or variant thereof (e.g., a protein encoded by NP_787063.1 or NM_175867.3, or a functional variant or fragment thereof, e.g., according to aa 274-386 of human DNMT3L according to a protein encoded by NP_787063.1 or NM_175867.3). In some embodiments, the DNMT3a / 3L comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the effector moiety described herein comprises SEQ ID NO: 15, or a sequence having 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 difference therefrom. DNMT3A / 3l(h) [ka]

[0439] In some embodiments, DNMT3a / 3L is encoded by the nucleotide sequence of SEQ ID NO: 16. In some embodiments, a nucleic acid described herein comprises the sequence of SEQ ID NO: 16, or a sequence having 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 difference therefrom. DNMT3A / 3l(h) [ka]

[0440] In some embodiments, the systems described herein include, or the methods described herein include, the use of an expression repressor or polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises EZH2 (e.g., according to the protein encoded by NP-004447.2 or NP_001190176.1 2, or NM_004456.5 or NM_001203247.2) or a functional variant or fragment thereof. In some embodiments, the EZH2 used in the expression repressors described herein is a variant comprising, e.g., one or more mutations, relative to EZH2, e.g., EZH2 according to the protein encoded by NP-004447.2 or NP_001190176.1 2, or NM_004456.5 or NM_001203247.2. In some embodiments, the EZH2 mutant comprises one or more amino acid substitutions, deletions, or insertions relative to wild-type EZH2. In some embodiments, the EZH2 comprises the amino acid sequence of SEQ ID NO: 17. EZH2 [ka]

[0441] In some embodiments, the EZH2 effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 18, or a sequence having 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 difference therefrom. EZH2 [ka] [ka]

[0442] In some embodiments, the EZH2 used in the polypeptides or expression repressors described herein is a variant containing, for example, one or more mutations relative to the EZH2 sequence of SEQ ID NO: 17. In some embodiments, the EZH2 variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 17.

[0443] In some embodiments, the polypeptide or expression repressor is a fusion protein comprising an effector moiety that is or comprises EZH2 and a targeting moiety. In some embodiments, the polypeptide or expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or expression repressor reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or expression repressor may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) described herein.

[0444] In some embodiments, the systems described herein include, or the methods described herein include the use of, an expression repressor or polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises HDAC8 (e.g., according to the protein encoded by NP_001159890 or NP_060956.1, or NM_001166418 or NM_018486.3) or a functional variant or fragment thereof. In some embodiments, the HDAC8 comprises the amino acid sequence of SEQ ID NO: 19. HDAC8 [ka]

[0445] In some embodiments, the HDAC8 effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 66. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 66, or a sequence having 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 difference therefrom. HDAC8 [ka]

[0446] In some embodiments, the HDAC8 used in the polypeptides or expression repressors described herein is a variant containing, for example, one or more mutations relative to the HDAC8 sequence of SEQ ID NO: 19. In some embodiments, the HDAC8 variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 19.

[0447] In some embodiments, the polypeptide or expression repressor is a fusion protein comprising an effector moiety that is or comprises HDAC8 and a targeting moiety. In some embodiments, the polypeptide or expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or expression repressor reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or expression repressor may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) described herein.

[0448] In some embodiments, the systems described herein include, or the methods described herein include the use of, an expression repressor or polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises G9A (e.g., according to a protein encoded by NP_001350618.1 or NM_001363689.1) or a functional variant or fragment thereof, e.g., aa 967-1250 comprising G9A (e.g., according to a protein encoded by NP_001350618.1 or NM_001363689.1). In some embodiments, G9A comprises the amino acid sequence of SEQ ID NO: 67. G9A [ka]

[0449] In some embodiments, the G9A effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 68. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 68, or a sequence having 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 difference therefrom. G9A [ka]

[0450] In some embodiments, the G9A used in the polypeptides or expression repressors described herein is a variant containing, for example, one or more mutations relative to the G9A sequence of SEQ ID NO: 67. In some embodiments, the G9A variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 67.

[0451] In some embodiments, the polypeptide or expression repressor is a fusion protein comprising an effector moiety that is or comprises G9A and a targeting moiety. In some embodiments, the polypeptide or expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or expression repressor reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or expression repressor may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) described herein.

[0452] In some embodiments, the nucleic acid sequence encoding the site-specific disrupting agent comprising a targeting moiety and / or one or more effector moieties may be part or all of a codon-optimized coding region optimized according to codon usage in a mammal, e.g., a human. In some embodiments, the nucleic acid sequence encoding the targeting moiety and / or one or more effector moieties is codon optimized to increase protein expression and / or to increase the duration of protein expression. In some embodiments, the protein produced by the codon-optimized nucleic acid sequence is at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% higher than the level of the protein when encoded by a nucleic acid sequence that is not codon-optimized.

[0453] In some embodiments, the systems described herein include, or the methods described herein include, the use of, a polypeptide comprising one or more (e.g., one) DNA-targeting moieties and one or more effector moieties, e.g., wherein the effector moiety is or comprises MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof. In some embodiments, the MQ1 is Spiroplasma monobiae MQ1, e.g., MQ1 from strain ATCC33825 and / or corresponding to Uniprot ID P15840. In some embodiments, the MQ1 effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 10, or a sequence having 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 difference therefrom.

[0454] In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, MQ1 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the effector domain described herein comprises SEQ ID NO: 11 or 12, or a sequence having 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 difference therefrom.

[0455] In some embodiments, the MQ1 used in the site-specific disrupting agents described herein is a mutant relative to wild-type MQ1 (e.g., SEQ ID NO: 11 or SEQ ID NO: 12), e.g., comprising one or more mutations. In some embodiments, the MQ1 mutant comprises one or more amino acid substitutions, deletions, or insertions relative to wild-type MQ1. In some embodiments, the MQ1 mutant comprises a K297P substitution. In some embodiments, the MQ1 mutant comprises an N299C substitution. In some embodiments, the MQ1 mutant comprises an E301Y substitution. In some embodiments, the MQ1 mutant comprises a Q147L substitution (e.g., having reduced DNA methyltransferase activity relative to wild-type MQ1). In some embodiments, the MQ1 mutant comprises K297P, N299C, and E301Y substitutions (e.g., having reduced DNA binding affinity relative to wild-type MQ1). In some embodiments, the MQ1 variant comprises Q147L, K297P, N299C, and E301Y substitutions (eg, has reduced methyltransferase activity and DNA binding affinity relative to wild-type MQ1).

[0456] In some embodiments, the site-specific disrupting agent comprises one or more linkers described herein, e.g., connecting a moiety / domain to another moiety / domain. In some embodiments, the site-specific disrupting agent comprises a targeting moiety that is or comprises a CRISPR / Cas molecule, e.g., a CRISPR / Cas protein, e.g., a dCas9 protein. In some embodiments, the site-specific disrupting agent is a fusion protein comprising an effector moiety that is or comprises MQ1 and a DNA-targeting moiety that is or comprises a CRISPR / Cas protein, e.g., a dCas9 protein; e.g., a CRISPR / Cas molecule, including dCas9m4. In some embodiments, the site-specific disrupting agent comprises an additional moiety described herein. In some embodiments, the site-specific disrupting agent reduces expression of a target gene or target multigene (e.g., a target gene or target multigene described herein). In some embodiments, the site-specific disrupting agent can be used in methods of regulating, e.g., reducing, gene expression, treating a disease state, or epigenetically modifying a target gene or a transcriptional control element described herein. In some embodiments, the system comprises two or more site-specific blocking agents.

