Methods and compositions for modulating ctnnb1 expression

Abstract

The present disclosure is directed to compositions and methods for reducing expression of the CTNNB1 gene in a cell, e.g., using an expression repressor that comprises a targeting moiety that binds a CTNNB1 promoter, anchor sequence, or super-enhancer and an effector domain that represses transcription or methylates DNA. Systems comprising two or more expression repressors are also disclosed. The compositions can be used, for example, to treat a CTNNB1-associated disease or disorder, such as cancer.

Description

[0001] METHODS AND COMPOSITIONS FOR MODULATING CTNNB1 EXPRESSION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the benefit of the filing date of U.S. Provisional Patent Application Ser. No. 63 / 514,488 filed July 19, 2023, and which is incorporated herein by reference.

[0004] BACKGROUND

[0005] P-catenin, encoded by the CTNNB1 gene, is a multifunctional protein with a central role in physiological homeostasis. P-catenin acts both as a transcriptional co-regulator and an adaptor protein for intracellular adhesion. Wnt-signaling is the primary regulator of P-catenin, which is a family of 19 glycoproteins to regulate both the P-catenin-dependent (canonical Wnt) and - P- catenin-independent (non-canonical Wnt) signaling pathways (van Ooyen A and Nusse R. Cell. 1984;39:233-240). In canonical Wnt pathway, Dsh, P-catenin, Glycogen Synthase Kinase 3 beta (GSK3P), adenomatous polyposis coli (APC), AXIN, and T-cell factor (TCF) / lymphoid enhancement factor (LEF) act as signal transducers of the canonical Wnt pathway, in which P- catenin acts as the transcriptional effector (Groenewald W, et al. Cells. 2023;12(7):990). In the absence of Wnt ligands, P-catenin is maintained at a low level through intracellular degradation.

[0006] Upon Wnt activation or genetic mutation within the Wnt pathway, P-catenin accumulates in the cytoplasm and then translocates into the nucleus. Consequently, it binds to other transcription factors, such as LEF-1 / TCF4, and activates transcription of target genes including proto-oncogenes Jun, c-Myc, and Cyclin D 1. As a result, aberrant high expression of P-catenin leads to various diseases including cancer. In addition, a high-level of cytoplasmic expression and consequent nuclear localization of P-catenin induces transformed phenotypes and promotes cancer cell proliferation and survival (Valkenburg KC, et al. Cancers (Basel) 2011;3:2050- 2079). Mutation and / or overexpression of P-catenin is associated with many cancers, including but not limited to hepatocellular carcinoma, colorectal carcinoma, lung cancer, malignant breast tumors, ovarian, and endometrial cancer (Morin PJ. BioEssays 1999;21(12): 1021-1030). SUMMARY

[0007] In some aspects, the disclosure provides an expression repressor targeting a gene encoding P-catenin (CTNNB1) comprising (i) a DNA targeting moiety that binds to a target sequence, wherein the target sequence is about 15-20 nucleotides of a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chromosome 3 (chr3); and (ii) an effector domain.

[0008] In some embodiments of the foregoing or related aspects, the region spans the region spans position 41,240,170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3. In some embodiments, the region spans position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the region spans position 41,240,170 to position 41,240,300; position 41,240,250 to position 41,240,350; position

[0009] 41.240.300 to position 41,240,400; position 41,240,350 to position 41,240,450; position

[0010] 41.240.400 to position 41,240,500; position 41,240,450 to position 41,240,550; position

[0011] 41.240.500 to position 41,240,600; position 41,240,550 to position 41,240,650; position 41,240,600 to position 41,240,700; position 41,240,650 to position 41,240,750; position 41,240,700 to position 41,240,800; position 41,240,750 to position 41,240,850; position 41,240,800 to position 41,240,900; position 41,240,850 to position 41,240,950; position 41,240,900 to position 41,241,000; position 41,240,950 to position 41,241,050; position 41,241,000 to position 41,241,100; position 41,241,050 to position 41,241,150; position 41,241,100 to position 41,241,200; position 41,241,150 to position 41,241,250; position 41,241,200 to position 41,241,300; position 41,241,250 to position 41,241,350; position

[0012] 41.241.300 to position 41,241,400; position 41,241,350 to position 41,241,450; position

[0013] 41.241.400 to position 41,241,523; position 41,241,450 to position 41,241,550; or position

[0014] 41.241.500 to position 41,241,623, each according to the hgl9 reference genome for chr3. In some embodiments, the CTNNB1 target sequence is 15, 16, 17, 18, 19, or 20 nucleotides in length.

[0015] In some aspects, the disclosure provides an expression repressor targeting CTNNB1 comprising (i) a DNA targeting moiety that binds to a target sequence, wherein the target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20; and (ii) an effector domain. In some embodiments of any of the foregoing or relates aspects, the target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the DNA-targeting moiety comprises a zinc finger (ZF) domain. In some embodiments, the DNA-targeting moiety comprises a transcription activator-like effector (TALE) domain. In some embodiments, the DNA-targeting moiety comprises a nuclease inactive Cas polypeptide (dCas) and a gRNA comprising a sequence complementary to the target sequence.

[0016] In some aspects, the disclosure provides an expression repressor targeting CTNNB1 comprising (i) a DNA targeting moiety that binds to a target sequence, wherein the DNA targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 13; and (ii) an effector domain. In some embodiments, the DNA targeting moiety comprises SEQ ID NO: 13. In some embodiments, the DNA targeting moiety consists of SEQ ID NO: 13.

[0017] In some aspects, the disclosure provides an expression repressor targeting CTNNB1 comprising (i) a DNA targeting moiety that binds to a target sequence of about 15-20 nucleotides in an insulated genomic domain (IGD) comprising CTNNB1 , wherein the DNA targeting moiety comprises a ZF domain or a TALE domain; and (ii) an effector domain. In some embodiments, the target sequence is in a region of about 500 bases to about 5,000 bases comprising a CpG island. In some embodiments, the target sequence is upstream of (e.g., up to about 200 bases upstream of), within, or downstream of (e.g., up to about 200 bases downstream of) a CpG island spanning position 41,198,672 to position 41,200,140, according to the hg38 reference genome for chr3. In some embodiments, the target sequence is upstream of (e.g., up to about 200 bases upstream of), within, or downstream of (e.g., up to about 200 bases downstream of) a CpG island spanning position 41,240,163 to position 41,241,631, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is within or up to 200 bases upstream a CpG island spanning position 41,198,672 to position 41,200,140, according to the hg38 reference genome for chr3. In some embodiments, the target sequence is within or up to 200 bases upstream a CpG island spanning position 41,240,163 to position 41,241,631, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is within or up to 200 bases downstream a CpG island spanning position 41,198,672 to position 41,200,140, according to the hg38 reference genome for chr3. In some embodiments, the target sequence is within or up to 200 bases downstream a CpG island spanning position 41,240,163 to position 41,241,631, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is (i) in a CpG island; or (ii) up to 200 bases upstream or downstream of a CpG island. In some embodiments, the target sequence is in or near a promoter region of CTNNB1. In some embodiments, the target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the DNA targeting moiety comprises a ZF domain. In some embodiments, the DNA targeting moiety comprises a TALE domain. In some embodiments, the DNA targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 13. In some embodiments, the DNA targeting moiety comprises SEQ ID NO: 13.

[0018] In some embodiments of any of the foregoing or related aspects, the expression repressor comprises a single effector domain. In some embodiments, the expression repressor comprises more than one effector domain. In some embodiments, the effector domain comprises a transcriptional repressor moiety. In some embodiments, the transcriptional repressor moiety comprises a Kruppel associated box (KRAB) domain or a functional variant or fragment thereof. In some embodiments, the transcriptional repressor moiety comprises a histone modifying enzyme selected from a histone methyltransferase, a histone deacetylase, and a histone demethylase. In some embodiments, the histone modifying enzyme is a histone deacetylase. In some embodiments, the histone deacetylase is 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 thereof. In some embodiments, the histone modifying enzyme is a histone methyltransferase. In some embodiments, the histone methyltransferase is SETDB1, SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof. In some embodiments, the transcriptional repressor moiety comprises a DNA methyltransferase. In some embodiments, the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof. In some embodiments, the DNA methyltransferase is MQ1, or a functional variant or fragment thereof. In some aspects, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding the expression repressor described herein. In some embodiments, the nucleic acid is an mRNA.

[0019] In some aspects, the disclosure provides an mRNA encoding an expression repressor described herein.

[0020] In some aspects, the disclosure provides a system for modulating expression of human CTNNB1 comprising (i) an expression repressor described herein, and (ii) a second expression repressor comprising: a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain. In some embodiments, the expression repressor and the second expression repressor are in the same composition. In some embodiments, the expression repressor and the second expression repressor are in different compositions. In some embodiments, the system comprises a first nucleic acid encoding the expression repressor and a second nucleic acid encoding the second expression repressor. In some embodiments, the first nucleic acid and the second nucleic acid are in the same composition. In some embodiments, the first nucleic acid and the second nucleic acid are in different compositions. In some embodiments, the first nucleic acid and the second nucleic acid are formulated in the same LNP. In some embodiments, the first nucleic acid and the second nucleic acid are formulated in different LNPs. In some embodiments, the system comprises a first recombinant expression vector comprising the first nucleic acid and a second recombinant expression vector comprising the second nucleic acid. In some embodiments, the first recombinant expression vector and the second recombinant expression vector are formulated in the same LNP. In some embodiments, the first recombinant expression vector and the second recombinant expression vector are formulated in different LNPs. In some embodiments, the system comprises a recombinant expression vector comprising the first nucleic acid and the second nucleic acid. In some embodiments, the recombinant expression vector is formulated in an LNP.

[0021] In some aspects, the disclosure provides a nucleic acid comprising a first nucleotide sequence encoding an expression repressor described herein, and a second nucleotide sequence encoding a second expression repressor comprising a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain.

[0022] In some aspects, the disclosure provides an mRNA that encodes: an expression repressor described herein; and a second expression repressor comprising: a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain. In some embodiments, the ORF comprises a ribosome skipping sequence between the first nucleotide sequence and the second nucleotide sequence.

[0023] In some aspects, the disclosure provides a method of treating a condition associated with CTNNB1 expression in a subject, comprising administering to the subject (i) an expression repressor described herein, and (ii) a second expression repressor comprising: a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain. In some embodiments, the condition is associated with a mutation in CTNNB1. In some embodiments, the conditions is associated with overexpression of CTNNB1. In some embodiments, the condition is cancer. In some embodiments, the method comprises administering the expression repressor and the second expression repressor in the same composition or in different compositions. In some embodiments, the method comprises administering a first nucleic acid encoding the expression repressor and a second nucleic acid encoding the second repression repressor. In some embodiments, the first nucleic acid is an mRNA encoding the expression repressor. In some embodiments, the second nucleic acid is an mRNA encoding the second expression repressor. In some embodiments, the method comprises administering the first nucleic acid and the second nucleic acid in the same composition or in different compositions. In some embodiments, the method comprises administering the first nucleic acid and the second nucleic acid formulated in the same LNP or in separate LNPs. In some embodiments, the method comprises administering a first recombinant expression vector comprising the first nucleic acid and a second recombinant expression vector comprising the second nucleic acid. In some embodiments, the first recombinant expression vector and the second recombinant expression vector are formulated in the same LNP or in separate LNPs. In some embodiments, the method comprises administering a recombinant expression vector comprising the first nucleic acid and the second nucleic acid. In some embodiments, the recombinant expression vector is formulated in an LNP.

[0024] In some embodiments of any of the foregoing or related aspects, the second target sequence is about 15-20 nucleotides in an insulated genomic domain (IGD) comprising CTNNB1. In some embodiments, the second target sequence is in a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chrl2. In some embodiments, the second target sequence is in a region spanning position 41,240, 170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3. In some embodiments, the second target sequence is in a region spanning position 41,240,553 to position 41,240,770 according to the hgl9 reference genome for chr3. In some embodiments, the region spans position 41,240,170 to position 41,240,300; position 41,240,250 to position 41,240,350; position

[0025] 41.240.300 to position 41,240,400; position 41,240,350 to position 41,240,450; position

[0026] 41.240.400 to position 41,240,500; position 41,240,450 to position 41,240,550; position

[0027] 41.240.500 to position 41,240,600; position 41,240,550 to position 41,240,650; position 41,240,600 to position 41,240,700; position 41,240,650 to position 41,240,750; position 41,240,700 to position 41,240,800; position 41,240,750 to position 41,240,850; position 41,240,800 to position 41,240,900; position 41,240,850 to position 41,240,950; position 41,240,900 to position 41,241,000; position 41,240,950 to position 41,241,050; position 41,241,000 to position 41,241,100; position 41,241,050 to position 41,241,150; position 41,241,100 to position 41,241,200; position 41,241,150 to position 41,241,250; position 41,241,200 to position 41,241,300; position 41,241,250 to position 41,241,350; position

[0028] 41.241.300 to position 41,241,400; position 41,241,350 to position 41,241,450; position

[0029] 41.241.400 to position 41,241,523; position 41,241,450 to position 41,241,550; or position

[0030] 41.241.500 to position 41,241,623, each according to the hgl9 reference genome for chr3. In some embodiments, the second target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the second target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the second target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments of any of the foregoing or related aspects, the second DNA- targeting moiety of the second fusion protein comprises a zinc finger (ZF) domain. In some embodiments, the second DNA targeting moiety comprises a ZF domain and the second target sequence is about 15-20 nucleotides in an IGD comprising CTNNB1. In some embodiments, the second DNA targeting moiety comprises a ZF domain and the second target sequence is about 15-20 nucleotides in a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the second DNA targeting moiety comprises a ZF domain and the second target sequence is about 15-20 nucleotides in a region spanning position 41,240,170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3. In some embodiments, the second DNA targeting moiety comprises a ZF domain and the second target sequence is about 15-20 nucleotides in a region spanning position 41,240,553 to position 41,240,770 according to the hgl9 reference genome for chr3. In some embodiments, the second DNA targeting moiety comprises a ZF domain and the second target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the second DNA targeting moiety comprises a ZF domain and the second target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the second DNA targeting moiety comprises a ZF domain and the second target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20.

[0031] In some embodiments of any of the foregoing or related aspects, the second DNA- targeting moiety of the second fusion protein comprises a TALE domain. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence is about 15-20 nucleotides in an IGD comprising CTNNB1. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence is about 15-20 nucleotides in a region spanning position 41,240,170 to position 41,241,623 according to the hgl9 reference genome for chr3. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence is about 15-20 nucleotides in a region spanning position 41,240,270 to position 41,241,523 according to the hgl9 reference genome for chr3. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence is about 15-20 nucleotides in a region spanning position 41,240,170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence is about 15-20 nucleotides in a region spanning position 41,240,553 to position 41,240,770 according to the hgl9 reference genome for chr3. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the second DNA targeting moiety comprises a TALE domain and the second target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20. In some embodiments, the second DNA targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 13. In some embodiments, the second DNA targeting moiety comprises SEQ ID NO: 13.

[0032] In some embodiments of any of the foregoing or related aspects, the second DNA- targeting moiety of the second fusion protein comprises a dCas and a gRNA comprising a sequence complementary to the second target sequence. In some embodiments, second DNA targeting moiety comprises a dCas and a gRNA comprising a sequence complementary to the second target sequence, wherein the second target sequence is in an insulated genomic domain (IGD) comprising CTNNB1. In some embodiments, second DNA targeting moiety comprises a dCas and a gRNA comprising a sequence complementary to the second target sequence, wherein the second target sequence is in in a region spanning position 41,240,170 to position 41,241,623 according to the hgl9 reference genome for chr3. In some embodiments, second DNA targeting moiety comprises a dCas and a gRNA comprising a sequence complementary to the second target sequence, wherein the second target sequence is in in a region spanning position 41,240,270 to position 41,241,523 according to the hgl9 reference genome for chr3. In some embodiments, second DNA targeting moiety comprises a dCas and a gRNA comprising a sequence complementary to the second target sequence, wherein the second target sequence is in a region spanning position 41,240,170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3. In some embodiments, second DNA targeting moiety comprises a dCas and a gRNA comprising a sequence complementary to the second target sequence, wherein the second target sequence is in a region spanning position 41,240,553 to position 41,240,770 according to the hgl9 reference genome for chr3.

[0033] In some embodiments of any of the foregoing or related aspects, the second effector domain comprises a second transcriptional repressor moiety. In some embodiments, the second transcriptional repressor moiety comprises a Kruppel associated box (KRAB) domain or a functional variant or fragment thereof. In some embodiments, the second transcriptional repressor moiety comprises a histone modifying enzyme selected from a histone methyltransferase, a histone deacetylase, and a histone demethylase. In some embodiments, the histone modifying enzyme is a histone deacetylase. In some embodiments, the histone deacetylase is 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 thereof. In some embodiments, the histone modifying enzyme is a histone methyltransferase. In some embodiments, the histone methyltransferase is SETDB1, SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof. In some embodiments, the second transcriptional repressor moiety comprises a DNA methyltransferase. In some embodiments, the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A1, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof. In some embodiments, the DNA methyltransferase is MQ1, or a functional variant or fragment thereof.

[0034] In some aspects, the disclosure provides a recombinant expression vector comprising a nucleic acid described herein.

[0035] In some aspects, the disclosure provides a lipid nanoparticle (LNP) comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, or an mRNA described herein.

[0036] In some aspects, the disclosure provides a pharmaceutical composition comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, or a system described herein and a pharmaceutically acceptable carrier.

[0037] In some aspects, the disclosure provides a cell comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein.

[0038] In some aspects, the disclosure provides a method of altering expression of CTNNB1 in a cell, comprising contacting the cell with an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein. In some embodiments, expression of CTNNB1 is decreased.

[0039] In some aspects, the disclosure provides a method of decreasing expression of CTNNB1 in a cell, comprising contacting the cell with expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein, wherein expression of CTNNB1 is decreased by at least about 15%. In some embodiments, expression of CTNNB1 is decreased as compared to a control cell not contacted with the expression repressor, system, nucleic acid, recombinant expression vector, mRNA, LNP, or pharmaceutical composition. In some embodiments, decreased expression of CTNNB1 is measured as a decrease in the level of an RNA transcript of CTNNB1 and / or P-catenin in the cell. In some embodiments, expression of CTNNB1 is decreased by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In some embodiments, expression of CTNNB1 is decreased by about 1.5-fold, about 2-fold, about 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as compared to a control cell not contacted with the expression repressor, system, nucleic acid, recombinant expression vector, mRNA, LNP, or pharmaceutical composition.