[0457] In some embodiments, the systems described herein include, or the methods described herein include the use of, site-specific disrupting agents or polypeptides comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises the Krueppel association box (KRAB) domain of zinc finger protein 10 (e.g., according to NP_056209.2), or the protein encoded by NM_015394.5, or a functional variant or fragment thereof. In some embodiments, the KRAB is a synthetic KRAB construct. In some embodiments, the KRAB used in the site-specific disrupting agents described herein is a variant compared to wild-type KRAB (e.g., according to the protein encoded by NP_056209.2, or NM_015394.5), e.g., comprising one or more mutations. In some embodiments, the KRAB variant comprises one or more amino acid substitutions, deletions, or insertions compared to wild-type KRAB. In some embodiments, the KRAB mutant comprises a L37P substitution. In some embodiments, the KRAB comprises the amino acid sequence of SEQ ID NO:13.

[0458] In some embodiments, the KRAB effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 14, or a sequence having 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 difference therefrom.

[0459] In some embodiments, the KRAB used in the polypeptides or site-specific disrupting agents described herein is a variant containing, for example, one or more mutations relative to the KRAB sequence of SEQ ID NO: 13. In some embodiments, the KRAB variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 13.

[0460] In some embodiments, the polypeptide or site-specific disrupting agent is a fusion protein, e.g., a CRISPR / Cas protein, comprising an effector moiety that is or comprises KRAB and a targeting moiety. In some embodiments, the polypeptide or site-specific disrupting agent comprises an additional moiety as described herein. In some embodiments, the polypeptide or site-specific disrupting agent reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or site-specific disrupting agent may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) as described herein.

[0461] In some embodiments, the systems described herein include, or the methods described herein include the use of, site-specific blocking agents or polypeptides comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises a DNMT3a / 3L complex, or a functional variant or fragment thereof. In some embodiments, the DNMT3a / 3L complex is a fusion construct. In some embodiments, the DNMT3a / 3L complex comprises DNMT3A, e.g., human DNMT3A (e.g., according to the protein encoded by NP_072046.2 or NM_022552.4), or a protein encoded by NM_022552.4 or a functional variant or fragment thereof, e.g., aa 679-912 of human DNMT3A (e.g., according to the protein encoded by NP_072046.2 or NM_022552.4). In some embodiments, the DNMT3a / 3L complex comprises human DNMT3L or a functional fragment or variant thereof (e.g., a protein encoded by NP_787063.1 or NM_175867.3, or a functional variant or fragment thereof, e.g., according to aa 274-386 of human DNMT3L according to a protein encoded by NP_787063.1 or NM_175867.3). In some embodiments, the DNMT3a / 3L comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the effector moiety described herein comprises SEQ ID NO: 15, or a sequence having 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 difference therefrom.

[0462] In some embodiments, DNMT3a / 3L is encoded by the nucleotide sequence of SEQ ID NO: 16. In some embodiments, a nucleic acid described herein comprises the sequence of SEQ ID NO: 16, or a sequence having 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 difference therefrom.

[0463] In some embodiments, the systems described herein include, or the methods described herein include, the use of a site-specific disrupting agent or polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises EZH2 (e.g., according to the protein encoded by NP-004447.2 or NP_001190176.1 2, or NM_004456.5 or NM_001203247.2) or a functional variant or fragment thereof. In some embodiments, the MQ1 used in the site-specific disrupting agents described herein is a variant comprising, for example, one or more mutations, relative to EZH2, e.g., EZH2 according to the protein encoded by NP-004447.2 or NP_001190176.1 2, or NM_004456.5 or NM_001203247.2. In some embodiments, the EZH2 mutant comprises one or more amino acid substitutions, deletions, or insertions relative to wild-type EZH2. In some embodiments, the EZH2 comprises the amino acid sequence of SEQ ID NO: 17.

[0464] In some embodiments, the EZH2 effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 18, or a sequence having 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 difference therefrom.

[0465] In some embodiments, the EZH2 used in the polypeptides or site-specific disrupting agents described herein is a variant that contains, for example, one or more mutations relative to the EZH2 sequence of SEQ ID NO: 17. In some embodiments, the EZH2 variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 17.

[0466] In some embodiments, the polypeptide or site-specific disrupting agent is EZH2 or is a fusion protein comprising an effector moiety comprising EZH2 and a targeting moiety. In some embodiments, the polypeptide or site-specific disrupting agent comprises an additional moiety described herein. In some embodiments, the polypeptide or site-specific disrupting agent reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or site-specific disrupting agent may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) described herein.

[0467] In some embodiments, the systems described herein include, or the methods described herein include the use of, site-specific blocking agents or polypeptides comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises HDAC8 (e.g., according to the protein encoded by NP_001159890 or NP_060956.1, or NM_001166418 or NM_018486.3) or a functional variant or fragment thereof. In some embodiments, HDAC8 comprises the amino acid sequence of SEQ ID NO: 19.

[0468] In some embodiments, the HDAC8 effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 66. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 66, or a sequence having 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 difference therefrom.

[0469] In some embodiments, the HDAC8 used in the polypeptides or site-specific disrupting agents described herein is a variant containing, for example, one or more mutations relative to the HDAC8 sequence of SEQ ID NO: 19. In some embodiments, the HDAC8 variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 19.

[0470] In some embodiments, the polypeptide or site-specific disrupting agent is a fusion protein comprising an effector moiety that is or comprises HDAC8 and a targeting moiety. In some embodiments, the polypeptide or site-specific disrupting agent comprises an additional moiety described herein. In some embodiments, the polypeptide or site-specific disrupting agent reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or site-specific disrupting agent may be used in methods of regulating, e.g., reducing, gene expression, treating a disease state, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) described herein.

[0471] In some embodiments, the systems described herein include, or the methods described herein include the use of, a site-specific blocking agent or a polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties, wherein the effector moiety is or comprises G9A (e.g., according to NP_001350618.1), or a protein encoded by NM_001363689.1, or a functional variant or fragment thereof, e.g., aa 967-1250, or comprises G9A (e.g., according to NP_001350618.1), or a protein encoded by NM_001363689.1. In some embodiments, G9A comprises the amino acid sequence of SEQ ID NO: 67.

[0472] In some embodiments, the G9A effector moiety is encoded by the nucleotide sequence of SEQ ID NO: 68. In some embodiments, the nucleotide sequence described herein comprises the sequence of SEQ ID NO: 68, or a sequence having 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 difference therefrom.

[0473] In some embodiments, the G9A used in the polypeptides or site-specific disrupting agents described herein is a variant containing, for example, one or more mutations relative to the G9A sequence of SEQ ID NO: 67. In some embodiments, the G9A variant contains one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 67.

[0474] In some embodiments, the polypeptide or site-specific disrupting agent is a fusion protein comprising an effector moiety that is or comprises G9A and a targeting moiety. In some embodiments, the polypeptide or site-specific disrupting agent comprises an additional moiety described herein. In some embodiments, the polypeptide or site-specific disrupting agent reduces expression of a target gene or multiple target genes. In some embodiments, the polypeptide or site-specific disrupting agent may be used in methods of regulating, e.g., reducing, gene expression, treating a disease condition, or epigenetically modifying a target gene or multiple target genes, e.g., a genomic regulatory element (e.g., a transcriptional control element) described herein.

[0475] system The systems of the present disclosure may include two or more expression repressors. In some embodiments, the expression repressor system includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more (and optionally, no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) expression repressors. In some embodiments, the system targets two or more different sequences (e.g., a first and a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and / or additional DNA sequences, and optionally no more than 20th, 19th, 18th, 17th, 16th, 15th, 14th, 13th, 12th, 11th, 10th, 9th, 8th, sixth, fifth, fourth, third, or second sequence). In some embodiments, the system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors does not detectably bind, e.g., does not bind, to another member of the plurality of expression repressors, hi some embodiments, the system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor does not detectably bind, e.g., does not bind, to the second expression repressor.

[0476] In some embodiments, the disclosed systems include two or more expression repressors, wherein the expression repressors are present together in a composition, pharmaceutical composition, or mixture. In some embodiments, the disclosed systems include two or more expression repressors, wherein one or more expression repressors are not mixed with at least one other expression repressor. In some embodiments, the systems 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, and the system achieves reduced expression of multiple genes through the non-overlapping presence of the first and second expression repressors. In some embodiments, the first expression repressor and the second expression repressor may act simultaneously or sequentially.

[0477] In some embodiments, each expression repressor of the system comprises a different targeting moiety (e.g., each of the first, second, third, or additional expression repressors comprises a different targeting moiety from each other). For example, the system may comprise a first expression repressor and a second expression repressor, where the first expression repressor comprises a first targeting moiety (e.g., a Zn finger domain, a Cas9 domain, or a TAL effector domain) and the second expression repressor comprises a second targeting moiety (e.g., a Zn finger domain, a Cas9 domain, or a TAL effector domain) that is different from the first targeting moiety. In some embodiments, different means comprising different types of targeting moieties, e.g., the first targeting moiety comprises a Cas9 domain and the second DNA targeting moiety comprises a Zn finger domain. In other embodiments, different means including different variants of the same type of targeting moiety, e.g., a first targeting moiety includes a first Cas9 domain (e.g., from a first species) and a second targeting moiety includes a second Cas9 domain (e.g., from a second species).

[0478] In one aspect, the system of the present disclosure may include one or more expression repressors and one or more site-specific blocking agents. In some embodiments, the system comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more (and optionally, up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) expression repressors. In some embodiments, the system comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more (and optionally, up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) site-specific blocking agents. In some embodiments, the system comprises two or more different sequences (e.g., a first and a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and / or additional D In some embodiments, the system targets one or more expression repressors and one or more site-specific disrupting agents, wherein each of the one or more expression repressors and each of the one or more site-specific disrupting agents does not detectably bind, e.g., does not bind, to another expression repressor and / or site-specific disrupting agent. In some embodiments, the system includes an expression repressor and a site-specific disrupting agent, wherein each of the expression repressors and site-specific disrupting agents does not detectably bind, e.g., does not bind, to each other.

[0479] In some embodiments, the system comprises one or more expression repressors and one or more site-specific disrupting agents, wherein each of the one or more expression repressors and each of the one or more site-specific disrupting agents independently binds to a different target. In some embodiments, the system comprises an expression repressor and a site-specific disrupting agent, wherein each of the expression repressors and the site-specific disrupting agents independently binds to a different target.

[0480] In some embodiments, the disclosed systems include one or more expression repressors and one or more site-specific disrupting agents, wherein the expression repressors and site-specific disrupting agents are present together in a composition, pharmaceutical composition, or mixture. In some embodiments, the disclosed systems include one or more expression repressors and one or more site-specific disrupting agents, wherein the one or more expression repressors and one or more site-specific disrupting agents are not mixed with at least one other expression repressor and / or site-specific disrupting agent. In some embodiments, the systems may include an expression repressor and a site-specific disrupting agent, wherein the presence of the expression repressor in the nucleus of a cell does not overlap with the presence of the site-specific disrupting agent in the nucleus of the same cell, and the system achieves reduced expression of multiple genes through the non-overlapping presence of the expression repressor and the site-specific disrupting agent. In some embodiments, the expression repressor and the site-specific disrupting agent may act simultaneously or sequentially.

[0481] In some embodiments, the expression repressors and site-specific blockers of the system each comprise a different targeting moiety (e.g., first, second, third, or additional expression repressors each comprise a different targeting moiety from one another, and / or first, second, third, or additional site-specific blockers each comprise a different targeting moiety from one another). In some embodiments, one or more expression repressors comprise a different targeting moiety from one or more site-specific blockers. For example, a system may include an expression repressor and a site-specific blocker, where the expression repressor comprises a first targeting moiety (e.g., a Zn finger domain, a Cas9 domain, or a TAL effector domain) and the site-specific blocker comprises a second targeting moiety (e.g., a Zn finger domain, a Cas9 domain, or a TAL effector domain) that is different from the first targeting moiety. In some embodiments, different means comprising different types of targeting moieties, e.g., the first targeting moiety comprises a Cas9 domain and the second DNA targeting moiety comprises a Zn finger domain. In other embodiments, different means including different variants of the same type of targeting moiety, e.g., a first targeting moiety includes a first Cas9 domain (e.g., from a first species) and a second targeting moiety includes a second Cas9 domain (e.g., from a second species).

[0482] In one embodiment, when a system includes two or more targeting moieties of the same type, e.g., two or more Cas9 or zinc finger domains, the targeting moieties specifically bind to two or more different sequences. For example, in a system including two or more Cas9 domains, two or more Cas9 domains may be selected or modified so that they specifically bind only to the gRNA corresponding to their target sequence (e.g., not specifically to the gRNA corresponding to the target of another Cas9 domain). In a further example, in a system including two or more effector moieties, two or more effector moieties may be selected or modified so that they specifically bind only to their target sequence (e.g., and not specifically to the target sequence of another effector moiety).

[0483] In some embodiments, the system includes three or more site-specific blocking agents, where two or more site-specific blocking agents include the same targeting moiety. For example, the system may include three site-specific blocking agents, where the first and second site-specific blocking agents both include a first targeting moiety and the third site-specific blocking agent includes a second, different targeting moiety. As a further example, the system may include four site-specific blocking agents, where the first and second site-specific blocking agents both include a first targeting moiety and the third and fourth site-specific blocking agents include a second, different targeting moiety. As a further example, the system may include five site-specific blocking agents, where the first and second site-specific blocking agents both include a first targeting moiety, the third and fourth site-specific blocking agents both include a second, different targeting moiety, and the fifth site-specific blocking agent includes a third, different targeting moiety. As noted above, different can mean including different types of targeting moieties or different variants of the same type of targeting moiety.

[0484] In some embodiments, the site-specific blocking agents of the system each bind to a different DNA sequence (e.g., a first, second, third, or additional site-specific blocking agents each bind to a different DNA sequence from each other). For example, a system may include a first site-specific blocking agent and a second site-specific blocking agent, where the first site-specific blocking agent binds to a first DNA sequence and the second site-specific blocking agent binds to a second DNA sequence. In some embodiments involving different DNA sequences, there is at least one position that is not identical between the DNA sequence to which one site-specific blocking agent binds and the DNA sequence to which another site-specific blocking agent binds, or there is at least one position in the DNA sequence to which one site-specific blocking agent binds that is not present in the DNA sequence to which another site-specific blocking agent binds.