[0040] In some aspects, the disclosure provides a method of introducing one or more epigenetic modifications to CTNNB1 in a cell, comprising contacting the cell with an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein. In some embodiments, the epigenetic modification is DNA methylation or histone methylation.

[0041] In some aspects, the disclosure provides a method of treating a condition associated with CTNNB1 expression in a subject, comprising administering to the subject an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein. In some embodiments, the condition is associated with a mutation in CTNNB1. In some embodiments, the condition is associated with overexpression of CTNNB1. In some embodiments, the condition is cancer.

[0042] In some aspects, the disclosure provides a method of treating cancer in a subject comprising administering to the subject an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein.

[0043] In some aspects, the disclosure provides a method of reducing tumor burden in a subject having cancer comprising administering to the subject an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein.

[0044] In some embodiments of any of the foregoing or related aspects, the cancer is associated with a mutation in CTNNB1. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colorectal cancer.

[0045] In some aspects, the disclosure provides a kit comprising a container comprising a pharmaceutical composition comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, or a system described herein, and a pharmaceutically acceptable carrier, and instructions for use in treating a condition associated with CTNNB1 expression in a subject. In some embodiments, the condition is associated with a mutation in CTNNB1. In some embodiments, the condition is associated with overexpression of CTNNB1. In some embodiments, the condition is cancer.

[0046] In some aspects, the disclosure provides a kit comprising a container comprising a pharmaceutical composition comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, or a system described herein, and a pharmaceutically acceptable carrier, and instructions for use in treating cancer in a subject. In some embodiments, the cancer is associated with a mutation in CTNNB1. In some aspects, the disclosure provides a kit comprising a container comprising a pharmaceutical composition comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, or a system described herein, and a pharmaceutically acceptable carrier, and instructions for use in reducing tumor burden in a subject having cancer. In some embodiments, the cancer is associated with a mutation in CTNNB1.

[0047] In some aspects, the disclosure provides use of an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein for treating a condition associated with CTNNB1 expression in a subject, comprising administering the expression repressor, the system, the nucleic acid, the recombinant expression vector, the mRNA, the LNP, or the pharmaceutical composition to the subject.

[0048] In some aspects, the disclosure provides use of an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein for treating cancer in a subject, comprising administering the expression repressor, the system, the nucleic acid, the recombinant expression vector, the mRNA, the LNP, or the pharmaceutical composition to the subject.

[0049] In some aspects, the disclosure provides use of an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein for reducing tumor burden in a subject, comprising administering the expression repressor, the system, the nucleic acid, the recombinant expression vector, the mRNA, the LNP, or the pharmaceutical composition to the subject.

[0050] In some aspects, the disclosure provides use of an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for treating a condition associated with CTNNB1 expression in a subject, comprising administering the medicament o the subject. In some aspects, the disclosure provides use of an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for treating cancer in a subject, comprising administering the medicament to the subject.

[0051] In some aspects, the disclosure provides use of an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for reducing tumor burden in a subject, comprising administering the medicament to the subject.

[0052] In some aspects, the disclosure provides a method of reducing cell viability in a population of cells, comprising contacting the population of cells with a nucleic acid described herein, a recombinant expression vector described herein, an mRNA described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein, thereby reducing cell viability in the population of cells.

[0053] In some embodiments, cell viability is reduced as compared to a population of control cells not contacted with the expression repressor, system, nucleic acid, recombinant expression vector, mRNA, LNP, or pharmaceutical composition. In some embodiments, reduced cell viability is measured as a decrease in cell proliferation.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 provides a schematic depicting a region of the human CTNNB1 gene containing a CpG island and target sequences for exemplary TALEs described herein. In the schematic, transcription occurs left-to-right and top-to-bottom. Indicated is the primary transcript of CTNNB1 and exon 1 of CTNNB1. The scale provides genomic coordinates according to the hgl9 reference genome (genomic coordinates of the CTNNB1 transcript (“CTNNB1”), CTNNB1 exon 1 (“CTNNB1-001 Exon 1”), CpG island, and target sequences as shown by the alignment are approximate).

[0056] FIG. 2 provides a graph depicting the level of B2M and CTNNB1 mRNA following treatment with MC3 LNP-formulated MR892 (TAL02-MQ1) mRNA in K-562 cells as measured by RT-qPCR. mRNA expression was normalized based upon ACTB (housekeeper) mRNA levels. Control cells were untreated. MC3 LNP-formulated mRNA encoding TALE targeting the B2M gene linked to MQ1 (“B2M TAL10”) was used as a positive control.

[0057] FIG. 3 provides boxplots depicting percent methylation of a region containing a CpG island in the CTNNB1 promoter as measured in K-562 cell lysate 72 hours following transfection with LNP-formulated MR883 (TAL01-MQ1), MR892 (TAL02-MQ1), and MR900 (TAL03- MQ1) mRNA. K-562 cells treated with MC3 LNP-formulated MR752 (“methylated control,” commercially available from Zymo Research, cat. no. D5014) was used as a positive control and negative control cells were untreated. DNA methylation was quantified by amplicon enzymatic methyl-seq (Em-Seq), and CpG methylation was determined by the ratio of methylated read coverage to total read coverage for each CpG. Shown are data for three biological replicates per treatment condition (3 technical replicates per each).

[0058] FIGs. 4A-4D provide plots depicting percent methylation of a region containing the CpG island in the CTNNB1 promoter as measured in K-562 cell lysate. DNA methylation was quantified by amplicon enzymatic methyl-seq (Em-Seq) at 72 hours following administration of PBS (FIG. 4A) or LNP-formulated MR883 (TAL01-MQ1) (FIG. 4B), MR892 (TAL02-MQ1) (FIG. 4C), or MR900 (TAL03-MQ1) (FIG. 4D) mRNA. CpG methylation was determined by the ratio of methylated read coverage to total read coverage for each CpG. Dot plot figures show the percent methylation versus relative position of each CpG across the amplicon. Within the dot plots, dot size corresponds to the read depth for that CpG, and color represents technical replicates and biological replicates for biological replicate and group plots, respectively.

[0059] FIG. 5 provides a line graph depicting CTNNB1 mRNA levels over time in K-562 cells following treatment with MC3 LNP-formulated mRNA encoding MR892 (TAL02-MQ1). CTNNB1 mRNA levels were normalized to ACTB (housekeeper) levels and quantified by RT- qPCR. Control cells were untreated.

[0060] FIGs. 6A-6C provide graphs depicting CTNNB1 (P-cat) mRNA or proteins levels in three HCC (Hepatocellular carcinoma cells) cell lines with Wnt / p-catenin pathway mutations, Hep3B (FIG. 6A), HepG2 (FIG. 6B), and SNU-398 (FIG. 6C), 48 or 72 hours following treatment with MC3 LNP-formulated mRNA encoding MR892 (TAL02-MQ1) at indicated concentrations. CTNNB1 (P-cat) mRNA levels were normalized to GAPDH (housekeeper) levels and quantified by RT-qPCR. Control cells were untreated or treated with SNC (short non- coding) mRNA.

[0061] FIGs. 7 -7B provide graphs depicting CTNNB1 (P-cat) mRNA levels and percent methylation of a region containing the CpG island in the CTNNB1 promoter as measured in cell lysates from two HCC cell lines with Wnt / p-catenin pathway mutations, Hep3B (FIG. 7A), and SNU-398 (FIG. 7B), 24 hours following treatment with MC3 LNP-formulated mRNA encoding MR892 (TAL02-MQ1) at indicated concentrations. CTNNB1 (P-cat) mRNA levels were normalized to GAPDH (housekeeper) levels and quantified by RT-qPCR. DNA methylation was quantified by amplicon enzymatic methyl-seq (Em-Seq).

[0062] FIGs. 8A-8C provide plots depicting CTNNB1 (P-cat) mRNA levels and percent cell viability in three HCC cell lines with Wnt / p-catenin pathway mutations, Hep3B (FIG. 8A), HepG2 (FIG. 8B), and SNU-398 (FIG. 8B), 48 to 120 hours following administration of MC3 LNP-formulated mRNA encoding MR892 (TAL02-MQ1) at varying concentrations. CTNNB1 (P-cat) mRNA levels were normalized to GAPDH (housekeeper) levels and quantified by RT- qPCR.

[0063] FIGs. 9A-9B provide graphs depicting IC50 values of viability (FIG. 9A) and CTNNB1 (P-cat) mRNA levels (FIG. 9B), following treatment with MC3 LNP-encapsulated mRNA encoding MR892 (TAL02-MQ1) in either WT or mutant Wnt / p-catenin cell lines (Table 6). CTNNB1 (P-cat) mRNA levels were normalized to GAPDH (housekeeper) levels and quantified by RT-qPCR.

[0064] FIGs. 10A-10C provide graphs depicting tumor volume (mm3) values (FIG. 10A), calculation of the area under curve of tumor volume (FIG. 10B), and percent mouse body weight change (FIG. 10C), following treatment with MC3 LNP-encapsulated mRNA encoding MR892 (TAL02-MQ1) at indicated concentrations and timepoints. Arrows represent specific timepoints of MR892 (TAL02-MQ1) administration.

[0065] FIG. 11 provides a graph depicting CTNNB1 (P-cat) mRNA levels in Hep3B tumors extracted from mice after 26 days following administration of MC3 LNP-formulated mRNA encoding MR892 (TAL02-MQ1) at varying concentrations. CTNNB1 (P-cat) mRNA levels were normalized to GAPDH (housekeeper) levels and quantified by RT-qPCR. DETAILED DESCRIPTION

[0066] The present disclosure is based, at least in part, on the discovery of a region of the genome comprising one or more transcriptional control elements for regulating expression of CTNNB1 (e.g., a region of the genome comprising a CTNNB1 promoter), wherein an expression repressor of the disclosure comprising (i) a DNA targeting moiety (e.g., a ZF, TALE, or dCas9) that binds to a target sequence in the region; and (ii) an effector domain capable of epigenetic modification (e.g., DNA methylation) functions to decrease CTNNB1 expression (e.g., by transcriptional repression) when introduced to a cell (e.g., in vitro or in vivo). As used herein, the term “expression repressor” refers to an agent or entity with one or more functionalities that decreases expression of a target gene in a cell and that specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene).

[0067] As demonstrated herein, introducing an mRNA encoding an exemplary expression repressor of the disclosure to a cell, wherein the exemplary expression repressor comprised (i) a DNA targeting moiety that binds a target sequence in a region of the genome comprising a CTNNB1 transcriptional control element (e.g., a CTNNB1 promoter); and (ii) an effector domain comprising a DNA methyltransferase, resulted in methylation of the genome within the region, thereby decreasing expression of CTNNB1. Such reduction results in reduced levels of CTNNB1 (e.g., in a tumor) for a prolonged period following treatment. Without being bound by theory, administering a dose (e.g., a single dose) of an expression repressor of the disclosure engineered to target a region of the genome comprising a CTNNB1 transcriptional control element (e.g., a CTNNB1 promoter), or a nucleic acid encoding the expression repressor, results in reduction of P-catenin levels, for treatment, alleviation, and / or prevention of P-catenin-expressing cancers.

[0068] Accordingly, in some aspects, the present disclosure provides an expression repressor comprising (i) a DNA targeting moiety that binds a target sequence in a region of the genome comprising a CTNNB1 transcriptional control element (e.g., a CTNNB1 promoter); and (ii) an effector domain. In some embodiments, the expression repressor comprises a single DNA targeting moiety. In some embodiments, the expression repressor comprises more than one DNA targeting moiety. In some embodiments, the expression repressor comprises a single effector domain. In some embodiments, the expression repressor comprises one or more effector domains. In some embodiments, the expression repressor comprises more than one effector domain. In some embodiments, the expression repressor comprises one to four effector domains. In some embodiments, the expression repressor comprises two effector domains. In some embodiments, the target sequence is a span of nucleotides (e.g., 10-50, 10-40, 10-30, 15-30, 15- 25, or 15-20 nucleotides) in or near an insulated genomic domain (IGD) comprising CTNNB1 (“P-catenin IGD”). As further described herein and understood by one of ordinary skill in the art, IGDs are units of genomic space with boundaries defined by factors that mechanistically drive functional insulation between gene transcription activities. Thus, IGDs are physical units that serve to parse chromosomes into discrete functional segments. For example, in some embodiments, an IGD comprises a DNA loop formed by interactions between two DNA sites bound by homodimerized CTCF and cohesin (see Dowen, et al (2014) Cell 159:374-87). In such an IGD, occupation of each of the DNA sites bound by CTCF and cohesin inhibits DNA-bound components on one chromosomal side of the DNA site from interacting with DNA-bound components on the opposite chromosomal side. Consequently, the DNA sites occupied by CTCF and cohesin in such DNA loops act as boundaries for the IGD. In some embodiments, the formation of such DNA loops facilitates (i) enhancer-promoter interactions in which both the enhancer and promoter are within the loop, (ii) inhibition of enhancer-promoter interactions in which one of those elements is within the loop and the other is outside the loop, or (iii) both (i) and (ii).

[0069] In some embodiments, the region of the genome comprising a CTNNB1 transcriptional control element (e.g., a CTNNB1 promoter) spans (i) position 41,197,499 to position 41,200,053 of chr3, according to the hg38 reference genome for chr3; and / or (ii) position 41,238,990 to position 41,241,544 of chr3, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides in the region (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides in the region). In some embodiments, the DNA targeting moiety comprises a polypeptide that binds the target sequence. In some embodiments, the DNA targeting moiety comprises a zinc finger (ZF) domain that binds the target sequence. In some embodiments, the DNA targeting moiety comprises a transcription activator-like effector (TALE) domain that binds the target sequence. In some embodiments, the DNA targeting moiety comprises a catalytically inactive site-directed nuclease (e.g., a catalytically inactive Cas nuclease) and a guide sequence, wherein the guide sequence is complementary, or substantially complementary, to the target sequence. In some embodiments, the effector domain comprises a polypeptide for suppressing gene transcription, e.g., by inducing one or more epigenetic changes. In some embodiments, the effector domain comprises a transcriptional repressor moiety. In some embodiments, the transcriptional repressor moiety recruits components of the endogenous transcriptional machinery to decrease expression of the target gene. In some embodiments, the transcriptional repressor moiety is a polypeptide, that upon binding to a transcriptional control element, recruits one or more corepressor proteins and / or transcription factors to inactivate, or substantially inactivate, gene transcription. In some embodiments, the transcriptional repressor moiety inhibits recruitment of transcription factors, thereby decreasing expression of the target gene. In some embodiments, the transcriptional repressor moiety comprises an epigenetic modifying moiety (e.g., a moiety for introducing an epigenetic modification in or near the target gene). In some embodiments, the transcriptional repressor moiety is an enzyme, that upon binding to a transcriptional control element, catalyzes one or more modifications of a genomic region comprising the transcriptional control element, wherein the one or more modifications inactivates, or substantially inactivates, gene transcription. In some embodiments, the one or more modifications are selected from a DNA modification and a histone modification.

[0070] In some aspects, the disclosure provides a nucleic acid encoding an expression repressor described herein. In some embodiments, the nucleic acid is an mRNA. In some aspects, the disclosure provides a recombinant expression vector comprising the nucleic acid. In some embodiments, the expression repressor, the nucleic acid (e.g., mRNA), or the recombinant expression vector is formulated in a lipid nanoparticle (LNP).

[0071] In some aspects, the disclosure provides a system comprising two or more expression repressors described herein. In some embodiments, the system comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 expression repressors described herein. In some embodiments, the system comprises two or more nucleic acids, wherein each nucleic acid encodes an expression repressor described herein. In some embodiments, the two or more nucleic acids are each mRNAs. In some embodiments, the system comprises two or more recombinant expression vectors, wherein each recombinant expression vector comprises a nucleic acid encoding an expression repressor described herein. In some embodiments, the two or more expression repressors, the two or more nucleic acids, or the two or more recombinant expression vectors are formulated in the same LNP or in different LNPs. In some aspects, the disclosure provides a nucleic acid encoding two or more expression repressors (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 expression repressors) described herein. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the disclosure provides a recombinant expression vector comprising the nucleic acid. In some embodiments, the nucleic acid or the recombinant expression vector is formulated in an LNP.

[0072] In some aspects, the disclosure provides a pharmaceutical composition comprising an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, or a system described herein, and a pharmaceutically acceptable carrier.

[0073] In some aspects, the disclosure provides a method of altering (e.g., decreasing) expression of P-catenin in a cell, comprising contacting the cell with an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein. In some embodiments, expression of P-catenin is decreased compared to a control cell not contacted with the expression repressor, nucleic acid, recombinant expression vector, LNP, system, or pharmaceutical composition.

[0074] In some aspects, the disclosure provides a method of introducing one or more epigenetic modifications to a region comprising a transcriptional control element of CTNNB1 in a cell, the method comprising contacting the cell with an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein. In some embodiments, the transcriptional control element comprises a promoter of CTNNB1. In some embodiments, the one or more epigenetic modifications comprises DNA methylation and / or histone modification.

[0075] In some aspects, the disclosure provides a method of treating a condition associated with CTNNB1 in a subject in need thereof, comprising administering to the subject an effective amount of an expression repressor described herein, a nucleic acid described herein, a recombinant expression vector described herein, an LNP described herein, a system described herein, or a pharmaceutical composition described herein. In some embodiments, the condition is associated with a mutation in CTNNB1. In some embodiments, the condition is associated with dysregulated expression (e.g., overexpression) of P-catenin. In some embodiments, the condition is cancer.

[0076] P-catenin Expression Repressors

[0077] In some embodiments, the disclosure provides an expression repressor for altering (e.g., decreasing) expression of P-catenin (e.g., human P-catenin). In some embodiments, the disclosure provides an expression repressor for decreasing expression of human P-catenin. As used herein, the term “human CTNNB1” (and corresponding protein “human P-catenin”) refers to a gene on human chromosome 3 encoding Catenin beta-1. In some embodiments, human CTNNB1 has the genomic coordinates (i) 41,194,741 to 41,260,096, according to human reference genome hg38 of chr3; and / or (ii) 41,236,232 -41,301,587, according to human reference genome hgl9 of chr3. The human CTNNB1 gene encodes a 781 amino acid protein. See also, e.g., Ensembl ENSG00000168036 providing human CTNNB1; Ensembl ENST00000349496 and NCBI Ref. Seq NM_001904 providing the human P-catenin mRNA sequence; and UniProt P35222 and NCBI Reference Sequence NP_001895 providing the corresponding human P-catenin polypeptide.

[0078] In some embodiments, an expression repressor of the disclosure has a targeting function and an effector function. In some embodiments, the targeting function localizes the effector function of the expression repressor to a region of the genome. In some embodiments, the region of the genome comprises the CTNNB1 IGD. In some embodiments, the region of the genome is in the CTNNB1 IGD. In some embodiments, the effector function comprises introducing one or more epigenetic modifications to the region of the genome.

[0079] In some embodiments, the expression repressor comprises a DNA targeting moiety and an effector domain. In some embodiments, the targeting function of the expression repressor is mediated by the DNA targeting moiety. In some embodiments, the targeting function is mediated by the DNA targeting moiety binding to a target sequence in the region of the genome.

[0080] In some embodiments, the effector domain is a transcriptional repressor moiety described herein. In some embodiments, the DNA targeting moiety binds to a target sequence in the CTNNB1 gene, whereupon the effector domain of the expression repressor functions to introduce one or more epigenetic modifications to a region in the CTNNB1 gene. In some embodiments, the DNA targeting moiety binds to a target sequence in a genomic region comprising the CTNNB1 IGD (e.g., the human CTNNB1 IGD), whereupon the effector domain of the expression repressor functions to introduce one or more epigenetic modifications to a region in or near the CTNNB1 IGD (e.g., the human CTNNB1 IGD). In some embodiments, the DNA targeting moiety binds to a target sequence in the CTNNB1 IGD (e.g., the human CTNNB1 IGD), whereupon the effector domain of the expression repressor functions to introduce one or more epigenetic modifications to a region in the CTNNB1 IGD (e.g., the human CTNNB1 IGD). In some embodiments, one or more epigenetic modifications is introduced to a transcriptional control element (e.g., promoter or enhancer) of CTNNB1 (e.g., human CTNNB1), or a portion thereof. In some embodiments, the one or more epigenetic modifications results in decreased expression of P-catenin (e.g., human P-catenin), e.g., as compared to a control cell not contacted with the expression repressor.

[0081] Target Sequences

[0082] In some embodiments, the DNA targeting moiety binds to a target sequence in the CTNNB1 gene. In some embodiments, the DNA targeting moiety binds to a target sequence in the human CTNNB1 gene. In some embodiments, the DNA targeting moiety binds to a target sequence in a genomic region comprising the CTNNB1 IGD. In some embodiments, the DNA targeting moiety binds to a target sequence in the CTNNB1 IGD. In some embodiments, the DNA targeting moiety binds to a target sequence in the human CTNNB1 IGD. In some embodiments, the human CTNNB1 IGD comprises the genomic coordinates (i) 40,574,145 to 41,897,813, according to human reference genome hg38 of chr3; and / or (ii) 40,615,636 - 41,939,305, according to human reference genome hgl9 of chr3.

[0083] In some embodiments, the DNA targeting moiety comprises a ZF that binds the target sequence in a genomic region comprising the CTNNB1 IGD. In some embodiments, the DNA targeting moiety comprises a ZF that binds the target sequence in the CTNNB1 IGD. In some embodiments, the DNA targeting moiety comprises a TALE that binds the target sequence in a genomic region comprising the CTNNB1 IGD. In some embodiments, the DNA targeting moiety comprises a TALE that binds the target sequence in the CTNNB1 IGD. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site- directed nuclease) that binds the target sequence in a genomic region comprising the CTNNB1 IGD. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) that binds the target sequence in the CTNNB1 IGD. In some embodiments, the site-directed nuclease comprises a Cas nuclease described herein (e.g., a catalytically inactive Cas nuclease) and a gRNA comprising a spacer sequence corresponding to the target sequence. The spacer sequence is a sequence that defines the target sequence in the CTNNB1 IGD. The target sequence is present in a double-stranded genomic DNA having one strand comprising the target sequence comprising a protospacer sequence adjacent to a PAM sequence that is referred to as the “PAM strand,” and a second strand that is referred to as the “non-PAM strand” and is complementary to the PAM strand. Both the gRNA spacer sequence and the target sequence are complementary to the non-PAM strand of the genomic DNA molecule. As used herein, a spacer sequence “corresponding to” a target sequence refers to a guide sequence that binds to the non-PAM strand of the target sequence by Watson-Crick basepairing, wherein the spacer sequence has sufficient complementarity to the non-PAM strand as to enable targeting of the Cas nuclease to the target sequence in the genomic DNA molecule. In some embodiments, the spacer sequence has up to 1, 2, or 3 mismatches relative to the target sequence in the genomic DNA molecule, wherein the spacer sequence has sufficient complementarity to the non-PAM strand as to enable targeting of the Cas nuclease to the target sequence in the genomic DNA molecule.

[0084] In some embodiments, the DNA targeting moiety binds to a target sequence in a genomic region comprising the human CTNNB1 IGD, wherein the target sequence is upstream of or in a 5 'boundary of the human CTNNB1 IGD. In some embodiments, the target sequence is between a 5' and 3 'boundary of the human CTNNB1 IGD. In some embodiments, the target sequence is downstream of or in the 3 'boundary of the human CTNNB1 IGD. In some embodiments, the DNA targeting moiety binds to a target sequence in the human CTNNB1 IGD, wherein the target sequence is in a region (e.g., a 0.5-2kb region) comprising a transcriptional control element (e.g., a promoter or enhancer). In some embodiments, the region comprises a promoter. In some embodiments, the target sequence is in a promoter. In some embodiments, the region comprises an enhancer. In some embodiments, the target sequence is in an enhancer. In some embodiments, the target sequence is in or near a CpG island in the human CTNNB1 IGD. In some embodiments, the target is in a region of about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, about 1,000 bases, about 1,100 bases, about 1,200 bases, about 1,300 bases, about 1,400 bases, about 1,500 bases, about 1,600 bases, about 1,700 bases, about 1,800 bases, about 1,900 bases, or about 2,000 bases comprising the CpG island. In some embodiments, the target is in a region of about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1,000 bases comprising the CpG island. In some embodiments, the target sequence is not more than about 300 bases, about 400 bases, or about 500 bases upstream or downstream the CpG island. In some embodiments, the target sequence is in the CpG island.

[0085] In some embodiments, the DNA targeting moiety binds to a target sequence in the human CTNNB1 IGD, wherein the target sequence is in a region (e.g., a 0.5-2kb region) comprising a transcriptional control element (e.g., a promoter) of human CTNNB1. In some embodiments, the target sequence is in a region comprising a human CTNNB1 promoter. As used herein, “a human CTNNB1 promoter” refers to a genomic region upstream of a transcriptional start sequence (TSS) of a CTNNB1 transcript. The promoter may include 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, or 1000 bp upstream of a TSS. The promoter may comprise or lie within (i) hg38 chr3: 41,197,499-41,200,053; and / or (ii) hgl9 chr3: 41,238,990 -41,241,544. Human P-catenin has multiple TSSs, and any TSS recognized in the art may be used to define a promoter sequence. For example, and without limitation, the TSS may comprise (i) hg38 chr3: 41,199,505; and / or (ii) hgl9 chr3: 41,250,996. In some embodiments, the target sequence is in a region comprising an enhancer of human P-catenin. In some embodiments, the target sequence is in a coding region of human P-catenin.

[0086] The length of the target sequence depends on the DNA targeting moiety used. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is 15 nucleotides. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is 16 nucleotides. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is 17 nucleotides. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is 18 nucleotides. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is 19 nucleotides. In some embodiments, the DNA targeting moiety comprises a ZF and the target sequence is 20 nucleotides.

[0087] In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is 15 nucleotides. In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is 16 nucleotides. In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is 17 nucleotides. In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is 18 nucleotides. In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is 19 nucleotides. In some embodiments, the DNA targeting moiety comprises a TALE and the target sequence is 20 nucleotides.

[0088] In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 15 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 16 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 17 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 18 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 19 nucleotides. In some embodiments, the DNA targeting moiety comprises a site-directed nuclease (e.g., a catalytically inactive site-directed nuclease) and the target sequence is 20 nucleotides.

[0089] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a genomic region comprising the CTNNB1 IGD (e.g., the human CTNNB1 IGD). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a genomic region comprising the CTNNB1 IGD (e.g., the human CTNNB1 IGD). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a genomic region comprising the CTNNB1 IGD (e.g., the human CTNNBHGD

[0090] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in the CTNNB1 IGD (e.g., the human CTNNB1 IGD). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in the CTNNB1 IGD (e.g., the human CTNNB1 IGD). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in the CTNNB1 IGD (e.g., the human CTNNB1 IGD).

[0091] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1 -2kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a transcriptional control element (e.g., a promoter or enhancer). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a transcriptional control element (e.g., a promoter or enhancer). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a transcriptional control element (e.g., a promoter or enhancer).

[0092] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a transcriptional control element (e.g., a promoter or enhancer) in the CTNNB1 IGD (e.g., human CTNNB1 IGD). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a transcriptional control element (e.g., a promoter or enhancer) in the CTNNB1 IGD (e.g., human CTNNB1 IGD). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a transcriptional control element (e.g., a promoter or enhancer) in the CTNNB1 IGD (e.g., human CTNNB1 IGD).

[0093] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a promoter. In some embodiments, the target sequence is within or overlapping the promoter. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a promoter. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a promoter.

[0094] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a promoter in the CTNNB1 IGD (e.g., human CTNNB1 IGD). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a promoter in the CTNNB1 IGD (e.g., human CTNNB1 IGD). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a promoter in the CTNNB1 IGD (e.g., human CTNNB1 IGD). In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1 -2kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises an enhancer. In some embodiments, the target sequence is within or overlapping the enhancer. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1 -2kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises an enhancer. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1 -2kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises an enhancer.

[0095] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in an enhancer in the CTNNB1 IGD (e.g., human CTNNB1 IGD). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in an enhancer in the CTNNB1 IGD (e.g., human CTNNB1 IGD). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in an enhancer in the CTNNB1 IGD (e.g., human CTNNB1 IGD).

[0096] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1 -2kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTCF binding site (e.g., a CTCF binding site at a boundary of the CTNNB1 IGD or a CTCF binding site in the CTNNB1 IGD). In some embodiments, the target sequence is within or overlapping the CTCF binding site. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTCF binding site (e.g., a CTCF binding site at a boundary of the CTNNB1 IGD or a CTCF binding site in the CTNNB1 IGD). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTCF binding site (e.g., a CTCF binding site at a boundary of the CTNNB1 IGD or a CTCF binding site in the CTNNB1 IGD).

[0097] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTNNB1 enhancer (e.g., a human CTNNB1 enhancer). In some embodiments, the target sequence is within or overlapping the CTNNB1 enhancer. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTNNB1 enhancer (e.g., a human CTNNB1 enhancer). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTNNB1 enhancer (e.g., a human CTNNB1 enhancer).

[0098] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CpG island. In some embodiments, the target sequence is within or overlapping the CpG island. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1 -2kb region) of the of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CpG island. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CpG island. In some embodiments, the region comprising a CpG island spans (i) position 41,198,672 to position 41,200,140, according to the hg38 reference genome for chr3; and / or (ii) position 41,240,163 to position 41,241,631, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a CTNNB1 enhancer (e.g., a human CTNNB1 enhancer). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a CTNNB1 enhancer (e.g., a human CTNNB1 enhancer). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a CTNNB1 enhancer (e.g., a human CTNNB1 enhancer).

[0099] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTNNB1 promoter (e.g., a human CTNNB1 promoter). In some embodiments, the target sequence is within or overlapping the CTNNB1 promoter. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTNNB1 promoter (e.g., a human CTNNB1 promoter). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of the CTNNB1 IGD (e.g., the human CTNNB1 IGD), wherein the region comprises a CTNNB1 promoter (e.g., a human CTNNB1 promoter).

[0100] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a CTNNB1 promoter (e.g., a human CTNNB1 promoter). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a CTNNB1 promoter (e.g., a human CTNNB1 promoter). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a CTNNB1 promoter (e.g., a human CTNNB1 promoter).

[0101] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region (e.g., a 0.1-2 kb region) of CTNNB1 (e.g., human CTNNB1 ). In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of CTNNB1 (e.g., human CTNNB1). In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region (e.g., a 0.1-2 kb region) of CTNNB1 (e.g., human CTNNBI).

[0102] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position 41,240,070 to position 41,241,723, according to the hgl9 reference genome for chr3.

[0103] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position

[0104] 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position

[0105] 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position

[0106] 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position

[0107] 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position

[0108] 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3.

[0109] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position 41,240,070 to position 41,240,487, according to the hgl9 reference genome for chr3.

[0110] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position

[0111] 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position

[0112] 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position

[0113] 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position

[0114] 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position

[0115] 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3.

[0116] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,453 to position 41,240,870, according to the hg!9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position

[0117] 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position

[0118] 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position

[0119] 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position

[0120] 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position

[0121] 41,240,453 to position 41,240,870, according to the hgl9 reference genome for chr3.

[0122] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position 41,240,553 to position 41,240,770, according to the hg!9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position

[0123] 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position

[0124] 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position

[0125] 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position

[0126] 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3.

[0127] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position

[0128] 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position

[0129] 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position

[0130] 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position

[0131] 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position

[0132] 41,241,306 to position 41,241,723, according to the hgl9 reference genome for chr3.

[0133] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides in a region spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in a region spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 15 nucleotides in a region spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 16 nucleotides in a region spanning position

[0134] 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 17 nucleotides in a region spanning position

[0135] 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 18 nucleotides in a region spanning position

[0136] 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 19 nucleotides in a region spanning position

[0137] 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is about 20 nucleotides in a region spanning position

[0138] 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3.

[0139] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,150 to position 41,240,250, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,200 to position 41,240,300, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,170 to position 41,240,300, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,250 to position 41,240,350, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15- 20 nucleotides) in a region spanning position 41,240,300 to position 41,240,400, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,350 to position 41,240,450, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,400 to position 41,240,500, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,450 to position 41,240,550, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,500 to position 41,240,600, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,550 to position 41,240,650, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,600 to position 41,240,700, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,650 to position 41,240,750, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,700 to position 41,240,800, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15- 20 nucleotides) in a region spanning position 41,240,750 to position 41,240,850, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,800 to position 41,240,900, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,850 to position 41,240,950, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,900 to position 41,241,000, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,240,950 to position 41,241,050, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,000 to position 41,241,100, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,050 to position 41,241,150, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,100 to position 41,241,200, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,150 to position 41,241,250, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15- 20 nucleotides) in a region spanning position 41,241,200 to position 41,241,300, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,250 to position 41,241,350, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,300 to position 41,241,400, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,350 to position 41,241,450, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,400 to position 41,241,523, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,450 to position 41,241,550, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,500 to position 41,241,600, according to the hgl9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,500 to position 41,241,623, according to the hg!9 reference genome for chr3. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) in a region spanning position 41,241,550 to position 41,241,650, according to the hgl9 reference genome for chr3.

[0140] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) comprising at least 10 contiguous nucleotides of a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the target sequence is about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 15 to about 20 nucleotides comprising at least 10 contiguous nucleotides of a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the target sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides comprising at least 10 contiguous nucleotides of a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the target sequence is (i) 15 nucleotides and comprises 10-15 contiguous nucleotides of the sequence; (ii) 16 nucleotides and comprises 10-16 contiguous nucleotides of the sequence; (iii) 17 nucleotides and comprises 10-17 contiguous nucleotides of the sequence; (iv) 18 nucleotides and comprises 10-18 contiguous nucleotides of the sequence; (v) 19 nucleotides and comprises 10-19 contiguous nucleotides of the sequence; or (vi) 20 nucleotides and comprises 10-20 contiguous nucleotides of the sequence.

[0141] In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15- 30, 15-25, or 15-20 nucleotides) comprising at least 10 contiguous nucleotides of SEQ ID NO: 8. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) comprising at least 10 contiguous nucleotides of SEQ ID NO: 14. In some embodiments, the target sequence is 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15- 20 nucleotides) comprising at least 10 contiguous nucleotides of SEQ ID NO: 20.

[0142] In some embodiments, the target sequence is 18-50 nucleotides (e.g., 18-40, 18-35, 18- 30, 18-25, or 18-20 nucleotides) comprising a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the target sequence is about 18 to about 50 nucleotides, about 18 to about 40 nucleotides, about 18 to about 30 nucleotides, or about 18 to about 20 nucleotides comprising a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the target sequence is about 18, about 19, or about 20 nucleotides comprising a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the target sequence is 18-50 nucleotides (e.g., 18-40, 18-35, 18- 30, 18-25, or 18-20 nucleotides) comprising SEQ ID NO: 8. In some embodiments, the target sequence is 18-50 nucleotides (e.g., 18-40, 18-35, 18-30, 18-25, or 18-20 nucleotides) comprising SEQ ID NO: 14. In some embodiments, the target sequence is 18-50 nucleotides (e.g., 18-40, 18-35, 18-30, 18-25, or 18-20 nucleotides) comprising SEQ ID NO: 20.

[0143] In some embodiments, the target sequence is about 10-50 nucleotides (e.g., 10-40, 10-30, 15-30, 15-25, or 15-20 nucleotides) within a region of about 200 or fewer nucleotides, wherein the region comprises at least 10 contiguous nucleotides of a sequence selected from SEQ ID NOs: 8, 14, or 20, and wherein the region is located within position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the region comprises 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous nucleotides of the sequence. In some embodiments, the region is about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 45, about 40, about 35, about 30, about 25, or about 20 nucleotides. In some embodiments, the target sequence is 15, 16, 17, 18, 19, or 20 nucleotides.

[0144] Exemplary target sequence of the disclosure in the human CTNNB1 IGD are set forth in Table 1.