[0485] In some embodiments, the first DNA sequence may be located on a first genomic DNA strand and the second DNA sequence may be located on a second genomic DNA strand. In some embodiments, the first DNA sequence may be located on the same genomic DNA strand as the second DNA sequence.

[0486] In some embodiments, the system includes three or more expression repressors, where two or more expression repressors bind to the same DNA sequence. For example, the system may include three expression repressors, where a first and a second expression repressor both bind to a first DNA sequence and a third expression repressor binds to a second, different DNA sequence. As a further example, the system may include four expression repressors, where a first and a second expression repressor both bind to a first DNA sequence and a third and a fourth expression repressor both bind to a second DNA sequence. As a further example, the system may include five expression repressors, where a first and a second expression repressor both bind to a first DNA sequence, a third and a fourth expression repressor both bind to a second DNA sequence, and a fifth expression repressor binds to a third DNA sequence. As noted above, different can mean that there is at least one position between the DNA sequence to which one expression repressor binds and the DNA sequence to which another expression repressor binds that is not identical, or that there is at least one position in the DNA sequence to which one expression repressor binds that is not present in the DNA sequence to which another expression repressor binds. Similarly, in some embodiments, the system comprises one or more expression repressors and one or more site-specific disrupting agents.

[0487] In some embodiments, the system includes two or more (e.g., two) expression repressors, and the plurality (e.g., two) of the expression repressors include targeting moieties that bind to different DNA sequences. In such embodiments, a first targeting moiety can bind to a first DNA sequence and a second targeting moiety can bind to a second DNA sequence, and the first and second DNA sequences are different and do not overlap. In some such embodiments, the first DNA sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and optionally 500, 400, 300, 200, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or In some such embodiments, the first DNA sequence is spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs or less (and optionally, 0 base pairs, e.g., the first and second sequences are adjacent to each other) from the second DNA sequence. In some embodiments, the first DNA sequence is located at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb away from the second DNA sequence.

[0488] In some embodiments, the system includes two or more (e.g., two) site-specific blocking agents, where the multiple (e.g., two) site-specific blocking agents include targeting moieties that bind to different DNA sequences. In such embodiments, a first targeting moiety can bind to a first DNA sequence, and a second DNA targeting moiety can bind to a second DNA sequence, where the first and second DNA sequences are different and do not overlap. In some such embodiments, the first DNA sequence is spaced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and optionally no more than 500, 400, 300, 200, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 base pairs) from the second DNA sequence. In some such embodiments, the first DNA sequence is spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs or less (and optionally, 0 base pairs, e.g., the first and second sequences are adjacent to each other) from the second DNA sequence.

[0489] In some embodiments, the expression repressors and / or site-specific disrupting agents of the system each independently comprise different effector moieties (e.g., first, second, third, or additional expression repressors each independently comprise effector moieties that are different from each other, and / or first, second, third, or additional site-specific disrupting agents each independently comprise effector moieties that are different from each other). For example, the system may include a first expression repressor and a second expression repressor, where the first expression repressor comprises a first effector moiety and the second expression repressor comprises a second effector moiety that is different from the first effector moiety. Further, the system may include an expression repressor and a site-specific disrupting agent, where the expression repressor comprises a first effector moiety and the site-specific disrupting agent comprises a second effector moiety that is different from the first effector moiety. In some embodiments, the different effector moieties comprise different types of effector moieties. In other embodiments, the different effector moieties comprise different variants of the same type of effector moiety.

[0490] In some embodiments, the present disclosure provides an expression repressor system comprising a first expression repressor and a second expression repressor. In some embodiments, the first expression repressor comprises a first targeting moiety. In some embodiments, the first targeting moiety comprises a zinc finger domain. In some embodiments, the first targeting moiety comprises a CRISPR / Cas (e.g., Cas9 or dCas9) domain. In some embodiments, the first targeting moiety comprises a TAL effector domain. In some embodiments, the first expression repressor comprises a first effector moiety. In some embodiments, the first effector moiety comprises a DNA methyltransferase, e.g., MQ1 or a functional fragment thereof, and / or a KRAB, e.g., a KRAB domain. In some embodiments, the second expression repressor comprises a second targeting moiety. In some embodiments, the second targeting moiety comprises a zinc finger domain. In some embodiments, the second expression repressor comprises a second effector moiety. In some embodiments, the second effector moiety comprises a DNA methyltransferase, eg, MQ1 or a functional fragment thereof, and / or a KRAB, eg, a KRAB domain.

[0491] In some embodiments, the expression repressor system is encoded by a first nucleic acid encoding a first expression repressor, e.g., a first targeting moiety and a first effector moiety, whose expression is driven by a first promoter or IRES, and a second nucleic acid encoding a second expression repressor, e.g., a second targeting moiety and a second effector moiety, whose expression is driven by a second promoter or IRES. In some embodiments, the expression repressor system is encoded by a nucleic acid, e.g., an mRNA, whose expression is not driven by a promoter or IRES. In some embodiments, a monocistronic sequence is used. 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. In some embodiments, the polycistronic sequence includes a sequence encoding a first expression repressor and a sequence encoding a second expression repressor. In some embodiments, the polycistronic sequence encodes a self-cleavable peptide sequence, e.g., a 2A peptide sequence, e.g., a T2A peptide sequence, a P2A sequence. In some embodiments, the polycistronic sequence encodes a T2A peptide sequence and a P2A peptide sequence. In some embodiments, the polycistronic sequence encodes a tandem 2A sequence, e.g., a tPT2A sequence. In some embodiments, the bicistronic construct further comprises a poly-A tail. In some embodiments, upon transcription of the bicistronic gene construct, a single mRNA transcript is produced encoding the first expression repressor and the second expression repressor, which upon translation is cleaved, e.g., after a glycine residue in the 2A peptide, to produce the first expression repressor and the second expression repressor as two separate proteins. In some embodiments, the first expression repressor and the second expression repressor are separated by "ribosomal skipping." In some embodiments, the first expression repressor and / or the second expression repressor retain a fragment of the 2A peptide after ribosomal skipping. In some embodiments, the expression levels of the first and second expression repressors are equal. In some embodiments, the expression levels of the first and second expression repressors are different.In some embodiments, the protein level of the first expressed repressor is within 1%, 2%, 5%, or 10% (greater than or less than) the protein level of the second expressed repressor.

[0492] In another aspect, the present disclosure provides a system comprising at least one expression repressor described herein and at least one site-specific disrupting agent (e.g., any site-specific disrupting agent described herein). In some embodiments, the system comprises a first expression repressor and a first site-specific disrupting agent. In some embodiments, the first expression repressor comprises a first targeting moiety. In some embodiments, the first targeting moiety comprises a zinc finger domain. In some embodiments, the first expression repressor comprises a first effector moiety. In some embodiments, the first effector moiety comprises a DNA methyltransferase, e.g., MQ1 or a functional fragment thereof, and / or KRAB, e.g., a KRAB domain. In some embodiments, the site-specific disrupting agent comprises a second targeting moiety, wherein the second targeting moiety targets an anchor sequence of the CXCL locus. In some embodiments, the site-specific disrupting agent comprises a second effector moiety (e.g., a site-specific disrupting agent effector moiety). In some embodiments, the second effector moiety (e.g., the site-specific blocker effector moiety) comprises a DNA methyltransferase, e.g., MQ1 or a functional fragment thereof, and / or KRAB, e.g., a KRAB domain. In some embodiments, the expression repressor effector moiety is the same as the site-specific blocker effector moiety. In some embodiments, the first effector moiety (e.g., the expression repressor effector moiety) is different from the second effector moiety (e.g., the site-specific blocker effector moiety). In some embodiments, the first effector moiety (e.g., the expression repressor effector moiety) and the second effector moiety (e.g., the site-specific blocker effector moiety) each independently comprise methyltransferase activity, e.g., comprise DNA methyltransferase activity.