[0145] In some embodiments, the DNA targeting moiety binds to the target sequence with submicromolar or nanomolar binding affinity (KD). Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluated and rank strengths of bimolecular interactions. As used herein, the term “KD” or “KD” refers to the equilibrium dissociation constant of a binding reaction between an DNA targeting moiety and a target sequence. The value of KD is a numeric representation of the ratio of the DNA targeting moiety off-rate constant (kd) to the on-rate constant (ka). The value of KD is inversely related to the binding affinity of the DNA targeting moiety and target sequence. The smaller the KD value the greater the affinity. As used herein, the term “kd” or “kd” (alternatively “koff” or “koff”) is intended to refer to the off- rate constant for the dissociation of the DNA targeting moiety from a complex of the DNA targeting moiety and the target sequence. The value of kd is a numeric representation of the fraction of complexes that decay or dissociate per second, and is expressed in units sec1. As used herein, the term “ka” or “ka” (alternatively “kon” or “kon”) is intended to refer to the on-rate constant for the association of the DNA targeting moiety and the target sequence. The value of ka is a numeric representation of the number of DNA targeting moiety / target DNA complexes formed per second in a 1 molar (IM) solution of the DNA targeting moiety and the target DNA, and is expressed in units M 'sec1. Methods to measure binding affinity (KD) of a DNA targeting moiety to the target sequence include, but are not limited to, DNA electrophoretic mobility shift assay (EMSA) and surface plasmon resonance.

[0146] In some embodiments, the DNA targeting moiety (e.g., TALE, ZF, dCas9 / gRNA) binds to the target sequence with an affinity (KD) of less than about 5pM, about 4.5pM, about 4pM, about 3.5pM, about 3pM, about 2.5pM, about 2pM, about 1 ,5pM, about I pM, about 0.5 pM, or about 0.1 pM. In some embodiments, the DNA targeting moiety (e.g., TALE, ZF, dCas9 / gRNA) binds to the target sequence with an affinity (KD) that is no greater than about 950, 900, 850, 800, 750, 700, 650, 600, 500, 500, 450, 400, 350, 300, 250, 200, 250, 200, 175, 150, 125, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 nM. In some embodiments, the DNA targeting moiety binds to the target sequence with an affinity (KD) of about 1 nM to about 100 nM, about 10 nM to about 500 nM, about 100 nM to about IpM, about 500 nM to about 1 pM, about 500 nM to about 2 pM, about 1 pM to about 2 pM, about 1 pM to about 3 pM, about 1 pM to about 4 pM, or about 1 pM to about 5 pM. In some embodiments, the DNA targeting moiety binds to the target sequence with an affinity (KD) of about 1 nM to about 10 nM, about 1 nM to about 20 nM, about 1 nM to about 30 nM, about 1 nM to about 40 nM, about 1 nM to about 50 nM, about 10 nM to about 50 nM, about 10 nM to about 100 nM, about 10 nM to about 200 nM, about 50 nM to about 200 nM, about 50 nM to about 300 nM, about 50 nM to about 400 nM, or about 50 nM to about 50 nM.

[0147] Table 1: Exemplary target sequences of the disclosure

[0148] * According to human reference genome hgl9 of chromosome 3

[0149] ** According to human reference genome hg38 of chromosome 3 DNA Targeting Moiety

[0150] The present disclosure provides, e.g., expression repressors comprising a DNA targeting moiety that specifically targets, e.g., binds, a genomic sequence element (e.g., a promoter, a TSS, or an anchor sequence) in, proximal to, and / or operably linked to a target gene. In some embodiments, the DNA targeting moiety specifically binds to a DNA sequence, e.g., a DNA sequence associated with a target gene, e.g., CTNNB1. Any molecule or compound that specifically binds a DNA sequence may be used as a DNA targeting moiety.

[0151] In some embodiments, the DNA targeting moiety targets, e.g., binds, a component of a genomic complex. In some embodiments, the DNA targeting moiety targets, e.g., binds, a transcriptional control sequence (e.g., a promoter or enhancer) operably linked to the target gene (e.g., CTNNB1). In some embodiments, the DNA targeting moiety targets, e.g., binds, a target gene or a part of a target gene (e.g., CTNNB1). The target of a DNA targeting moiety may be referred to as its targeted component. A targeted component may be any genomic sequence element operably linked to a target gene, or the target gene itself, including but not limited to a promoter, enhancer, anchor sequence, exon, intron, UTR encoding sequence, a splice site, or a transcription start site. In some embodiments, the DNA targeting moiety binds specifically to one or more target anchor sequences (e.g., within a cell) and not to non-targeted anchor sequences (e.g., within the same cell).

[0152] In some embodiments, the DNA targeting moiety comprises a CRISPR / Cas domain (e.g., a catalytically inactive CRISPR / Cas domain), a TAL effector domain, a Zn finger domain, a peptide nucleic acid (PNA), or a nucleic acid molecule.

[0153] In some embodiments, an expression repressor of the disclosure comprises one DNA targeting moiety. In some embodiments, the expression repressor comprises a plurality of DNA targeting moieties, wherein each DNA targeting moiety does not detectably bind, e.g., does not bind, to another DNA targeting moiety.

[0154] In some embodiments, the DNA targeting moiety binds to its target sequence with a KD of less than or equal to 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 (and optionally, a KD 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). In some embodiments, the DNA targeting moiety binds to its target sequence with a KD of 0.001 nM to 500 nM, e.g., 0.1 nM to 5 nM, e.g., about 0.5 nM. In some embodiments, a DNA targeting moiety binds to a non-target sequence with a KD of at least 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, or 100,000 nM (and optionally, does not appreciably bind to a non-target sequence). In some embodiments, the DNA targeting moiety does not substantially bind to a non-target sequence.

[0155] CRISPR / Cas Domains

[0156] In some embodiments, the DNA targeting moiety comprises a CRISPR / Cas domain. A CRISPR / Cas protein can comprise a CRISPR / Cas effector and optionally one or more other domains. A CRISPR / Cas domain typically has structural and / or functional similarity to a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., a Cas protein. The CRISPR / Cas domain optionally comprises a guide RNA, e.g., single guide RNA (sgRNA). In some embodiments, the gRNA comprised by the CRISPR / Cas domain is noncovalently bound by the CRISPR / Cas domain.

[0157] CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e.g., Cas9 or Cpfl) to cleave foreign DNA. For example, in a typical CRISPR / Cas system, an endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome that is to be sequence-edited) by sequence specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “guide RNA”, typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence. crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure which is cleaved by Rnase III, resulting in a crRNA / tracrRNA hybrid. A crRNA / tracrRNA hybrid then directs Cas9 endonuclease to recognize and cleave a target DNA sequence. A target DNA sequence must generally be adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences appear throughout a given genome. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5’-NGG (Streptococcus pyogenes'), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), and 5’-NNNGATT (Neisseria meningiditis). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5’-NGG, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5’ from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpfl , which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system requires only Cpfl nuclease and a crRNA to cleave a target DNA sequence. Cpfl endonucleases, are associated with T-rich PAM sites, e.g., 5’-TTN. Cpfl can also recognize a 5’-CTA PAM motif. Cpfl cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5 -nucleotide 5’ overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) from a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759-771.

[0158] A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Casl, Cas8, Cas9, Cas 10, Cpfl, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be 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, a DNA-targeting moiety includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus (e.g., an S. pyogenes, or an S. thermophilus), a Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.

[0159] In some embodiments, a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and / or function. In some embodiments, the PAM is or comprises, from 5' to 3', NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G. In some embodiments, a Cas protein is a protein listed in Table 2. In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises DI 135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions.

[0160] Table 2: Exemplary Cas Proteins of the Disclosure

[0161]

[0162] In some embodiments, the Cas protein is modified to deactivate the nuclease, e.g., nuclease-deficient Cas. In some embodiments, the Cas protein is a Cas9 protein. Whereas wildtype Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, dCas binding to a DNA sequence may interfere with transcription at that site by steric hindrance. In some embodiments, a DNA-targeting moiety is or comprises a catalytically inactive Cas, e.g., dCas. Many catalytically inactive Cas proteins are known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A mutations.

[0163] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a DI 1A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H969A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a N995A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises DI 1A, H969A, and N995A mutations or analogous substitutions to the amino acids corresponding to said positions.

[0164] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H557A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D10A and H557A mutations or analogous substitutions to the amino acids corresponding to said positions.

[0165] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D839A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H840A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a N863A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D10A, D839A, H840A, and N863A mutations or analogous substitutions to the amino acids corresponding to said positions.

[0166] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a E993A mutation or an analogous substitution to the amino acid corresponding to said position.

[0167] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a E1006A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D1255A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D917A, E1006A, and D1255A mutations or analogous substitutions to the amino acids corresponding to said positions.

[0168] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D587A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H588A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises an N611A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D16A, D587A, H588A, and N611A mutations or analogous substitutions to the amino acids corresponding to said positions.

[0169] In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) DNA targeting moiety and one or more effector domain, wherein the one or more DNA targeting moiety is or comprises a CRISPR / Cas domain comprising a Cas protein, e.g., catalytically inactive Cas9 protein, e.g., dCas9, or a functional variant or fragment thereof. In some embodiments, dCas9 comprises an amino acid sequence of SEQ ID NO:35.

[0170] In some embodiments, the dCas9 is encoded by a nucleic acid sequence of SEQ ID NO:36.

[0171] In some embodiments, a DNA targeting moiety comprises a Cas domain comprising or linked (e.g., covalently linked) to a gRNA. A gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas-protein binding and a user-defined about 20 nucleotide targeting sequence for a genomic target. In practice, guide RNA sequences are generally designed to have a length of between 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to the targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. Gene editing has also been achieved using a chimeric “single guide RNA” (“sgRNA”), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective for use with Cas proteins; see, for example, Hendel et al. (2015) Nature Biotechnol, 985 - 991.

[0172] In some embodiments, a gRNA comprises a nucleic acid sequence that is complementary to a target sequence described herein. In some embodiments, a gRNA comprises a nucleic acid sequence that is at least 90, 95, 99, or 100% complementary to a target sequence described herein. In some embodiments, a gRNA for use with a DNA-targeting moiety that comprises a Cas molecule is an sgRNA.

[0173] TAL Domains

[0174] In some embodiments, a DNA-targeting moiety is or comprises a TAL effector (also sometimes referred to herein as a “TALE”) domain. A TAL effector domain, e.g., a TAL effector domain that specifically binds a DNA sequence, comprises a plurality of TAL effector repeats or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effector repeats (e.g., N- and / or C-terminal of the plurality of TAL effector domains) wherein each TAL effector repeat recognizes a nucleotide. In some embodiments, a TAL effector protein can comprise a TAL effector domain and optionally one or more other domains. Many TAL effector domains are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.

[0175] TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanthomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat variable di-residues, RVD domain).

[0176] Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats ranges from 1.5 to 33.5 repeats and the C-terminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “halfrepeat”. Each repeat of the TAL effector features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one basepair on the target gene sequence). Generally, the smaller the number of repeats, the weaker the protein-DNA interactions. A number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).

[0177] Repeat to repeat variations occur predominantly at amino acid positions 12 and 13, which have therefore been termed “hypervariable” and which are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 3 listing exemplary repeat variable di-residues (RVD) and their correspondence to nucleic acid base targets. Table 3: RVDs and Nucleic Acid Base Specificity

[0178] Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5' base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXalO and AvrBs3.

[0179] Accordingly, in some embodiments, the TAL effector repeat of the TAL effector domain of the present disclosure may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain strain 756C and Xanthomonas oryzae pv. Oryzicolastrain BLS256 (Bogdanove et al. 2011). As used herein, the TAL effector domain in accordance with the present disclosure comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and / or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. In some embodiments, it may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector domain. The TAL effector domain of the present disclosure is designed to target a given DNA sequence based on the above code and others known in the art. The number of TAL effector repeats (e.g., monomers or modules) and their specific sequence(s) are selected based on the desired DNA target sequence. For example, TAL effector repeats may be removed or added in order to suit a specific target sequence. In an embodiment, the TAL effector domain of the present disclosure comprises between 6.5 and 33.5 TAL effector repeats. In an embodiment, TAL effector domain of the present disclosure comprises between 8 and 33.5 TAL effector repeats, e.g., between 10 and 25 TAL effector repeats, e.g., between 10 and 14 TAL effector repeats. In some embodiments, the TAL effector domain comprises TAL effector repeats that correspond to a perfect match to the DNA target sequence. In some embodiments, a mismatch between a repeat and a target base-pair on the DNA target sequence is permitted as along as it allows for the function of the expression repression system, e.g., the expression repressor comprising the TAL effector domain. In general, TALE binding is inversely correlated with the number of mismatches. In some embodiments, the TAL effector domain of an expression repressor of the present disclosure comprises no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, 2 mismatches, or 1 mismatch, and optionally no mismatch, with the target DNA sequence. Without wishing to be bound by theory, in general the smaller the number of TAL effector repeats in the TAL effector domain, the smaller the number of mismatches will be tolerated while still allowing for the function of the expression repressor or expression repressor system, e.g., the expression repressor comprising the TAL effector domain. The binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, TAL effector domains having 25 TAL effector repeats or more may be able to tolerate up to 7 mismatches.

[0180] In addition to the TAL effector repeats, in some embodiments, the TAL effector domain of the present disclosure may comprise additional sequences derived from a naturally occurring TAL effector. The length of the C-terminal and / or N-terminal sequence(s) included on each side of the TAL effector repeat portion of the TAL effector domain can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL- effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of the N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector repeats of the naturally occurring TAL effector are included in the TAL effector domain of an expression repressor of the present disclosure. Accordingly, in an embodiment, a TAL effector domain of the present disclosure comprises 1) one or more TAL effector repeats derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector repeats; and / or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side of the TAL effector repeats.

[0181] In some embodiments, a modulating agent comprises a DNA targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a TAL effector comprising a TAL effector repeat that binds to a target sequence, e.g., a promoter or transcription start site (TSS)) sequence operably linked to a target gene (e.g., CTNNB1), e.g., a sequence proximal to the transcription regulatory element, e.g., an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene (e.g., CTNNB1), e.g., a sequence proximal to the anchor sequence. In some embodiments, the TAL effector binds to a target sequence described herein. In some embodiments, the TAL effector domain can be engineered to carry epigenetic effector domains to target sites.

[0182] The amino acid sequences of exemplary DNA targeting moieties disclosed herein are listed in Table 4. In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence set forth in Table 4, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0183] Table 4: Protein Sequences for Exemplary TAL Effector Domains of the Disclosure and Corresponding Target Sequence in the human CTNNB1 IGD

[0184] * According to human reference genome hgl9 of chromosome 3

[0185] ** According to human reference genome hg38 of chromosome 3 In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence set forth in SEQ ID NO:5. In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the sequence set forth in SEQ ID NO:5. In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence with at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the sequence set forth in SEQ ID NO:5.

[0186] In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence set forth in SEQ ID NO: 13. In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 13. In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence with at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the sequence set forth in SEQ ID NO:13.

[0187] In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence set forth in SEQ ID NO: 19. In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 19. In some embodiments, an expression repressor or system described herein comprises a DNA targeting moiety comprising a sequence with at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the sequence set forth in SEQ ID NO:19.

[0188] Zn Finger domains

[0189] In some embodiments, a DNA-targeting moiety is or comprises a Zn finger domain. A Zn finger domain comprises a Zn finger, e.g., a naturally occurring Zn finger or engineered Zn finger, or fragment thereof. Many Zn fingers are known to those of skill in the art and are commercially available, e.g., from Sigma- Aldrich. Generally, a Zn finger domain comprises a plurality of Zn fingers, wherein each Zn finger recognizes three nucleotides. A Zn finger protein can comprise a Zn finger domain and optionally one or more other domains.

[0190] In some embodiments, a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol. 20: 135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties.

[0191] An engineered Zn finger may have a novel binding specificity, compared to a naturally occurring Zn finger. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.

[0192] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; and WO 01 / 88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02 / 077227.

[0193] In addition, as disclosed in these and other references, zinc fingers and / or multi-fingered zinc finger domains may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in International Patent Publication No. WO 02 / 077227.

[0194] Zn fingers and methods for design and construction of expression repressors (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos. WO 95 / 19431; WO 96 / 06166; WO 98 / 53057; WO 98 / 54311; WO 00 / 27878; WO 01 / 60970; WO 01 / 88197; WO 02 / 099084; WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO 03 / 016496.

[0195] In certain embodiments, the DNA-targeting moiety comprises a Zn finger domain comprising an engineered zinc finger that binds (in a sequence-specific manner) to a target DNA sequence. In some embodiments, the Zn finger domain comprises one Zn finger or fragment thereof. In some embodiments, the Zn finger domain comprises a plurality of Zn fingers (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn fingers (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn fingers). In some embodiments, the Zn finger domain comprises at least three Zn fingers. In some embodiments, the Zn finger domain comprises four, five or six Zn fingers. In some embodiments, the Zn finger domain comprises 8, 9, 10, 11 or 12 Zn fingers. In some embodiments, a Zn finger domain comprising three Zn fingers recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger domain comprising four Zn fingers recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger domain comprising six Zn fingers recognizes a target DNA sequence comprising 18 to 21 nucleotides.

[0196] In some embodiments, a DNA targeting domain comprises a two-handed Zn finger protein. Two handed zinc finger proteins are those proteins in which two clusters of zinc fingers are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two-handed type of zinc finger binding protein is SIP1, where a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three Zn fingers is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these domains is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides. In some embodiments, an expression repressor comprises a DNA targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a Zn finger domain comprising a Zn finger (ZFN) that binds to a target sequence, e.g., a promoter or transcription start site (TSS)) sequence operably linked to a target gene (e.g., CTNNB1), e.g., a sequence proximal to the transcription regulatory element, e.g., an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene (e.g., CTNNB1), e.g., a sequence proximal to the anchor sequence. In some embodiments, the ZFN binds to a target sequence described herein. In some embodiments, the ZFN can be engineered to carry epigenetic effector molecules to target sites.

[0197] Effector Domain

[0198] In some embodiments, expression repressors of the present disclosure comprise one or more effector domains. In some embodiments, an effector domain, when used as part of an expressor repressor or an expression repression system described herein, decreases expression of a target gene in a cell. In some embodiments, the expression repressor comprises a single effector domain. In some embodiments, the expression repressor comprises one or more effector domains. In some embodiments, the expression repressor comprises more than one effector domain. In some embodiments, the expression repressor comprises one to four effector domains. In some embodiments, the expression repressor comprises two effector domains. In some embodiments, the expression repressor comprises three effector domains. In some embodiments, the expression repressor comprises four effector domains.

[0199] In some embodiments, the effector domain has functionality unrelated to the binding of the DNA targeting moiety. For example, effector domains may target, e.g., bind, a genomic sequence element or genomic complex component proximal to the genomic sequence element targeted by the DNA targeting moiety or recruit a transcription factor. As a further example, an effector domain may comprise an enzymatic activity, e.g., a genetic modification functionality.

[0200] In some embodiments, the effector domain is any one described in Int Pub No. WO2022 / 132195; Int Pub No W02022 / 067033; or US Pat No. 11,312,955 (herein incorporated by reference).

[0201] In some embodiments, an effector domain comprises a transcriptional repressor moiety. In some embodiments, an effector domain comprises a DNA modifying functionality, e.g., a DNA methyltransferase. In some embodiments, the effector domain comprises a polypeptide that induces DNA methylation. In some embodiments, the effector domain comprises a polypeptide that induces DNA methylation of a CpG island (i.e., a region of the genome comprising a high concentration of CpG residues). In some embodiments, the effector domain comprises a DNA methyltransferase enzyme (DNMT). In some embodiments, the effector domain comprises a polypeptide that induces histone modification. In some embodiments, the effector domain comprises a histone modifying enzyme. In some embodiments, the histone modifying enzyme is selected from a histone acetyltransferase, a histone deacetylase (HD AC), a histone lysine methyltransferase, and a histone lysine demethylase. In some embodiments, the effector domain comprises a polypeptide that forms a complex for epigenetic modification. In some embodiments, the polypeptide forms a complex that induces DNA modification and / or histone modification. In some embodiments, the effector domain comprises a Kriippel-associated box (KRAB) domain.

[0202] In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) DNA methyltransferase or a functional variant or fragment thereof. In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) DNA methyltransferase or a functional variant or fragment thereof and at least one additional effector domain. In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) histone modifying enzyme or functional variant or fragment thereof. In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) histone modifying enzyme or functional variant or fragment thereof and a least one additional effector domain. In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) polypeptide that forms a complex for epigenetic modification (e.g., a KRAB domain or functional variant or fragment thereof). In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) polypeptide that forms a complex for epigenetic modification (e.g., a KRAB domain or functional variant or fragment thereof) and at least one additional domain. In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) DNA methyltransferase or a functional variant or fragment thereof and at least one (e.g., 1, 2, or 3) histone modifying enzyme or functional variant or fragment thereof. In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) DNA methyltransferase or a functional variant or fragment thereof and at least one (e.g., 1, 2, or 3) polypeptide that forms a complex for epigenetic modification (e.g., a KRAB domain or functional variant or fragment thereof). In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) polypeptide that forms a complex for epigenetic modification (e.g., a KRAB domain or functional variant or fragment thereof and at least one (e.g., 1, 2, or 3) histone modifying enzyme or functional variant or fragment thereof. In some embodiments, the expression repressor comprises at least one (e.g., 1, 2, or 3) DNA methyltransferase or a functional variant or fragment thereof, at least one (e.g., 1, 2, or 3) polypeptide that forms a complex for epigenetic modification (e.g., a KRAB domain or functional variant or fragment thereof), and at least one (e.g., 1, 2, or 3) histone modifying enzyme or functional variant or fragment thereof.

[0203] In some embodiments, an effector domain is or comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof.

[0204] In some embodiments, an effector domain comprises a transcription repressor that stimulates or promotes transcription, e.g., of the target gene. In some embodiments, the transcription repressor recruits a factor that inhibits transcription, e.g., of the target gene. In some embodiments, an effector domain, e.g., transcription repressor, is or comprises a protein chosen from KRAB, MeCP2, HP1, RBBP4, REST, F0G1, SUZ12, or a functional variant or fragment of any thereof.

[0205] In some embodiments an effector domain promotes epigenetic modification, e.g., directly or indirectly. For example, an effector domain can indirectly promote epigenetic modification by recruiting an endogenous protein that epigenetically modifies the chromatin. An effector domain can directly promote epigenetic modification by catalyzing epigenetic modification, wherein the effector domain comprises enzymatic activity and directly places an epigenetic mark on the chromatin.

[0206] In some embodiments, an effector domain comprises a histone modifying functionality, e.g., a histone methyltransferase, histone demethylase, or histone deacetylase activity. In some embodiments, an effector domain is or comprises a protein chosen from SETDB1, SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof. In some embodiments, an effector domain is or comprises a protein chosen from KDM1A (i.e., ESDI), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional variant or fragment of any thereof. In some embodiments, an effector domain is or comprises a protein chosen from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC1 1, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof.

[0207] In some embodiments, an effector domain comprises a protein having a functionality described herein. In some embodiments, an effector domain is or comprises a protein selected from: KRAB (e.g., as according to NP_056209.2 or the protein encoded by NM_015394.5); a SET domain (e.g., the SET domain of: SETDB1 (e.g., as according to NP 001353347.1 or the protein encoded by NM_001366418.1); EZH2 (e.g., as according to NP-004447.2 or the protein encoded by NM_004456.5); G9A (e.g., as according to NP_001350618.1 or the protein encoded by NM_001363689.1); or SUV39H1 (e.g., as according to NP_003164.1 or the protein encoded by NM_003173.4)); histone demethylase LSD1 (e.g., as according to NP 055828.2 or the protein encoded by NM 015013.4); FOG1 (e.g., the N-terminal residues of FOG1) (e.g., as according to NP_722520.2 or the protein encoded by NM_153813.3); or KAP1 (e.g., as according to NP_005753.1 or the protein encoded by NM_005762.3); a functional fragment or variant of any thereof, or a polypeptide with a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the above-referenced sequences.

[0208] In some embodiments, an effector domain is or comprises a protein selected from: DNMT3A (e.g., human DNMT3A) (e.g., as according to NP_072046.2 or the protein encoded by NM_022552.4); DNMT3B (e.g., as according to NP 008823.1 or the protein encoded by NM_006892.4); DNMT3L (e.g., as according to NP_787063.1 or the protein encoded by NM_175867.3); DNMT3A / 3L complex, bacterial MQ1 (e.g., as according to CAA35058.1 or P15840.3); a functional fragment of any thereof, or a polypeptide with a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the above-referenced sequences.

[0209] In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) DNA targeting moiety and one or more effector domains, wherein the one or more effector domains are or comprise Kriippel-associated box (KRAB) e.g., as according to NP_056209.2 or the protein encoded by NM_015394.5 or a functional variant or fragment thereof. In some embodiments, KRAB is a synthetic KRAB construct. In some embodiments, KRAB comprises an amino acid sequence of SEQ ID NO:39. In some embodiments, the KRAB effector domain comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the KRAB effector domain is encoded by a nucleotide sequence of SEQ ID NO:40. In some embodiments, a nucleotide sequence described herein comprises a sequence of SEQ ID NO:40 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

[0210] In some embodiments, KRAB for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to the KRAB sequence of SEQ ID NO:39. In some embodiments, a KRAB variant comprises one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO:39. In some embodiments, the effector domain comprises an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, or about 90% identity to SEQ ID NO:39. In some embodiments, the effector domain comprises an amino acid sequence having at least about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NO:39.

[0211] In some embodiments, the polypeptide or the expression repressor is a fusion protein comprising an effector domain that is or comprises KRAB and a DNA-targeting moiety. In some embodiments, the DNA targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR / Cas domain, e.g., comprising a CRISPR / Cas protein, e.g., a dCas9 protein. In some embodiments, the polypeptide or the expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or the expression repressor decreases expression of a target gene, e.g., CTNNB1. In some embodiments, the polypeptide or the expression repressor may be used in methods of modulating, e.g., decreasing, gene expression, methods of treating a condition, or methods of epigenetically modifying a target gene, e.g., CTNNB1 or transcription control element described herein, e.g., in place of an expression repressor system. In some embodiments, an expression repressor system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector domain comprising the KRAB sequence of SEQ ID NO:39, or a functional variant or fragment thereof.

[0212] In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) DNA targeting moiety and one or more effector domains, wherein the one or more effector domains are or comprise MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof. In some embodiments, MQ1 is Mollicutes spiroplasma MQ1. In some embodiments, MQ1 is Spiroplasma monobiae MQ1. In some embodiments, MQ1 is MQ1 derived from strain ATCC 33825 and / or corresponding to Uniprot ID P15840. In some embodiments, MQ1 comprises an amino acid sequence of SEQ ID N0:7. In some embodiments, MQ1 comprises an amino acid sequence of SEQ ID NO:37. In some embodiments, an effector domain described herein comprises SEQ ID NO: 7 or 37, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

[0213] In some embodiments, MQ1 is encoded by a nucleotide sequence of SEQ ID NO: 6 or 38. In some embodiments, a nucleic acid described herein comprises a sequence of SEQ ID NO: 6, 38, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

[0214] In some embodiments, MQ1 for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to a wildtype MQ1 (e.g., SEQ ID NO:7). In some embodiments, an MQ1 variant comprises one or more amino acid substitutions, deletions, or insertions relative to a wildtype MQ1, e.g., the MQ1 of SEQ ID NO:7. In some embodiments, an MQ1 variant comprises a K297P substitution. In some embodiments, an MQ1 variant comprises a N299C substitution. In some embodiments, an MQ1 variant comprises a E301 Y substitution. In some embodiments, an MQ1 variant comprises a Q147L substitution (e.g., and has reduced DNA methyltransferase activity relative to wildtype MQ1). In some embodiments, an MQ1 variant comprises K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA binding affinity relative to wildtype MQ1). In some embodiments, an MQ1 variant comprises Q147L, K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA methyltransferase activity and DNA binding affinity relative to wildtype MQ1). In any of the above embodiments, the wildtype MQ1 may comprise an N-terminal methionine, e.g., SEQ ID NO:37.

[0215] In some embodiments, the polypeptide or the expression repressor is a fusion protein comprising an effector domain that is or comprises MQ1 and a DNA targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR / Cas domain, a dCas9 domain. In some embodiments, the polypeptide or the expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or the expression repressor decreases expression of a target gene, e.g., P-catenin. In some embodiments, the polypeptide or the expression repressor may be used in methods of modulating, e.g., decreasing, gene expression, methods of treating a condition, or methods of epigenetically modifying a target gene, e.g., CTNNB1 or transcription control element described herein, e.g., in place of an expression repressor system. In some embodiments, an expression repressor system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector domain comprising MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof.

[0216] In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) DNA targeting moiety and one or more effector domains, wherein the one or more effector domains are or comprise DNMT1, e.g., human DNMT1, or a functional variant or fragment thereof. In some embodiments, DNMT1 is human DNMT1, e.g., corresponding to Gene ID 1786, e.g., corresponding to UniProt ID P26358.2. In some embodiments, DNMT1 comprises an amino acid sequence of SEQ ID NO:41. In some embodiments, an effector domain described herein comprises a sequence according to SEQ ID NO:41 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

[0217] In some embodiments, DNMT1 is encoded by a nucleotide sequence of SEQ ID NO:42. In some embodiments, a nucleic acid described herein comprises a sequence of SEQ ID NO:42 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

[0218] In some embodiments, DNMT1 for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to a DNMT sequence of SEQ ID NO:41. In some embodiments, the effector domain comprises one or more amino acid substitutions, deletions, or insertions relative to wild type DNMT1. In some embodiments, the polypeptide is a fusion protein comprising a repressor domain that is or comprises DNMT1 and a DNA targeting moiety. In some embodiments, the DNA targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR / Cas domain, e.g., a dCas9 domain. In some embodiments, an expression repression system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector domain comprising DNMT1, or a functional variant or fragment thereof.

[0219] In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) DNA targeting moiety and one or more effector domains, wherein the one or more effector domains are or comprise DNMT3a / 3Lcomplex, or a functional variant or fragment thereof. In some embodiments, the one or more effector domains are or comprise a DNMT3a / 3L complex fusion construct. In some embodiments, the DNMT3a / 3L complex comprises DNMT3A (e.g., human DNMT3A) (e.g., as according to NP_072046.2 or the protein encoded by NM_022552.4). In some embodiments, the DNMT3a / 3L complex comprises DNMT3L (e.g., as according to NP_787063.1 or the protein encoded by NM_175867.3). In some embodiments, DNMT3a / 3L comprises an amino acid sequence of SEQ ID NO:43 or SEQ ID NO:44. In some embodiments, an effector domain described herein comprises SEQ ID NO:43 or SEQ ID NO:44, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

[0220] In some embodiments, DNMT3a / 3L is encoded by a nucleotide sequence of SEQ ID NO:45. In some embodiments, a nucleic acid described herein comprises a sequence of SEQ ID NO:45 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

[0221] In some embodiments, DNMT3a / 3L for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to the DNMT3a / 3L of SEQ ID NO:43 or SEQ ID NO:44. In some embodiments, a DNMT3a / 3L variant comprises one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 43 or SEQ ID NO: 44. In some embodiments, the polypeptide or the expression repressor is a fusion protein comprising an effector domain that is or comprises DNMT3a / 3L and a DNA targeting moiety. In some embodiments, the DNA targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR / Cas domain e.g., a dCas9 domain. In some embodiments, an expression repressor system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector domain comprising DNMT3a / 3L, or a functional variant or fragment thereof. In some embodiments, an effector domain is or comprises a polypeptide. In some embodiments, an effector domain is or comprises a nucleic acid. In some embodiments, an effector domain is a chemical, e.g., a chemical that modulates a cytosine I or an adenine(A) (e.g., Na bisulfite, ammonium bisulfite). In some embodiments, an effector domain has enzymatic activity (e.g., methyl transferase, demethylase, nuclease (e.g., Cas9), or deaminase activity). An effector domain may be or comprise one or more of a small molecule, a peptide, a nucleic acid, a nanoparticle, an aptamer, or a pharmaco-agent with poor PK / PD.

[0222] In some embodiments, an effector domain may comprise a peptide ligand, a full-length protein, a protein fragment, an antibody, an antibody fragment, and / or a targeting aptamer. In some embodiments, the protein may bind a receptor such as an extracellular receptor, neuropeptide, hormone peptide, peptide drug, toxic peptide, viral or microbial peptide, synthetic peptide, or agonist or antagonist peptide.

[0223] In some embodiments, an effector domain may comprise antigens, antibodies, antibody fragments such as, e.g. single domain antibodies, ligands, or receptors such as, e.g., glucagon- like peptide- 1 (GLP-1), GLP-2 receptor 2, cholecystokinin B (CCKB), or somatostatin receptor, peptide therapeutics such as, e.g., those that bind to specific cell surface receptors such as G protein-coupled receptors (GPCRs) or ion channels, synthetic or analog peptides from naturally- bioactive peptides, anti-microbial peptides, poreforming peptides, tumor targeting or cytotoxic peptides, or degradation or self-destruction peptides such as an apoptosis-inducing peptide signal or photosensitizer peptide.

[0224] Peptide or protein moieties for use in effector domains as described herein may also include small antigen-binding peptides, e.g., antigen binding antibody or antibody-like fragments, such as, e.g., single chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076-1 13). Such small antigen binding peptides may bind, e.g., a cytosolic antigen, a nuclear antigen, or an intra-organellar antigen.

[0225] In some embodiments, an effector domain comprises a dominant negative component (e.g., dominant negative moiety), e.g., a protein that recognizes and binds a sequence (e.g., an anchor sequence, e.g., a CTCF binding motif), but with an inactive (e.g., mutated) dimerization domain, e.g., a dimerization domain that is unable to form a functional anchor sequence- mediated conjunction), or binds to a component of a genomic complex (e.g., a transcription factor subunit, etc.) preventing formation of a functional transcription factor, etc. For example, the Zinc Finger domain of CTCF can be altered so that it binds a specific anchor sequence (by adding zinc fingers that recognize flanking nucleic acids), while the homo-dimerization domain is altered to prevent the interaction between engineered CTCF and endogenous forms of CTCF. In some embodiments, a dominant negative component comprises a synthetic nucleating polypeptide with a selected binding affinity for an anchor sequence within a target anchor sequence-mediated conjunction. In some embodiments, binding affinity may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher or lower than binding affinity of an endogenous nucleating polypeptide (e.g., CTCF) that associates with a target anchor sequence. In some embodiments, A synthetic nucleating polypeptide may have between 30-90%, 30-85%, 30-80%, 30-70%, 50-80%, 50-90% amino acid sequence identity to a corresponding endogenous nucleating polypeptide. A nucleating polypeptide may modulate (e.g., disrupt), such as through competitive binding, e.g., competing with binding of an endogenous nucleating polypeptide to its anchor sequence.

[0226] In some embodiments, an effector domain comprises an antibody or antigen-binding fragment thereof. In some embodiments, target gene (e.g., P-catenin) expression is altered via use of effector domains that are or comprise one or more antibodies or antigen-binding fragments thereof. In some embodiments, gene expression is altered via use of effector domains that are or comprise one or more antibodies (or antigen-binding fragments thereof) and dCas9.

[0227] In some embodiments, an antibody or antigen-binding fragment thereof for use in an effector domain may be monoclonal. An antibody may be a fusion, a chimeric antibody, a nonhumanized antibody, a partially or fully humanized antibody, a single chain antibody, Fab fragment, Fv fragment, F(ab')2 fragment, scFv fragment, etc. As will be understood by one of skill in the art, format of antibody(ies) used may be the same or different depending on a given target.

[0228] In some embodiments, an effector domain comprises one or more RNAs (e.g., gRNA) and dCas9. In some embodiments, one or more RNAs is / are targeted to a genomic sequence element via dCas9 and target-specific guide RNA. As will be understood by one of skill in the art, RNAs used for targeting may be the same or different depending on a given target. An effector domain may comprise an aptamer, such as an oligonucleotide aptamer or a peptide aptamer. Aptamer moieties are oligonucleotide or peptide aptamers. An effector domain may comprise an oligonucleotide aptamer. Oligonucleotide aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind to pre-selected targets including proteins and peptides with high affinity and specificity.

[0229] Oligonucleotide aptamers are nucleic acid species that may be engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers provide discriminate molecular recognition and can be produced by chemical synthesis. In addition, aptamers possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

[0230] Both DNA and RNA aptamers show robust binding affinities for various targets. For example, DNA and RNA aptamers have been selected for t lysozyme, thrombin, human immunodeficiency virus trans-acting responsive element (HIV TAR), hemin, interferon y, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), dopamine, and the non-classical oncogene, heat shock factor 1 (HSF1).

[0231] An effector domain may comprise a peptide aptamer moiety. Peptide aptamers have one (or more) short variable peptide domains, including peptides having low molecular weight, 12 — 14 Da. Peptide aptamers may be designed to specifically bind to and interfere with proteinprotein interactions inside cells.