[0493] In some embodiments, the expression repressor system is encoded by a first nucleic acid encoding a first expression repressor, e.g., a first targeting moiety and a first effector moiety, whose expression is driven by a first promoter or IRES, and a second nucleic acid encoding a site-specific disrupting agent, e.g., a second targeting moiety and a second effector moiety, whose expression is driven by a second promoter or IRES. In some embodiments, the expression repressor system is encoded by a nucleic acid, e.g., an mRNA, whose expression is not driven by a promoter or IRES. In some embodiments, a monocistronic sequence is used. 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. In some embodiments, the multicistronic sequence includes a sequence encoding a first expression repressor and a sequence encoding a site-specific disrupting agent. In some embodiments, the polycistronic sequence encodes a self-cleaving peptide sequence, e.g., a 2A peptide sequence, e.g., a T2A peptide sequence, a P2A sequence. In some embodiments, the polycistronic sequence encodes a T2A peptide sequence and a P2A peptide sequence. In some embodiments, the polycistronic sequence encodes a tandem 2A sequence, e.g., a tPT2A sequence. In some embodiments, the bicistronic construct further comprises a polyA tail. In some embodiments, upon transcription of the bicistronic gene construct, a single mRNA transcript encoding the first expressed repressor and the site-specific disrupting agent is produced, which upon translation is cleaved, e.g., after a glycine residue in the 2A peptide, to produce the first expressed repressor and the site-specific disrupting agent as two separate proteins. In some embodiments, the first expressed repressor and the site-specific disrupting agent are separated by "ribosomal skipping." In some embodiments, the first expressed repressor and / or the site-specific disrupting agent retain a fragment of the 2A peptide after ribosomal skipping. In some embodiments, the expression levels of the first expressed repressor and the site-specific disrupting agent are equal. In some embodiments, the expression levels of the first expressed repressor and the site-specific disrupting agent are different.In some embodiments, the protein level of the first expression repressor is within 1%, 2%, 5%, or 10% (greater or lesser) of the protein level of the site-specific blocking agent.

[0494] Targeting section The targeting moiety can specifically bind to a DNA sequence, for example, a DNA sequence associated with the target multiple genes, for example, a genomic regulatory element or an anchor sequence of ASMC containing the target multiple genes. Any molecule or compound that specifically binds to a DNA sequence can be used as a targeting moiety. In some embodiments, the targeting moiety comprises, for example, a nucleic acid having a sequence complementary to an enhancer sequence, for example, a sequence operably linked to the target multiple genes. In some embodiments, the targeting moiety of the site-specific blocking agent comprises, for example, a nucleic acid having a sequence complementary to an anchor sequence, for example, an anchor sequence of ASMC containing the target multiple genes. In some embodiments, the nucleic acid is an oligonucleotide that physically / sterically blocks the binding of a factor (e.g., a transcription factor, for example, P65, or a nucleation polypeptide, for example, CTCF) to a sequence (e.g., an enhancer sequence or anchor sequence). In some embodiments, the nucleic acid comprises, for example, a guide RNA (gRNA) compatible with a CRISPR / Cas molecule. In some embodiments, the targeting moiety comprises a CRISPR / Cas molecule, a TAL effector molecule, a Zn finger molecule, a tetR domain, a meganuclease, a peptide nucleic acid (PNA), or a nucleic acid.

[0495] In some embodiments, the targeting moiety specifically binds to a nucleic acid sequence within the E1 or E2 cRE of the CXCL locus. In some embodiments, the targeting moiety specifically binds to a nucleic acid sequence within the E1 cRE of the CXCL locus. In certain embodiments, the targeting moiety (e.g., an E1 targeting moiety) specifically binds to a region within the nucleic acid sequence of SEQ ID NO: 162, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a nucleic acid sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 difference therefrom. In some embodiments, the targeting moiety specifically binds to a nucleic acid sequence comprising the E2 cRE of the CXCL locus. In certain embodiments, the targeting moiety (e.g., an E2 targeting moiety) specifically binds to a region within the nucleic acid sequence of SEQ ID NO: 163, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity thereto, or a nucleic acid sequence having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 difference therefrom.

[0496] Exemplary sequence of the E1 cRE of the CXCL locus, SEQ ID NO: 162: [ka]

[0497] Exemplary sequence of the E2 cRE of the CXCL locus, SEQ ID NO: 163: [ka]

[0498] In some embodiments, the targeting moiety specifically binds to a nucleic acid sequence within the IL8 promoter. In some embodiments, the target site (e.g., the target site within the IL8 promoter) is within genomic coordinates chr4:74606112-74606462 (hg19).In some embodiments, the target site (e.g., a target site within the IL8 promoter) is one of the following: chr:74606112-74606462 (e.g., 74606112-74606662, chr4:74606112-74606862, chr4:74606112-74607062, chr4:74606112-74607262, chr4:74606112-74607462, chr4:74605912-74606462, chr4:74605712-7460646 2, chr4:74605512-74606462, chr4:74605312-74606462, chr4:74605112-74606462, chr4:74605912-74606662, chr4:74605912-74 606862, chr4:74605912-74607062, chr4:74605912-74607262, chr4:74605912-74607462, chr4:74605712-74606662, chr4:746057 12-74606862, chr4:74605712-74607062, chr4:74605712-74607262, chr4:74605712-74607462, chr4:74605512-74606662, chr4:7 4605512-74606862, chr4:74605512-74607062, chr4:74605512-74607262, chr4:74605512-74607462, chr4:74605312-74606662, c Located within 1 kb of hr4:74605312-74606862, chr4:74605312-74607062, chr4:74605312-74607262, chr4:74605312-74607462, chr4:74605112-74606662, chr4:74605112-74606862, chr4:74605112-74607062, chr4:74605112-74607262, or chr4:74605112-74607462).

[0499] In some embodiments, the target site (e.g., a target site within the IL8 promoter) is located 500 bp upstream from the transcription start site. In certain embodiments, the target site (e.g., a target site within the IL8 promoter) is located at chr4:74605723-74606223. In some embodiments, the target sites (e.g., target sites within the IL8 promoter) are the following: chr4:74605723-74606426, chr4:74605723-74606626, chr4:74605723-74606826, chr4:74605723-74607026, chr4:74605723-74607226, chr4:74605523-74606226, chr4:74605323-74606226, chr4:7460512 3-74606226, chr4:74604923-74606226, chr4:74604723-74606226, chr4:74605523-74606426, chr4:74605523-74606626, chr 4:74605523-74606826, chr4:74605523-74607026, chr4:74605523-74607226, chr4:74605323-74606426, chr4:74605323-7460 6626, chr4:74605323-74606826, chr4:74605323-74607026, chr4:74605323-74607226, chr4:74605123-74606426, chr4:7460 5123-74606626, chr4:74605123-74606826, chr4:74605123-74607026, chr4:74605123-74607226, chr4:74604923-74606426, c Located at hr4:74604923-74606626, chr4:74604923-74606826, chr4:74604923-74607026, chr4:74604923-74607226, chr4:74604723-74606426, chr4:74604723-74606626, chr4:74604723-74606826, chr4:74604723-74607026, or chr4:74604723-74607226.