[0232] Peptide aptamers are artificial proteins selected or engineered to bind specific target molecules. These proteins include one or more peptide complexes of variable sequence. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind cellular protein targets and exert biological effects, including interference with the normal protein interactions of their targeted molecules with other proteins. In particular, a variable peptide aptamer complex attached to a transcription factor binding domain is screened against a target protein attached to a transcription factor activating domain. In vivo binding of a peptide aptamer to its target via this selection strategy is detected as expression of a downstream yeast marker gene. Such experiments identify particular proteins bound by aptamers, and protein interactions that aptamers disrupt, to cause a given phenotype. In addition, peptide aptamers derivatized with appropriate functional moieties can cause specific post-translational modification of their target proteins or change subcellular localization of the targets. Peptide aptamers can also recognize targets in vitro. They have found use in lieu of antibodies in biosensors and may be used to detect active isoforms of proteins from populations containing both inactive and active protein forms. Derivatives known as tadpoles, in which peptide aptamer “heads” are covalently linked to unique sequence double-stranded DNA “tails”, allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative real-time polymerase chain reaction) of their DNA tails.

[0233] Peptide aptamer selection can be made using different systems, but the most commonly used is currently a yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers. Peptides panned from combinatorial peptide libraries have been stored in a special database with named MimoDB.

[0234] An exemplary effector domain may include, but is not limited to: ubiquitin, bicyclic peptides as ubiquitin ligase inhibitors, transcription factors, DNA and protein modification enzymes such as topoisomerases, topoisomerase inhibitors such as topotecan, DNA methyltransferases such as the DNMT family (e.g., DNMT3A, DNMT3B, DNMT3a / 3L, MQ1), protein methyltransferases (e.g., viral lysine methyltransferase (vSET), protein-lysine N- methyltransferase (SMYD2), deaminases (e.g., APOBEC, UG1), histone methyltransferases such as enhancer of zeste homolog 2 (EZH2), PRMT1, histone-lysine-N-methyltransferase (Setdbl), histone methyltransferase (SET2), euchromatic histone-lysine N-methyltransferase 2 (G9a), histone-lysine N-methyltransferase (SUV39H1), and G9a), histone deacetylase (e.g., HDAC1, HDAC2, HDAC3), enzymes with a role in DNA demethylation (e.g., the TET family enzymes catalyze oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidative derivatives), protein demethylases such as KDM1A and lysine-specific histone demethylase 1 (LSD1), helicases such as DHX9, deacetylases (e.g., sirtuin 1, 2, 3, 4, 5, 6, or 7), kinases, phosphatases, DNA-intercalating agents such as ethidium bromide, SYBR green, and proflavine, efflux pump inhibitors such as peptidomimetics like phenylalanine arginyl P-naphthylamide or quinoline derivatives, nuclear receptor activators and inhibitors, proteasome inhibitors, competitive inhibitors for enzymes such as those involved in lysosomal storage diseases, protein synthesis inhibitors, nucleases (e.g., Cpfl, Cas9, zinc finger nuclease), specific domains from proteins, such as a KRAB domain, and fusions of one or more thereof (e.g., dCas9-DNMT, dCas9-MQl, dCas9-KRAB).

[0235] In some embodiments, a candidate effector domain may be determined to be suitable for use as an effector domain by methods known to those of skill in the art. For example, a candidate effector domain may be tested by assaying whether, when the candidate effector domain is present in the nucleus of a cell and appropriately localized (e.g., to a target gene or transcription control element operably linked to said target gene, e.g., via a DNA targeting moiety), the candidate effector domain decreases expression of the target gene in the cell, e.g., decreases the level of RNA transcript encoded by the target gene (e.g., as measured by RNASeq or Northern blot) or decreases the level of protein encoded by the target gene (e.g., as measured by ELISA).

[0236] In some embodiments, an expression repressor comprises a plurality of effector domains, wherein each effector domain does not detectably bind, e.g., does not bind, to another effector domain. In some embodiments, an expression repressor system comprises a first expression repressor comprising a first effector domain and a second expression repressor comprising a second effector domain, wherein the first effector domain does not detectably bind, e.g., does not bind, to the second effector domain.

[0237] In some embodiments, an expression repressor system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors comprises an effector domain, wherein each effector domain does not detectably bind, e.g., does not bind, to another effector domain. In some embodiments, an expression repressor system comprises a first expression repressor comprising a first effector domain and a second expression repressor comprising a second effector domain, wherein the first effector domain does not detectably bind, e.g., does not bind, to the second effector domain. In some embodiments, an expression repressor system comprises a first expression repressor comprising a first effector domain and a second expression repressor comprising a second effector domain, wherein the first effector domain does not detectably bind, e.g., does not bind, to another first effector domain, and the second effector domain does not detectably bind, e.g., does not bind, to another second effector domain. In some embodiments, an effector domain for use in the compositions and methods described herein is functional in a monomeric, e.g., non-dimeric, state.

[0238] In some embodiments, an effector domain is or comprises a transcriptional repressor moiety. In some embodiments, the transcriptional repressor moiety e.g., modulates the two- dimensional structure of chromatin ( / .<?., modulates structure of chromatin in a way that would alter its two-dimensional representation).

[0239] Transcriptional repressor moieties useful in methods and compositions of the present disclosure include agents that affect epigenetic markers, e.g., DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA- associated silencing. Exemplary epigenetic enzymes that can be targeted to a genomic sequence element as described herein include DNA methylases (e.g., DNMT3a, DNMT3b, DNMT3a / 3L, MQ1), DNA demethylation (e.g., the TET family), histone methyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3), sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine-specific histone demethylase 1 (LSD1), histone-lysine-N-methyltransferase (Setdbl), euchromatic histone-lysine N-methyltransferase 2 (G9a), histone-lysine N-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), viral lysine methyltransferase (vSET), histone methyltransferase (SET2), and protein-lysine N-methyltransferase (SMYD2). Examples of such epigenetic modifying agents are described, e.g., in de Groote et al. Nuc. Acids Res. (2012): 1-18.

[0240] In some embodiments, an expression repressor, e.g., comprising an epigenetic modifying moiety, useful herein comprises or is a construct described in Koferle et al. Genome Medicine 7.59 (2015): 1-3 incorporated herein by reference. For example, in some embodiments, an expression repressor comprises or is a construct found in Table 1 of Koferle et al., e.g., histone deacetylase, histone methyltransferase, DNA demethylation, or H3K4 and / or H3K9 histone demethylase described in Table 1 (e.g., dCas9-p300, TALE-TET1, ZF-DNMT3A, or TALE- LSD1).

[0241] In some embodiments, an effector domain comprises a component of a gene editing system, e.g., a CRISPR / Cas domain, e.g., a Zn Finger domain, e.g., a TAL effector domain. In some embodiments, a transcriptional repressor moiety may comprise a polypeptide (e.g., peptide or protein moiety) linked to a gRNA and a targeted nuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A), a catalytically inactive Cas9 (dCas9), eSpCas9, Cpfl, C2C1, or C2C3, or a nucleic acid encoding such a nuclease.

[0242] In some embodiments, an effector domain comprises a biologically active fragment of the effector domain. As used herein, a “biologically active fragment of an effector domain” is a portion that maintains function (e.g., completely, partially, minimally) of an effector domain (e.g., a “minimal” or “core” domain). In some embodiments, fusion of a dCas9 with all or a portion of one or more effector domains of an epigenetic modifying agent (such as a DNA methylase or enzyme with a role in DNA demethylation, e.g., DNMT3a, DNMT3b, DNMT3L, a DNMT inhibitor, combinations thereof, TET family enzymes, protein acetyl transferase or deacetylase, dCas9-DNMT3a / 3L, dCas9-DNMT3a / 3L / KRAB, dCas9 / VP64) creates a chimeric protein that is linked to the polypeptide and useful in the methods described herein. An effector domain comprising such a chimeric protein is referred to as either a genetic modifying moiety (because of its use of a gene editing system component, Cas9) or a transcriptional repressor moiety (because of its use of an effector domain of a transcriptional repressor agent).

[0243] In some embodiments, provided technologies are described as comprising a gRNA that specifically targets a target gene. In some embodiments, the target gene is CTNNB1.

[0244] Additional Moieties

[0245] An expression repressor may further comprise one or more additional moieties (e.g., in addition to one or more targeting moieties and one or more effector domains). In some embodiments, an additional moiety is selected from a tagging or monitoring moiety, a cleavable moiety (e.g., a cleavable moiety positioned between a DNA-targeting moiety and an effector domain or at the N- or C-terminal end of a polypeptide), a small molecule, a membrane translocating polypeptide, or a pharmaco- agent moiety.

[0246] Linkers

[0247] An expression repressor or an expression repressor system as disclosed herein 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, e.g., one or more covalent bonds or non-covalent bonds. In some embodiments, a linker is covalent. In some embodiments, a linker is non-covalent. In some embodiments, a linker is a peptide linker. Such a linker may be between 2-30, 5-30, 10-30, 15- 30, 20-30, 25-30, 2-25, 5-25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2- 10, 5-10, or 2-5 amino acids in length, or greater than or equal to 2, 5, 10, 15, 20, 25, or 30 amino acids in length (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length). In some embodiments, a linker can be used to space a first domain or moiety from a second domain or moiety, e.g., a DNA-targeting moiety from an effector moiety. In some embodiments, for example, a linker can be positioned between a DNA-targeting moiety and an effector moiety, e.g., to provide molecular flexibility of secondary and tertiary structures. A linker may comprise flexible, rigid, and / or cleavable linkers described herein. In some embodiments, a linker includes at least one glycine, alanine, and serine amino acids to provide for flexibility. In some embodiments, a linker is a hydrophobic linker, such as including a negatively charged sulfonate group, polyethylene glycol (PEG) group, or pyrophosphate diester group. In some embodiments, a linker is cleavable to selectively release a moiety (e.g., polypeptide) from a modulating agent, but sufficiently stable to prevent premature cleavage.

[0248] In some embodiments, one or more moieties and / or domains of an expression repressor described herein are linked with one or more linkers. In some embodiments, an expression repression may comprise a linker situated between the targeting moiety and the effector moiety. In some embodiments, the linker may have a sequence of ASGSGGGSGGARD (SEQ ID NO:21), or ASGSGGGSGG (SEQ ID NO:46). In some embodiments, a system comprising a first and second repressor may comprise a first linker situated between the first targeting moiety and the first effector moiety, and a second linker situated between the second targeting moiety and the second effector moiety. In some embodiments, the first and the second linker may be identical. In some embodiments, the first and the second linker may be different. In some embodiments, the first linker may comprise an amino acid sequence according to SEQ ID NO:21 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto and the second linker may comprise an amino acid sequence according to SEQ ID NO:46 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

[0249] As will be known by one of skill in the art, commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). Flexible linkers may be useful for joining domains / moieties that require a certain degree of movement or interaction and may include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of a linker in aqueous solutions by forming hydrogen bonds with water molecules, and therefore reduce unfavorable interactions between a linker and moieties / domains. In some embodiments, the linker is a GS linker or a variant thereof e.g., G4S (SEQ ID NO:47).

[0250] Rigid linkers are useful to keep a fixed distance between domains / moieties and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of domains is critical to preserve the stability or bioactivity of one or more components in the fusion. Rigid linkers may have an alpha helix-structure or Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.

[0251] Cleavable linkers may release free functional domains in vivo. In some embodiments, linkers may be cleaved under specific conditions, such as presence of reducing reagents or proteases. In vivo cleavable linkers may utilize reversible nature of a disulfide bond. One example includes a thrombin-sensitive sequence (e.g., PRS) between the two Cys residues. In vitro thrombin treatment of CPRSC results in the cleavage of a thrombin-sensitive sequence, while a reversible disulfide linkage remains intact. Such linkers are known and described, e.g., in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivo cleavage of linkers in fusions may also be carried out by proteases that are expressed in vivo under certain conditions, in specific cells or tissues, or constrained within certain cellular compartments. Specificity of many proteases offers slower cleavage of the linker in constrained compartments. In some embodiment, the cleavable linker may be a self-cleaving linker, e.g., a T2A peptide linker. In some embodiments, the linker may comprise a “ribosome skipping” sequence, e.g., a tPT2A linker.

[0252] Examples of molecules suitable for use in linkers described herein include a negatively charged sulfonate group; lipids, such as a poly (— CH2— ) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof; noncarbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecule capable of covalently linking two or more components of an expression repressor. Non-covalent linkers are also included, such as hydrophobic lipid globules to which the polypeptide is linked, for example through a hydrophobic region of a polypeptide or a hydrophobic extension of a polypeptide, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues. Components of an expression repressor may be linked using charge-based chemistry, such that a positively charged component of an expression repressor is linked to a negative charge of another component.

[0253] Exemplary CTNNB1 Expression Repressors In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain that binds a target sequence described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof.

[0254] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof.

[0255] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to an KRAB domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3.

[0256] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3.

[0257] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to an KRAB domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,170 to position 41,240,387, according to the hgl9 reference genome for chr3.

[0258] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3.

[0259] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to an KRAB domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3.

[0260] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3.

[0261] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to an KRAB domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence of about 15-20 nucleotides in a region of the genome spanning position 41,241,406 to position 41,241,623, according to the hgl9 reference genome for chr3.

[0262] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, or 20.

[0263] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, or 20.

[0264] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain described herein operably linked to an MQ1 effector domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence selected from SEQ ID NOs: 8, 14, or 20.

[0265] In some embodiments, the disclosure provides an expression repressor comprising a TAL effector domain operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the TAL effector domain binds to a target sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a ZFN effector domain that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the ZFN effector domain binds to a target sequence selected from SEQ ID NOs: 8, 14, or 20. In some embodiments, the disclosure provides an expression repressor comprising a dCas9 effector domain comprising a guide sequence that binds a target sequence described herein operably linked to a KRAB domain, or a functional variant or fragment thereof, wherein the effector domain comprises a guide sequence that binds to a target sequence selected from SEQ ID NOs: 8, 14, or 20.

[0266] In some embodiments, an expression repressor of the present disclosure comprises a fusion protein comprising an MQ1 effector domain and a TAL effector domain, wherein the TAL effector domain targets a nucleotide sequence in the human CTNNB1 gene set forth as SEQ ID NO:8. In some embodiments, the MQ1 effector domain comprises an mRNA sequence as set forth in SEQ ID NO:6. In some embodiments, the MQ1 effector domain comprises an amino acid sequence as set forth in SEQ ID NO:7. In some embodiments, the TAL effector domain comprises an amino acid sequence as set forth in SEQ ID NO:5. In some embodiments, the fusion protein comprises a linker sequence between the MQ1 effector domain and the TAL effector domain. In some embodiments, the linker sequence comprises an amino acid sequence selected from SEQ ID NOs: 21 and 46.

[0267] In some embodiments, an expression repressor of the present disclosure comprises a fusion protein comprising an MQ1 effector domain and a TAL effector domain, wherein the TAL effector domain targets a nucleotide sequence in the human CTNNB1 gene set forth as SEQ ID NO: 14. In some embodiments, the MQ1 effector domain comprises an mRNA sequence as set forth in SEQ ID NO:6. In some embodiments, the MQ1 effector domain comprises an amino acid sequence as set forth in SEQ ID NO:7. In some embodiments, the TAL effector domain comprises an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the fusion protein comprises a linker sequence between the MQ1 effector domain and the TAL effector domain. In some embodiments, the linker sequence comprises an amino acid sequence selected from SEQ ID NOs: 21 and 46.

[0268] In some embodiments, an expression repressor of the present disclosure comprises a fusion protein comprising an MQ1 effector domain and a TAL effector domain, wherein the TAL effector domain targets a nucleotide sequence in the human CTNNB1 gene set forth as SEQ ID NO:20. In some embodiments, the MQ1 effector domain comprises an mRNA sequence as set forth in SEQ ID NO:6. In some embodiments, the MQ1 effector domain comprises an amino acid sequence as set forth in SEQ ID NO:7. In some embodiments, the TAL effector domain comprises an amino acid sequence as set forth in SEQ ID NO: 19. In some embodiments, the fusion protein comprises a linker sequence between the MQ1 effector domain and the TAL effector domain. In some embodiments, the linker sequence comprises an amino acid sequence selected from SEQ ID NOs: 21 and 46.

[0269] In some embodiments, an expression repressor of the present disclosure comprises a fusion protein comprising an MQ1 effector domain and a TAL effector domain, wherein the TAL effector domain targets a nucleotide sequence in the human CTNNB1 gene set forth as SEQ ID NO:29. In some embodiments, the MQ1 effector domain comprises an mRNA sequence as set forth in SEQ ID NO:6. In some embodiments, the MQ1 effector domain comprises an amino acid sequence as set forth in SEQ ID NO:7. In some embodiments, the TAL effector domain comprises an amino acid sequence as set forth in SEQ ID NO:28. In some embodiments, the fusion protein comprises a linker sequence between the MQ1 effector domain and the TAL effector domain. In some embodiments, the linker sequence comprises an amino acid sequence selected from SEQ ID NOs: 21 and 46.

[0270] Expression Repressor Systems

[0271] In some embodiments, the disclosure provides an expression repression system comprising two or more expression repressors described herein. In some embodiments, an expression repression system comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expression repressors (and optionally no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2).

[0272] In some embodiments, the expression repressor system comprises a first expression repressor comprising (i) a first DNA targeting moiety that binds a first target sequence described herein, and (ii) a first effector domain; and at least one additional expression repressor comprising (i) a second DNA targeting moiety that binds a second target sequence described herein, and (ii) a second effector domain. In some embodiments, the first target sequence is different from the second target sequence.

[0273] In some embodiments, the expression repressor system comprises a first expression repressor comprising (i) a first DNA targeting moiety that binds a first target sequence described herein, and (ii) a first effector domain; and at least one additional expression repressor comprising (i) a second DNA targeting moiety that binds a second target sequence described herein, and (ii) a second effector domain, wherein the first target sequence is different from the second target sequence. In some embodiments, the first effector domain is the same as the second effector domain. In some embodiments, the first effector domain is different from the second effector domain.

[0274] In some embodiments, the expression repressor system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional expression repressors. In some embodiments, the expression repressor system comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional expression repressors

[0275] In some embodiments, the expression repressor system comprises a first expression repressor comprising (i) a first DNA targeting moiety that binds a first target sequence described herein, and (ii) a first effector domain; and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional expression repressors, wherein each of the additional expression repressors comprises (i) a DNA targeting moiety that binds a target sequence described herein; and (ii) an effector domain, wherein the target sequence of each of the additional expression repressors is different from one another and from the first target sequence. In some embodiments, the first effector domain and the effector domain of each of the additional expression repressors are the same or different.

[0276] In some embodiments, each of the expression repressors of the expression repressor system binds to a different target sequence described herein.

[0277] In some embodiments, each of the expression repressors of the expression repressor system are formulated in the same composition. In some embodiments, each of the expression repressors of the expression repressor system are formulated in different compositions.

[0278] In some embodiments, the expression repressors of an expression repressor system each comprise a different DNA targeting moiety (e.g., the first, second, third, or further expression repressors each comprise different targeting moieties from one another). For example, an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor comprises a first targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain), and the second expression repressor comprises a second targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain) different from the first targeting moiety. In some embodiments, different is comprising distinct types of targeting moiety, e.g., the first targeting moiety comprises a Cas9 domain, and the second DNA-targeting moiety comprises a Zn finger domain. In some embodiments, different is comprising distinct variants of the same type of targeting moiety, e.g., the first targeting moiety comprises a first Cas9 domain (e.g., from a first species) and the second targeting moiety comprises a second Cas9 domain (e.g., from a second species). In an embodiment, when an expression repressor system comprises two or more targeting moieties of the same type, e.g., two or more Cas9 or ZF domains, the targeting moieties specifically bind two or more different target sequences. For example, in an expression repressor system comprising two or more Cas9 domains, the two or more Cas9 domains may be chosen or altered such that they only appreciably bind the gRNA corresponding to their target sequence (e.g., and do not appreciably bind the gRNA corresponding to the target of another Cas9 domain). In a further example, in an expression repressor system comprising two or more effector moieties, the two or more effector moieties may be chosen or altered such that they only appreciably bind to their target sequence (e.g., and do not appreciably bind the target sequence of another effector moiety). In some embodiments, an expression repressor system comprises three or more expression repressors and two or more expression repressors comprise the same DNA targeting moiety. For example, an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both comprise a first DNA targeting moiety and the third expression repressor comprises a second different DNA targeting moiety. For a further example, an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both comprise a first DNA targeting moiety and the third and fourth expression repressors comprises a second different DNA targeting moiety. For a further example, an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both comprise a first DNA targeting moiety, the third and fourth expression repressors both comprise a second different DNA targeting moiety, and the fifth expression repressor comprises a third different DNA targeting moiety. As described above, different can mean comprising different types of DNA- targeting moieties or comprising distinct variants of the same type of targeting moiety.

[0279] In some embodiments, the expression repressors of an expression repressor system each bind to a different target sequence described herein (e.g., the first, second, third, or further expression repressors each bind DNA sequences that are different from one another). For example, an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor binds to a first target sequence described herein, and the second expression repressor binds to a second target sequence described herein. In some embodiments, different can mean that: there is at least one position that is not identical between the target sequence bound by one expression repressor and the target sequence bound by another expression repressor, or that there is at least one position present in the target sequence bound by one expression repressor that is not present in the target sequence bound by another expression repressor.

[0280] In some embodiments, the expression repressors of an expression repressor system each comprise a different effector domain (e.g., the first, second, third, or further expression repressors each comprise a different effector domain from one another). For example, an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor comprises a first effector moiety (e.g., comprising a DNA methyltransferase or functional fragment thereof), and the second expression repressor comprises a second effector moiety (e.g., comprising a transcription repressor (e.g., KRAB) or functional fragment thereof) different from the first effector moiety. In some embodiments, different can mean comprising distinct types of effector moiety. In other embodiments, different can mean comprising distinct variants of the same type of effector moiety, e.g., the first effector moiety comprises a first DNA methyltransferase (e.g., having a first site specificity or amino acid sequence) and the second effector moiety comprises a second DNA methyltransferase (e.g., having a second site specificity or amino acid sequence).

[0281] In some embodiments, an expression repressor system comprises a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety, wherein the first effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, F0G1, SUZ12 or a functional variant or fragment thereof, and the second effector moiety comprises a different protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, F0G1, SUZ12 , or a functional variant or fragment thereof.

[0282] In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof, and the other effector moiety comprises a transcription repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, F0G1, SUZ12, or a functional variant or fragment of any thereof), the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a histone deacetylase activity (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof). In some embodiments, the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof). In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a transcription repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, F0G1, SUZ12, or a functional variant or fragment of any thereof). In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises a different transcription repressor activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises the same DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises a DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises a different histone demethylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises the same histone demethylase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a different histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises the same histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a different DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises the same DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a different DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises the same DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises a different transcription repressor activity. In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises the same transcription repressor activity.

[0283] In some embodiments, an expression repressor system comprises three or more expression repressors and two or more expression repressors comprise the same DNA-targeting moiety. For example, an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both comprise a first effector moiety and the third expression repressor comprises a second different effector moiety. For a further example, an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both comprise a first effector moiety and the third and fourth expression repressors comprises a second different effector moiety. For a further example, an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both comprise a first effector moiety, the third and fourth expression repressors both comprise a second different effector moiety, and the fifth expression repressor comprises a third different effector moiety. As described above, different can mean comprising different types of effector moiety or comprising distinct variants of the same type of effector moiety.

[0284] In some embodiments, two or more (e.g., all) expression repressors of an expression repressor system are not covalently associated with each other, e.g., each expression repressor is not covalently associated with any other expression repressor. In another embodiment, two or more expression repressors of an expression repressor system are covalently associated with one another. In an embodiment, an expression repression system comprises a first expression repressor and a second expression repressor disposed on the same polypeptide, e.g., as a fusion molecule, e.g., connected by a peptide bond and optionally a linker. In some embodiments, the peptide is a self-cleaving peptide, e.g., a T2A self-cleaving peptide. In an embodiment, an expression repression system comprises a first expression repressor and a second expression repressor that are connected by a non-peptide bond, e.g., are conjugated to one another.

[0285] Methods of Making Expression Repressors

[0286] In some embodiments, a protein or polypeptide of compositions of the present disclosure can be biochemically synthesized by employing standard solid phase techniques. Such methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods can be used when a peptide is relatively short (e.g., 10 kDa) and / or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

[0287] Solid phase synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses, 2nd Ed., Pierce Chemical Company, 1984; and Coin, I., et al., Nature Protocols, 2:3247-3256, 2007. For longer peptides, recombinant methods may be used. Methods of making a recombinant therapeutic polypeptide are routine in the art. See, e.g., Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0288] Exemplary methods for producing an expression repressor or polypeptide described herein involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under control of appropriate promoters. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, splice, and poly adenylation sites may be used to provide other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0289] In some embodiments, large amounts of the expression repressor or polypeptide are desired, it can be generated using techniques such as described by Brian Bray, Nature Reviews Drug Discovery, 2:587-593, 2003; and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463.

[0290] Various mammalian cell culture systems can be employed to express and manufacture recombinant protein. Examples of mammalian expression systems include, without limitation, CHO cells, COS cells, HeLA and BHK cell lines. Processes of host cell culture for production of protein therapeutics are described, for example, in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologies Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). Compositions described herein may include a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein. In some embodiments, a vector, e.g., a viral vector, may comprise a nucleic acid encoding a recombinant protein. Compositions described herein may include a lipid nanoparticle encapsulating a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein. In some embodiments, a lipid nanoparticle encapsulating a vector, e.g., a viral vector, may comprise a nucleic acid encoding a recombinant protein. Purification of protein therapeutics is described, for example, in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0291] Proteins comprise one or more amino acids. Amino acids include any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)L COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide. Nucleic Acids of the Disclosure

[0292] In another aspect, provided herein are nucleic acids encoding an expression repressor or an expression repressor system of the present disclosure. In some embodiments, an expression repressor may be provided via a composition comprising a nucleic acid encoding the expression repressor, wherein the nucleic acid is associated with sufficient other sequences to achieve expression in a system of interest (e.g., in a particular cell, tissue, organism, etc.).

[0293] In some embodiments, the present disclosure provides compositions of nucleic acids that encode an expression repressor or fragment thereof. In some embodiments, nucleic acids may be or may include DNA, RNA, or any other nucleic acid moiety or entity as described herein and may be prepared by any technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.). In some embodiments, provided nucleic acids that encode an expression repressor or fragment thereof may be operationally associated with one or more replication, integration, and / or expression signals appropriate and / or sufficient to achieve integration, replication, and / or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).

[0294] In some embodiments, a composition for delivering an expression repressor or an expression repressor system described herein is or comprises a vector, e.g., a viral vector, comprising one or more nucleic acids encoding an expression repressor or one or more components of an expression repressor as described herein.

[0295] In some embodiments, the present disclosure provides compositions of nucleic acids that encode an expression repressor, one or more expression repressors, or fragments thereof. In some embodiments, provided nucleic acids may be or include DNA, RNA, or any other nucleic acid moiety or entity as described herein, and may be prepared by any technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.). The nucleic acid sequence may include, for example and without limitation, DNA, RNA, modified oligonucleotides (e.g., chemical modifications, such as modifications that alter the backbone linkages, sugar molecules, and / or nucleic acid bases), and artificial nucleic acids. In some embodiments, the nucleic acid sequence includes, for example and without limitation, genomic DNA, cDNA, peptide nucleic acids (PNA) or peptide oligonucleotide conjugates, locked nucleic acids (LNA), bridged nucleic acids (BNA), polyamides, triplex forming oligonucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules. In some embodiments, provided nucleic acids encoding an expression repressor, one or more expression repressors, or polypeptide fragments thereof may be operationally associated with one or more replication, integration, and / or expression signals appropriate and / or sufficient to achieve integration, replication, and / or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.').

[0296] In some embodiments, the nucleic acid sequence has a length from about 2 to about 5000 nts, about 10 to about 100 nts, about 50 to about 150 nts, about 100 to about 200 nts, about 150 to about 250 nts, about 200 to about 300 nts, about 250 to about 350 nts, about 300 to about 500 nts, about 10 to about 1000 nts, about 50 to about 1000 nts, about 100 to about 1000 nts, about 1000 to about 2000 nts, about 2000 to about 3000 nts, about 3000 to about 4000 nts, about 4000 to about 5000 nts, or any range therebetween.

[0297] In some embodiments, a composition for delivering an expression repressor or an expression repressor system described herein is or comprises RNA, e.g., mRNA, comprising one or more nucleic acids encoding an expression repressor as described herein. In some embodiments, a composition for delivering an expression repressor or an expression repressor system described herein is or comprises RNA, e.g., mRNA, comprising one or more components of an expression repressor, as described herein.

[0298] In some embodiments, a nucleic acid of the disclosure comprises nucleosides, e.g., purines or pyrimidines, e.g., adenine, cytosine, guanine, thymine, and uracil. In some embodiments, the nucleic acid sequence includes one or more nucleoside analogs. The nucleoside analog includes, but is not limited to, a nucleoside analog, such as 5 -fluorouracil; 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 4- methylbenzimidazole, 5 -(carboxy hydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5 -methylcytosine, N6- adenine, 7-methylguanine, 5 -methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5 -methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine, 3 -nitropyrrole, inosine, thiouridine, queuosine, wyosine, diaminopurine, isoguanine, isocytosine, diaminopyrimidine, 2,4- difluorotoluene, isoquinoline, pyrrolo[2,3-P]pyridine, and any others that can base pair with a purine or a pyrimidine side chain. Additional modifications are known and described, e.g., in WO 2012 / 019168; WO 2015 / 038892; WO 2015 / 038892; WO 2015 / 089511; WO 2015 / 196130; WO 2015 / 196118, and WO 2015 / 196128. mRNA

[0299] In one aspect, provided herein is an RNA, e.g., an mRNA, encoding an expression repressor or an expression repressor system as described herein. In some embodiments, an mRNA comprises an open reading frame (ORF), e.g., a sequence of codons that is translatable into a peptide or protein, e.g., into an expression repressor or an expression repressor system.

[0300] Open Reading Frames ( ORFs)

[0301] An open reading frame includes a start codon at its 5'-end and a subsequent nucleotide region which usually exhibits a length which is a multiple of 3 nucleotides. In some embodiments, an ORF is terminated by a stop-codon (e.g., TAA, TAG, or TGA). In certain embodiments, the ORF may be isolated, or it may be incorporated in a longer nucleic acid sequence, e.g., in a vector or an mRNA. An ORF may also be known in the art as a protein coding region.

[0302] In some embodiments, an mRNA of the disclosure comprises an ORF, e.g.. encoding a DN A targeting moiety and / or an effector domain of an expression repressor or an expression repressor system described herein. In certain embodiments, an ORF comprises a sequence that has been sequence optimized. Sequence-optimized nucleotide sequences disclosed herein are distinct from the corresponding wild-type nucleotide acid sequences and from other known sequence-optimized nucleotide sequences, e.g., these sequence-optimized nucleic acids have unique compositional characteristics.

[0303] In some embodiments, the mRNA comprises a bicistronic RNA. As used herein, a bicistronic RNA is typically an RNA, preferably an mRNA, comprising two ORFs. In some embodiments, the mRNA comprises a multicistronic RNA. As used herein, a multicistronic RNA is typically an RNA, preferably an mRNA, comprising more than two ORFs.

[0304] In some embodiments, the nucleic acid encoding the expression repressor system is a multicistronic sequence. In some embodiments, the multicistronic sequence is a bicistronic sequence. In some embodiments, the multicistronic sequence comprises a sequence encoding the first expression repressor and a sequence encoding the second expression repressor. In some embodiments, the multicistronic sequence encodes a self-cleavable peptide sequence, e.g., a 2A peptide sequence, e.g., a T2A peptide sequence or a P2A sequence. In some embodiments, the multicistronic sequence encodes a T2A peptide sequence and a P2A peptide sequence.

[0305] In some embodiments, a bicistronic construct further comprises a polyA tail. In some embodiments, upon transcription of a bicistronic gene construct, a single mRNA transcript encoding the first expression repressor, and the second expression repressor are produced, which upon translation gets cleaved, e.g., after the glycine residue within the 2A peptide, to yield the first expression repressor and the second expression repressor as two separate proteins. In some embodiments, the first and the second expression repressor are separated by “ribosomeskipping.” In some embodiments, the first expression repressor and / or the second expression repressor retains a fragment of the 2A peptide after ribosome skipping. In some embodiments, the expression level of the first and second expression repressor are equal. In some embodiments, the expression level of the first and the second expression repressor are different. In some embodiments, the protein level of the first expression repressor is within about 1%, 2%, 5%, or 10% of (greater than or less than) the protein level of the second expression repressor.

[0306] In some embodiments, a system encoded by a bicistronic nucleic acid decreases expression of a target gene (e.g., CTNNB1) at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, in a cell, as compared to an otherwise similar system wherein the first and second expression repressor are encoded by monocistronic nucleic acids.

[0307] Untranslated Regions (UTRs)

[0308] In certain embodiments, a polynucleotide (e.g., mRNA) encoding an expression repressor or an expression repressor system of the present disclosure further comprises a 5' UTR and / or a translation initiation sequence. Natural 5 'UTRs bear features which function in initiation of protein translation. They harbor signatures, e.g., Kozak sequences, which are commonly involved in ribosomal initiation of translation of many genes. 5 'UTRs also may form secondary structures that function in elongation factor binding to further facilitate translation. The skilled person would recognize that engineering these features may enhance the stability and protein production of the polynucleotides of the disclosure. Untranslated regions useful in the design and manufacture of polynucleotides include, for example and without limitation, those disclosed in International Patent Publication No. WO 2014 / 164253 (see also US 2016 / 0022840).

[0309] Other non-UTR sequences may be used as regions or subregions within the polynucleotides. For example, and without limitation, introns or fragments of introns sequences can be incorporated into regions of the polynucleotides. In some embodiments, incorporation of one or more intronic sequences may increase protein production and / or polynucleotide levels.

[0310] Combinations of features can be included in flanking regions and can be contained within other features. For example, an ORF can be flanked by a 5' UTR which can contain a strong Kozak translational initiation signal and / or a 3' UTR which can include an oligo(dT) sequence for templated addition of a poly-A tail. 5' UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes such as the 5'UTRs described in U.S. Patent Application Publication No. 2010 / 0293625. In some embodiments, a 5'UTR may comprise a sequence as set forth in SEQ ID NO:30.

[0311] A UTR, or a fragment thereof, can be placed in the same orientation as in the transcript from which it was selected, or can be altered in orientation and / or location. For example, a 5' or 3' UTR can be inverted, shortened, lengthened, or made with one or more other 5' UTRs or 3' UTRs. In some embodiments, a UTR sequence can be changed in some way relative to a reference sequence, e.g., an endogenous UTR. For example, a 3' or 5' UTR can be altered relative to a wild-type or native UTR by a change in orientation or location, by inclusion of additional nucleotides, deletion of nucleotides, or swapping or transposition of nucleotides.

[0312] In some embodiments, two copies of the same UTR are encoded either in series or substantially in series. In some embodiments, more than two copies of the same UTR are encoded either in series or substantially in series.

[0313] In some embodiments, flanking regions, e.g., flanking an ORF, can be heterologous. In some embodiments, a 5' untranslated region can be derived from a different species than a 3' untranslated region. The untranslated region can also include translation enhancer elements (TEE). For example and without limitation, TEEs are described in U.S. Patent Application

[0314] Publication No. 2009 / 0226470.

[0315] In certain embodiments, a polynucleotide (e.g., an mRNA) encoding an expression repressor or an expression repressor system further comprises a 3' UTR. A 3'-UTR is the section of mRNA immediately following the translation termination codon. In some embodiments, a 3'- UTR includes regulatory regions that post-transcriptionally influence gene expression. Such regulatory regions within a 3'-UTR can influence polyadenylation, translation efficiency, localization, and / or stability of the mRNA. In some embodiments, a 3'-UTR comprises a binding site for regulatory proteins and / or microRNAs. In some embodiments, the 3'-UTR has a silencer region, which binds to repressor proteins and inhibits the expression of the mRNA. In other embodiments, a 3'-UTR comprises an AU-rich element (ARE). Proteins may bind AREs to affect the stability and / or decay rate of mRNA. In some embodiments, a 3'-UTR comprises a sequence SEQ ID NO:31 that directs addition of adenine residues in a poly(A) tail to the end of the mRNA transcript. In some embodiments, a poly(A)tail comprises a sequence as set forth in SEQ ID NO:32.