[0500] In some embodiments, the target site (e.g., a target site within the IL8 promoter) is located 1000 bp upstream from the transcription start site. In certain embodiments, the target site (e.g., a target site within the IL8 promoter) is located at chr4:74605223-74606223. In some embodiments, the target sites (e.g., target sites within the IL8 promoter) are the following: chr4:74605226-74606426, chr4:74605226-74606626, chr4:74605226-74606826, chr4:74605226-74607026, chr4:74605226-74607226, chr4:74605026-74606226, chr4:74604826-74606226, chr4:7460462 6-74606226, chr4:74604426-74606226, chr4:74604226-74606226, chr4:74605026-74606426, chr4:74605026-74606626, chr 4:74605026-74606826, chr4:74605026-74607026, chr4:74605026-74607226, chr4:74604826-74606426, chr4:74604826-7460 6626, chr4:74604826-74606826, chr4:74604826-74607026, chr4:74604826-74607226, chr4:74604626-74606426, chr4:7460 4626-74606626, chr4:74604626-74606826, chr4:74604626-74607026, chr4:74604626-74607226, chr4:74604426-74606426, c Located at hr4:74604426-74606626, chr4:74604426-74606826, chr4:74604426-74607026, chr4:74604426-74607226, chr4:74604226-74606426, chr4:74604226-74606626, chr4:74604226-74606826, chr4:74604226-74607026, or chr4:74604226-74607226.

[0501] In some embodiments, the targeting moiety has a K of 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001 nM or less. D (and optionally a K of at least 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001 nM) D In some embodiments, the targeting moiety binds to its target sequence with a K of 0.001 nM to 500 nM, e.g., 0.1 nM to 5 nM, e.g., about 0.5 nM. D In some embodiments, the targeting moiety binds to its target sequence with a K of at least 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, or 100,000 nM. D In some embodiments, the targeting moiety binds to the non-target sequence (and optionally does not explicitly bind to the non-target sequence). In some embodiments, the targeting moiety does not bind to the non-target sequence.

[0502] In some embodiments, the targeting moiety of the expression repressor or site-specific disrupting agent comprises a nucleic acid comprising a sequence complementary to a sequence selected from Table 8 or 8A, or a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto, or differing therefrom at no more than 1, 2, 3, 4, or 5 positions. In some embodiments, the targeting moiety of the expression repressor or site-specific disrupting agent comprises a nucleic acid having a sequence selected from Table 8 or 8A, or a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto, or a sequence that differs therefrom at no more than 1, 2, 3, 4, or 5 positions. In some embodiments, the targeting moiety of the expression repressor or site-specific disrupting agent binds to a target site having a sequence in Table 8 or 8A. It is understood that in some embodiments, the targeting moiety comprises an RNA sequence in which each position designated as T in Table 8 or 8A is occupied by U.

[0503] [Table 8]

[0504] [Table 8A]

[0505] In some embodiments, the targeting moiety comprises a nucleic acid having a sequence selected from Table 9 or 9A, or a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto, or a sequence which differs therefrom at no more than 1, 2, 3, 4, or 5 positions. In some embodiments, the targeting moiety comprises a nucleic acid having a spacer sequence within a sequence of Table 9 or 9A, or a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto, or a sequence which differs therefrom at no more than 1, 2, 3, 4, or 5 positions. In some embodiments, the targeting moiety is understood to comprise an RNA sequence in which each position designated as T in Table 9 or 9A is occupied by U.

[0506] [Table 9-1]

[0507] [Table 9-2]

[0508] [Table 9-3]

[0509] [Table 9A-1]

[0510] [Table 9A-2]

[0511] [Table 9A-3]

[0512] In some embodiments, the targeting moiety comprises a nucleic acid that comprises a sequence complementary to at least a portion of the sequence of a cRE (e.g., an E1 cRE) or has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions non-complementary thereto.

[0513] In some embodiments, the targeting moiety comprises a nucleic acid that includes a sequence complementary to at least a portion of the sequence of a non-human cRE (e.g., a non-human E1 cRE) (e.g., a mouse cRE) that is homologous to a human cRE, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions that are non-complementary thereto.

[0514] In some embodiments, the targeting moiety is located at genomic coordinates GRCh37:chr4:74591768-74591790, GRCh37:chr4:74591844-74591866, GRCh37:chr4:74591892-74591914, GRCh37:chr4:74592088-74592110, GRCh3 7:chr4:74982748-74982770, GRCh37:chr4:74982841-74982863, GRCh37:chr4:74982882-74982904, GRCh37:chr4:74982960-74982982, GRCh37:chr4:74983108-74983130, GRCh37: chr4:74983181-74983203, or GRCh37:chr4:74606162-74606184.

[0515] In some embodiments, the targeting moiety is located at genomic positions GRCh37:chr4:74591768-74591790, GRCh37:chr4:74591844-74591866, GRCh37:chr4:74591892-74591914, GRCh37:chr4:74592088-74592110, GRCh37:chr4:74982748-74982770, GRCh37: Binding to sequences at chr4:74982841-74982863, GRCh37:chr4:74982882-74982904, GRCh37:chr4:74982960-74982982, GRCh37:chr4:74983108-74983130, GRCh37:chr4:74983181-74983203, or GRCh37:chr4:74606162-74606184.

[0516] In some embodiments, the targeting moiety binds to a cRE (eg, an E1 cRE) or a site proximal to a cRE (eg, an E1 cRE), eg, a cRE operably linked to a target gene.

[0517] Site-specific blocking agent gRNA In some embodiments, the targeting moiety comprises a nucleic acid having a sequence selected from Table 7, or a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto, or a sequence which differs therefrom at no more than 1, 2, 3, 4, or 5 positions. It is understood that in some embodiments, the targeting moiety comprises an RNA sequence in which each position designated as T in Table 7 is occupied by U.

[0518] [Table 7]

[0519] In some embodiments, the targeting moiety comprises a nucleic acid comprising a sequence selected from Table 6, or a sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto, or differing therefrom at no more than 1, 2, 3, 4, or 5 positions. It is understood that in some embodiments, the targeting moiety comprises an RNA sequence in which each position designated as T in Table 6 is occupied by U.

[0520] [Table 6-1]

[0521] [Table 6-2]

[0522] [Table 6-3]

[0523] In some embodiments, the targeting moiety comprises a nucleic acid that comprises a sequence complementary to, or has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer positions non-complementary to, an anchor sequence, e.g., an anchor sequence of an ASMC comprising a target multi-gene.

[0524] In some embodiments, the targeting moiety is located at genomic coordinates chr4:74595464-74595486, chr4:74595457-74595479, chr4:74595460-74595482, chr4:74595472-74595494, chr4:75000088-75000110, chr4:75000091-75000113, chr4:75000085-75000107, chr4:75000157-75000179, chr4:75000156-75000178, chr4:745 95215-74595237, chr4:74595370-74595392, chr4:74595560-74595582 , chr4:74595642-74595664, chr4:74595787-74595809, chr4:74528428- 74528450, chr4:74528567-74528589, chr4:74528609-74528631, chr4:7 4789132-74789154, chr4:74789250-74789272, chr4:74789312-7478933 4, chr4:74964853-74964875, chr4:74964906-74964928, chr4:74965538-74965560, chr4:74965737-74965759, chr4:75000031-75000053, chr4 :75000115-75000137, chr4:75000231-75000253, chr4:74975146-74975168, chr4:74975369-74975391, chr4:74976318-74976340, chr4:74570 348-74570370, chr4:74570503-74570525, chr4:74570526-74570548, c hr5:90785724-90785746, chr5:90788137-90788159, chr5:90908926-90 908948, chr5:90661492-90661514, chr5:90661646-90661668, chr5:906 61744-90661766, chr5:90785610-90785632, chr5:90909047-90909069,or chr6:113076028-113076047, or a sequence that is within 5, 10, 15, 20, 30, 40, or 50 nucleotides of said region, or has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the sequence of said genomic region, or a sequence that differs therefrom at no more than 1, 2, 3, 4, or 5 positions.

[0525] In some embodiments, the targeting moiety is located at genomic positions chr4:74595464-74595486, chr4:74595457-74595479, chr4:74595460-74595482, chr4:74595472-74595494, chr4:75000088-75000110, chr4:75000091-75000113, chr4:75000085-75000107, chr4:75000157-75000179, chr4:75000156-75000178, chr4:7459 5215-74595237, chr4:74595370-74595392, chr4:74595560-74595582, chr4:74595642-74595664, chr4:74595787-74595809, chr4:74528428-7 4528450, chr4:74528567-74528589, chr4:74528609-74528631, chr4:7 4789132-74789154, chr4:74789250-74789272, chr4:74789312-7478933 4, chr4:74964853-74964875, chr4:74964906-74964928, chr4:74965538-74965560, chr4:74965737-74965759, chr4:75000031-75000053, chr4 :75000115-75000137, chr4:75000231-75000253, chr4:74975146-74975168, chr4:74975369-74975391, chr4:74976318-74976340, chr4:74570 348-74570370, chr4:74570503-74570525, chr4:74570526-74570548, c hr5:90661492-90661514, chr5:90661646-90661668, chr5:90661744-90 661766, chr5:90785610-90785632, chr5:90909047-90909069, chr5:907 85724-90785746, chr5:90788137-90788159, chr5:90908926-90908948,or binds to the sequence chr6:113076028-113076047.

[0526] In some embodiments, the targeting moiety binds to an anchor sequence or a site adjacent to an anchor sequence, for example, an anchor sequence that is part of an ASMC that fully or partially contains the target multiple genes.

[0527] CRISPR / Cas targeting moiety In some embodiments, the targeting moiety comprises a CRISPR / Cas molecule. In some embodiments, the effector moiety comprises a CRISPR / Cas molecule. A CRISPR / Cas molecule comprises a protein involved in the clustered regularly interspaced short palindromic repeats (CRISPR) system, e.g., a Cas protein, and optionally a guide RNA, e.g., a single guide RNA (sgRNA).

[0528] The CRISPR system is an adaptive defense system first discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases called CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpf1) to cleave foreign DNA. For example, in a typical CRISPR / Cas system, the endonuclease is directed to the target nuclease sequence (e.g., the site in the genome where the sequence is to be edited) by a sequence-specific, non-coding "guide RNA" that targets a single- or double-stranded DNA sequence. Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). Class II CRISPR systems include a type II Cas endonuclease, e.g., Cas9, a CRISPR RNA ("crRNA"), and a trans-acting crRNA ("tracrRNA"). The crRNA includes a "guide RNA," which is typically an approximately 20-nucleotide RNA sequence that corresponds to the target DNA sequence. The crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure, which is cleaved by RNase III to yield a crRNA / tracrRNA hybrid. The crRNA / tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave the target DNA sequence. The target DNA sequence must generally be adjacent to a "protospacer adjacent motif" ("PAM") specific to a given Cas endonuclease; however, PAM sequences occur throughout a given genome.CRISPR endonucleases identified from diverse prokaryotic species have unique PAM sequence requirements; example PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endoglucanases, such as Cas9 endonucleases, bind to G-rich PAM sites, e.g., 5'-NGG (e.g., TGG, e.g., CGG, e.g., AGG), and perform blunt-end cleavage of target DNA three nucleotides upstream (5') of the PAM site. Another class II CRISPR system contains a V-type endonuclease, Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays are processed into mature crRNA without the need for tracrRNA; in other words, the Cpf1 system only requires Cpf1 nuclease and crRNA to cleave the target DNA sequence. Cpf1 endonuclease binds to T-rich PAM sites, such as 5'-TTN. Cpf1 also recognizes the 5'-CTA PAM motif. Cpf1 cleaves target DNA by introducing offset or staggered double-stranded breaks with 4- or 5-nucleotide 5' overhangs, e.g., cleaving the target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream (3') from the PAM on the coding strand and 23 nucleotides downstream from the PAM on the complementary strand; the 5-nucleotide overhangs created by such offset cuts allow for more precise genome editing by DNA insertion via homologous recombination compared to insertion with blunt-cut DNA.See, for example, Zetsche et al. (2015) Cell, 163:759-771.

[0529] A wide variety of CRISPR-associated (Cas) genes or proteins can be used in the techniques provided herein, and the choice of Cas protein can vary depending on the specific requirements of the method. Specific examples of Cas proteins include Class II systems, including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, the Cas protein, e.g., the Cas9 protein, can be derived from any of a variety of prokaryotic species. In some embodiments, a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a particular protospacer adjacent motif (PAM) sequence. In some embodiments, the targeting moiety includes a sequence-targeting polypeptide, e.g., a Cas protein, e.g., Cas9. In certain embodiments, the Cas protein, e.g., the Cas9 protein, can be obtained from bacteria or archaea or synthesized using known methods. In certain embodiments, the Cas protein can be derived from Gram-positive or Gram-negative bacteria.In certain embodiments, the Cas protein is selected from the group consisting of Streptococcus (e.g., S. pyogenes or S. thermophilus), Francisella (e.g., F. novicida), Staphylococcus (e.g., S. aureus), Acidaminococcus (e.g., Acidaminococcus spp.), and the like. sp. BV3L6), Neisseria (e.g., N. meningitidis), Cryptococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter.

[0530] In some embodiments, Cas proteins require a protospacer adjacent motif (PAM) to be present in or adjacent to the target DNA sequence for the Cas protein to bind and / or function. In some embodiments, the PAM is or includes, from 5' to 3', NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N represents any nucleotide, Y represents C or T, R represents A or G, and V represents A, C, or G. In some embodiments, the Cas protein is a protein listed in Table 1. In some embodiments, the Cas protein includes one or more mutations that alter its PAM. In some embodiments, the Cas protein includes E1369R, E1449H, and R1556A mutations, or similar substitutions for the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises E782K, N968K, and R1015H mutations, or analogous substitutions at the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises D1135V, R1335Q, and T1337R mutations, or analogous substitutions at the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises S542R and K607R mutations, or analogous substitutions at the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises S542R, K548V, and N552R mutations, or analogous substitutions at the amino acids corresponding to said positions.

[0531] [Table 1-1]

[0532] [Table 1-2]

[0533] In some embodiments, the Cas protein is catalytically active and cleaves one or both strands of the target DNA site, and in some embodiments, cleavage of the target DNA site is followed by the formation of an alteration, e.g., an insertion or deletion, e.g., by cellular repair mechanisms.

[0534] In some embodiments, the Cas protein is modified to inactivate the nuclease (e.g., nuclease-deleted Cas9). While wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by the gRNA, several CRISPR endonucleases are available with modified functionality, for example: "nickase" versions of Cas9 generate only single-strand breaks; catalytically inactive Cas9 ("dCas9") does not cleave the target DNA. In some embodiments, binding of dCas9 to a DNA sequence interferes with transcription at that site due to steric hindrance. In some embodiments, binding of dCas9 to an anchor sequence can interfere with (e.g., reduce or prevent) the formation and / or maintenance of a genome complex (e.g., ASMC). In some embodiments, the targeting moiety comprises a catalytically inactive Cas9, e.g., dCas9, e.g., Cas9m4. Many catalytically inactive Cas9 proteins are known in the art. In some embodiments, the dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A mutations.

[0535] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D11A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a H969A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a N995A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises D11A, H969A, and N995A mutations or an analogous substitution at the amino acid corresponding to said position.

[0536] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10A mutation or an analogous substitution at the corresponding amino acid at said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a H557A mutation or an analogous substitution at the corresponding amino acid at said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10A and H557A mutation or an analogous substitution at the corresponding amino acid at said position.

[0537] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D839A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a H840A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a N863A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises D10A, D839A, H840A, and N863A mutations or an analogous substitution at the amino acid corresponding to said position.

[0538] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises an E993A mutation or an analogous substitution of the amino acid corresponding to said position.

[0539] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises an E1006A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D1255A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises D917A, E1006A, and D1255A mutations or an analogous substitution at the amino acid corresponding to said position.

[0540] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D587A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a H588A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a N611A mutation or an analogous substitution at the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises D16A, D587A, H588A, and N611A mutations or an analogous substitution at the amino acid corresponding to said position.

[0541] In some aspects, the systems described herein comprise, or the methods described herein comprise the use of, an expression repressor or site-specific disrupting agent, or a polypeptide comprising one or more (e.g., one) targeting moieties and one or more effector moieties (e.g., one or two effector moieties), wherein the one or more targeting moieties is or comprises a Cas protein, e.g., a CRISPR / Cas molecule comprising a catalytically inactive Cas9 protein, e.g., sadCas9, dCas9, e.g., dCas9m4, or a functional variant or fragment thereof. In some embodiments, the dCas9 comprises the amino acid sequence of SEQ ID NO: 5, 6, or 7. Cas9 [ka] dCas9(Cas9m4) [ka] Sa-dCas9 [ka]

[0542] In some embodiments, the dCas9 is encoded by the nucleic acid sequence of SEQ ID NO: 8 or 9. [ka] [ka] [ka]

[0543] [ka] [ka] 【054...

Claims

1. A first targeting portion that binds to a target site, wherein the target site is located within the E1 cis-acting regulatory element of the CXCL gene locus or the E2 cis-acting regulatory element of the CXCL gene locus; Optionally, the first effect pedal section and, An expression repressor that includes, An expression repressor that can reduce the expression of the CXCL gene.

2. The expression repressor according to claim 1, wherein the target site is located within genomic coordinates chr4:74591400–74593000 or chr4:74982639–74983600 (based on the HG19 human genome reference assembly).

3. A first targeting moiety that binds to a target site containing at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides of the sequence of sequence number 162 or 163, Optionally, the first effect pedal section and, An expression repressor that includes, An expression repressor that can reduce the expression of the CXCL gene.

4. A first targeting portion that binds to a target site, wherein the target site is located within the IL-8 promoter, Optionally, the first effect pedal section and, An expression repressor that includes, The expression repressor is capable of reducing the expression of IL-8.

5. The first effector portion is an effector as described herein, for example, KRAB, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, EZH2, HDAC8, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39 The expression repressor according to claim 1, comprising H1, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or DNMT3, or any functional variant or fragment thereof.

6. The expression repressor according to claim 1, wherein the first targeting portion includes a zinc finger domain or a TAL domain.

7. An expression repressor according to claim 1, comprising one amino acid sequence of sequence numbers 152-161 or 164-169, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity therewith, or a sequence having positional differences of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less therewith.

8. a) The first expression repressor according to claim 1, b) A secondary expression repressor, for example, a secondary expression repressor that reduces the expression of the CXCL gene, A system that includes this.

9. The second expression repressor is as follows: A second target site that binds to a second target site within the CXCL gene locus, Optionally, the second effector section and The system according to claim 8, including the above.

10. The target site includes a sequence according to sequence number 134; The first effector section includes a KRAB array; The second target site comprises a sequence according to sequence number 292; The second effector section includes a KRAB array, The system according to claim 9.

11. A nucleic acid encoding an expression repressor according to claim 1, wherein the nucleic acid optionally further encodes a second expression repressor, for example, a second expression repressor that reduces the expression of the CXCL gene.

12. A vector comprising the nucleic acid described in claim 11.

13. A pharmaceutical composition comprising the expression repressor described in claim 1.

14. A human cell comprising an expression repressor according to any one of claims 1 to 7, a system according to any one of claims 8 to 10, a nucleic acid according to claim 11, or a vector according to claim 12.

15. Human cells in which the expression of the CXCL gene is reduced, wherein the cells are produced by a method comprising contacting the cells with an expression repressor according to any one of claims 1 to 7, a system according to any one of claims 8 to 10, a nucleic acid according to claim 11, or a vector according to claim 12.

16. A composition or system for use in a method for reducing the expression of one or more CXCL genes in cells, comprising an expression repressor according to any one of claims 1 to 7, a system according to any one of claims 8 to 10, a composition comprising a nucleic acid according to claim 11, or a vector according to claim 12, wherein the method comprises contacting the cells with the expression repressor, the system, the nucleic acid, or the vector.

17. A reaction mixture comprising cells (e.g., human cells, e.g., primary human cells) and an expression repressor or system according to any one of claims 1 to 10.

18. A composition comprising an expression repressor according to any one of claims 1 to 7, a system according to any one of claims 8 to 10, a composition comprising a nucleic acid according to claim 11, a composition comprising a vector according to claim 12, or a pharmaceutical composition according to claim 13, for use in the treatment of a subject having an inflammatory disorder or cancer.

19. Use of an expression repressor, system, nucleic acid, vector, or pharmaceutical composition according to any one of claims 1 to 13 in the manufacture of a drug for treating a subject having an inflammatory disorder or cancer.

20. The reaction mixture according to claim 17 for use in the treatment of a subject having an inflammatory disorder or cancer.

21. A nucleic acid encoding an expression repressor according to claim 1, wherein the nucleic acid further encodes a second expression repressor of the system according to any one of claims 8 to 10.

22. Use of the reaction mixture according to claim 17 in the manufacture of a drug for treating a subject having an inflammatory disorder or cancer.

Images