[0316] Terminal Modifications

[0317] In some embodiments, an mRNA described herein comprises one or more terminal modifications, e.g., a 5'Cap structure and / or a poly-A tail (e.g., between 100-200 nucleotides in length). The 5' cap structure may be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8 -oxo-guanosine, 2- amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the modified RNAs also contains a 5' UTR comprising at least one Kozak sequence, and a 3' UTR. Such modifications are known and are described, e.g., in WO 2012 / 135805 and WO 2013 / 052523. Additional terminal modifications are described, e.g., in WO 2014 / 164253, WO 2016 / 011306, WO 2012 / 045075, and WO 2014 / 093924.

[0318] The polynucleotide comprising an mRNA encoding an expression repressor or an expression repressor system of the present disclosure can further comprise a 5' cap. The 5' cap can bind the mRNA Cap Binding Protein (CBP), thereby increasing mRNA stability. The cap can further assist the removal of 5' proximal introns removal during mRNA splicing. In some embodiments, a polynucleotide comprising an mRNA encoding an expression repressor or an expression repressor system of the present disclosure comprises a non- hydrolyzable cap structure preventing decapping. In some embodiments, a non-hydrolyzable cap structure increases mRNA half-life. Cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages; thus, modified nucleotides can be used during the capping reaction. Modified guanosine nucleotides may also be suitable for use in the present disclosure, e.g., a- thio-guanosine, a-methyl-phosphonate, and seleno-phosphate nucleotides.

[0319] In certain embodiments, a 5' cap comprises 2'-0-methylation of the ribose sugars at 5 '-terminal and / or 5'-anteterminal nucleotides at the 2'-hydroxyl group of the sugar ring. In some embodiments, a cap may include cap analogs, i.e., synthetic cap analogs, chemical caps, chemical cap analogs, or structural / functional cap analogs differing from naturally occurring (i.e., endogenous, wild-type, or physiological) 5'-caps in chemical structure. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to the polynucleotides of the disclosure.

[0320] In certain embodiments, an mRNA encoding an expression repressor or an expression repressor system of the present disclosure can be capped after manufacture (e.g., IVT or chemical synthesis), using enzymes, to generate 5 '-cap structures.

[0321] In certain embodiments, 5' terminal caps can include endogenous caps or cap analogs. In certain embodiments, a 5' terminal cap can comprise a guanine analog. Suitable guanine analogs include, for example and without limitation, inosine, N 1 -methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2- amino-guanosine, LNA-guanosine, and 2-azido- guanosine.

[0322] In some embodiments, an mRNA encoding an expression repressor or an expression repressor system of the present disclosure further comprises a poly-A tail. In some embodiments, one or more terminal groups on the poly-A tail can be incorporated for stabilization. Such poly-A tails can also include structural moieties or 2'-0-methyl modifications, for example, as taught by Li et al. (2005) Current Biology 15: 1501-1507.

[0323] In some embodiments, a poly-A tail when present is greater than 30 nucleotides in length. In some embodiments, a poly-A tail is greater than 35 nucleotides in length (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,

[0324] 2,500, or 3,000 nucleotides)

[0325] In some embodiments, a poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. For example, this can be based on the length of a coding region, the length of a particular feature or region, or based on the length of the product expressed from the polynucleotide. Accordingly, in some embodiments, a poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or fragment thereof.

[0326] In some embodiments, one or more polynucleotides may be linked together by a Poly-A binding protein (PABP) by the 3'-end of the PABP, using modified nucleotides at the 3'-terminus of a poly-A tail.

[0327] In some embodiments, an mRNA encoding an expression repressor or an expression repressor of the present disclosure comprises, consists essentially of, or consists of a 5' terminal cap, a 5' UTR, an open reading frame (ORF), a 3' UTR, and a polyA tail.

[0328] In some embodiments, a modified mRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5'-end binding proteins. The mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5 '- / 3' -linkage may be intramolecular or intermolecular. Such modifications are described, e.g., in WO 2013 / 151736.

[0329] Recombinant Expression Vectors

[0330] Nucleic acids as described herein or nucleic acids encoding an expression repressor or an expression repressor system described herein may be incorporated into a vector. Vectors, including those derived from retroviruses such as lentivirus, are suitable tools to achieve longterm gene transfer since they allow long-term, stable integration of a transgene, and its propagation in daughter cells. Examples of suitable vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In some embodiments, an expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and described in a variety of virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

[0331] Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. Vectors can be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence. Additional promoter elements, e.g., enhancing sequences, may regulate frequency of transcriptional initiation. Typically, these sequences are located in a region 30-110 bp upstream of a transcription start site, although a number of promoters have recently been shown to contain functional elements downstream of transcription start sites as well.

[0332] In some embodiments, an expression repressor or an expression repressor system described herein acts at an enhancing sequence. In some embodiments, the enhancing sequence is an enhancer, a stretch enhancer, a shadow enhancer, a locus control region (LCR), or a super enhancer. In some embodiments, the super enhancer comprises a cluster of enhancers and other regulatory elements. In some embodiments, these sequences are located in a region .2- 2 Mb upstream or downstream of a transcription start site. In some embodiments, the region is a noncoding region. In some embodiments, the region is associated with long-range regulation of a target gene, e.g., CTNNB1. In some embodiments, the regions are cell- type specific. In some embodiments, a super-enhancer modifies (e.g., increases or decreases) target gene expression, e.g., CTNNB1 expression, by recruiting the target gene promoter, e.g., CTNNB1 promoter. In some embodiments, the super enhancer interacts with a target gene promoter, e.g., CTNNB1 promoter, through an enhancer docking site. In some embodiments, the enhancer docking site is an anchor sequence. In some embodiments, the enhancer docking site is located at least 100 bp, 200 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, or 3000 bp away from the target gene promoter, e.g., CTNNB1 promoter. In some embodiments, a super enhancer region is at least 100 bp, at least 200 bp, at least 300 bp, at least 500 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, or at least 25 kb long.

[0333] Spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. For example, in a thymidine kinase (tk) promoter, spacing between promoter elements can be increased to about 50 bp apart before activity begins to decline. Without wishing to be bound by theory, it is hypothesized that depending upon the promoter, individual elements can function either cooperatively or independently to activate transcription.

[0334] One example of a suitable promoter for use in the present disclosure is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. In some embodiments, a suitable promoter is Elongation Growth Factor- la (EF-la). Alternatively, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters including, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0335] The present disclosure should not be interpreted to be limited to use of any particular promoter or category of promoters (e.g., constitutive promoters). For example, in some embodiments, inducible promoters are contemplated as part of the present disclosure. In some embodiments, use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence to which it is operatively linked, when such expression is desired. In some embodiments, use of an inducible promoter provides a molecular switch capable of turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0336] In some embodiments, an expression vector to be introduced can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some aspects, a selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate transcriptional control sequences to enable expression in the host cells. Useful selectable markers may include, for example, antibiotic -resistance genes, such as neomycin, etc. In some embodiments, reporter genes may be used for identifying potentially transfected cells and / or for evaluating the functionality of transcriptional control sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient source (of a reporter gene) and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity or visualizable fluorescence. Expression of a reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui- Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, a construct with a minimal 5’ flanking region that shows highest level of expression of reporter gene is identified as a promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for ability to modulate promoter-driven transcription.

[0337] Cells

[0338] The present disclosure is further directed, in part, to cells comprising an expression repressor or expression repressor system described herein. Any cell, e.g., cell line, e.g., a cell line suitable for expression of a recombinant polypeptide, known to one of skill in the art is suitable to comprise an expression repressor or an expression repressor system described herein. In some embodiments, a cell, e.g., cell line, may be used to express an expression repressor or an expression repressor system, e.g., expression repressor(s), described herein. In some embodiments, a cell, e.g., cell line, may be used to express or amplify a nucleic acid, e.g., a vector, encoding an expression repressor or an expression repressor system, e.g., expression repressor(s), described herein. In some embodiments, a cell comprises a nucleic acid encoding an expression repressor or an expression repressor system, e.g., expression repressor(s), described herein.

[0339] In some embodiments, a cell comprises a first nucleic acid encoding a first component of an expression repressor system, e.g., a first expression repressor, and a second nucleic acid encoding a second component of the expression repressor system, e.g., a second expression repressor. In some embodiments, wherein a cell comprises nucleic acid encoding an expression repressor system comprising two or more expression repressors, the sequences encoding each expression repressor are disposed on separate nucleic acid molecules, e.g., on different vectors, e.g., a first vector encoding a first expression repressor and a second vector encoding a second expression repressor. In some embodiments, the sequences encoding each expression repressor are disposed on the same nucleic acid molecule, e.g., on the same vector. In some embodiments, some or all of the nucleic acid encoding the expression repressor system is integrated into the genomic DNA of the cell. In some embodiments, the nucleic acid encoding a first expression repressor of an expression repressor system is integrated into the genomic DNA of a cell, and the nucleic acid encoding a second expression repressor of an expression repressor system is not integrated into the genomic DNA of a cell (e.g., is situated on a vector). In some embodiments, the nucleic acid(s) encoding a first and a second expression repressor of an expression repressor system are integrated into the genomic DNA of a cell, e.g., at the same (e.g., adjacent or colocalized) or different sites in the genomic DNA.

[0340] Examples of cells that may comprise and / or express an expression repressor system or an expression repressor described herein include, but are not limited to, hepatocytes, neuronal cells, endothelial cells, myocytes, and lymphocytes.

[0341] Methods of Making RNA

[0342] Methods of making and purifying modified RNAs are known and disclosed in the art. For example and without limitation, modified RNAs are made using in vitro transcription (IVT) enzymatic synthesis. Methods of making IVT polynucleotides are known in the art and are described in WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151671, WO 2013 / 151672, WO 2013 / 151667, and WO 2013 / 151736. Methods of purification include purifying an RNA transcript comprising a polyA tail by contacting the sample with a surface linked to a plurality of thymidines or derivatives thereof and / or a plurality of uracils or derivatives thereof (polyT / U) under conditions such that the RNA transcript binds to the surface and eluting the purified RNA transcript from the surface (WO 2014 / 152031); using ion (e.g., anion) exchange chromatography that allows for separation of longer RNAs up to 10,000 nucleotides in length via a scalable method (WO 2014 / 144767); and subjecting a modified RMNA sample to DNAse treatment (WO 2014 / 152030). Modified RNAs encoding proteins in the fields of human disease, antibodies, viruses, and a variety of in vivo settings are known and are disclosed in for example, Table 6 of International Publication Nos. WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151736; Tables 6 and 7 International Publication No. WO 2013 / 151672; Tables 6, 178 and 179 of International Publication No. WO 2013 / 151671; and Tables 6, 185 and 186 of International Publication No WO 2013 / 151667. Any of the foregoing may be synthesized as an IVT polynucleotide, chimeric polynucleotide or a circular polynucleotide and linked to the polypeptide described herein, and each may comprise one or more modified nucleotides or terminal modifications.

[0343] In some embodiments, an expression repressor comprises or consists of a protein and may thus be produced by methods of making proteins as known in the art, for example, as provided in the present disclosure. In some embodiments, an expression repressor system, e.g., the expression repressor(s) of an expression repressor system, comprise one or more proteins and may thus be produced by methods of making proteins. As will be appreciated by one of skill in the art, methods of making proteins or polypeptides (which may be included in modulating agents as described herein) are routine in the art. See, e.g., Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); see also Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0344] Delivery

[0345] Lipid Particles

[0346] Expression repressors or expression repressor systems as described herein can be delivered using any biological delivery system / formulation including a particle, for example, a nanoparticle delivery system. Nanoparticles include particles with a dimension e.g., diameter) between about 1 and about 1000 nanometers, between about 1 and about 500 nanometers in size, between about 1 and about 100 nm, between about 30 nm and about 200 nm, between about 50 nm and about 300 nm, between about 75 nm and about 200 nm, between about 100 nm and about 200 nm, and any range therebetween. A nanoparticle has a composite structure of nanoscale dimensions. In some embodiments, nanoparticles are typically spherical although different morphologies are possible depending on the nanoparticle composition. The portion of the nanoparticle contacting an environment external to the nanoparticle is generally identified as the surface of the nanoparticle. In some embodiments, nanoparticles have a greatest dimension ranging between 25 nm and 200 nm. Nanoparticles as described herein comprise delivery systems that may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal nanoparticles. A nanoparticle delivery system may include but is not limited to lipid-based systems, liposomes, micelles, micro-vesicles, exosomes, or gene gun. In one embodiment, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP is a particle that comprises a plurality of lipid molecules physically associated with each other by intermolecular forces.

[0347] In some embodiments, an LNP may comprise multiple components, e.g., 3-4 components. In one embodiment, the expression repressor or a pharmaceutical composition comprising said expression repressor (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repressor nucleic acid) is encapsulated in an LNP. In one embodiment, the expression repressor system or a pharmaceutical composition comprising said expression repressor system (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repressor system nucleic acid) is encapsulated in an LNP. In some embodiments, the nucleic acid encoding the first expression repressor and the nucleic acid encoding the second expression repressor are present in the same LNP. In some embodiments, the nucleic acid encoding the first expression repressor and the nucleic acid encoding the second expression repressor are present in different LNPs. Preparation of LNPs and the modulating agent encapsulation may be used / and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. In some embodiments, lipid nanoparticle compositions disclosed herein are useful for expression of a protein encoded by mRNA. In some embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

[0348] In some embodiments, the LNP formulations may include a CCD lipid, a neutral lipid, and / or a helper lipid. In some embodiments, the LNP formulation comprises an ionizable lipid. In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, or an amine- containing lipid that can be readily protonated. In some embodiments, the lipid is a cationic lipid that can exist in a positively charged or neutral form depending on pH. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyl lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids.

[0349] In some embodiments, LNP formulation (e.g., MC3 and / or SSOP) includes cholesterol, PEG, and / or a helper lipid. The LNPs may be, e.g., microspheres (including unilamellar and multilamellar vesicles, lamellar phase lipid bilayers that, in some embodiments, are substantially spherical).

[0350] In some embodiments, the LNP can comprise an aqueous core, e.g., comprising a nucleic acid encoding an expression repressor or a system as disclosed herein and referred to herein as “cargo.” In some embodiments of the present disclosure, the cargo for the LNP formulation includes at least one guide RNA. In some embodiments, the cargo, e.g., a nucleic acid encoding an expression repressor, or a system as disclosed herein, may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the cargo, e.g., a nucleic acid encoding an expression repressor, or a system as disclosed herein, may be associated with the LNP. In some embodiments, the cargo, e.g., a nucleic acid encoding an expression repressor, or a system as disclosed herein, may be encapsulated, e.g., fully encapsulated and / or partially encapsulated in an LNP.

[0351] In some embodiments, an LNP comprising a cargo may be administered for systemic delivery, e.g., delivery of a therapeutically effective dose of cargo that can result in a broad exposure of an active agent within an organism. Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example and without limitation, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery. In some embodiments, an LNP comprising a cargo may be administered for local delivery, e.g., delivery of an active agent directly to a target site within an organism.

[0352] In some embodiments, an LNP may be locally delivered into a disease site, e.g., a tumor, or other target site, e.g., a site of inflammation, or to a target organ, e.g., the liver, lung, stomach, colon, pancreas, uterus, breast, lymph nodes, and the like. In some embodiments, an LNP as disclosed herein may be locally delivered to a specific cell, e.g., lung, pancreas, and / or epithelial cells. In some embodiments, an LNP as disclosed herein may be locally delivered to a specific site, e.g., a tumor site, e.g., by subcutaneous or orthotopic administration. The LNPs may be formulated as a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. In some embodiments, the LNPs are biodegradable. In some embodiments, the LNPs do not accumulate to cytotoxic levels or cause toxicity in vivo at a therapeutically effective dose. In some embodiments, the LNPs do not accumulate to cytotoxic levels or cause toxicity in vivo after repeat administrations at a therapeutically effective dose. In some embodiments, the LNPs do not cause an innate immune response that leads to a substantially adverse effect at a therapeutically effective dose.

[0353] In some embodiments, the LNP used comprises the formula (6Z,9Z,28Z,31Z)- heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate or ssPalmO-phenyl-P4C2 (ssPalmO-Phe, SS-OP). In some embodiments, the LNP formulation comprises the formula, (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate(MC3), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), Cholesterol, l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000(PEG2k-DMG), e.g., MC3 LNP or ssPalmO-phenyl-P4C2 (ssPalmO-Phe, SS-OP), l,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), Cholesterol, 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(PEG2k-DMG), e.g., SSOP-LNP.

[0354] Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011).

[0355] Vesicles can be made from several different types of lipids; however, phospholipids are most used to generate liposomes as drug carriers. Vesicles may comprise, for example and without limitation, DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Methods for preparation of multilamellar vesicle lipids are known in the art (see, for example, U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al, Nature Biotech, 15:647-652, 1997, the teachings of which relate to extruded lipid preparation are incorporated herein by reference.

[0356] Viral Vectors

[0357] In some embodiments, viral vector systems which can be utilized with the methods and compositions described herein. Suitable viral vector systems for use include, for example and without limitation, (a) adenovirus vectors (e.g., an Ad5 / F35 vector); (b) retrovirus vectors, including but not limited to lentiviral vectors (including integration competent or integrationdefective lentiviral vectors), moloney murine leukemia virus, etc.', (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells’ genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors. See, e.g., U.S. Patent Nos.6, 534, 261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the entire contents of each of which is incorporated by reference herein. Vectors, including those derived from retroviruses such as lentivirus, are suitable tools to achieve long- term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In certain embodiments, an expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is known in the art and described in a variety of virology and molecular biology manuals.

[0358] In some embodiments, a suitable viral vector for use in the present invention is an adeno- associated viral vector, such as a recombinant adeno-associated viral vector. Recombinant adeno-associated virus vectors (rAAV) are gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. In some embodiments, the vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al., Lancet 351:91171702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)). AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9, can be used in accordance with the present invention. Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titer and readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and / or E3 genes; subsequently, the replication defective vector is propagated in a suitable cell system, e.g., HEK293 and variants thereof, that supply deleted gene function in trans.

[0359] Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells, such as those found in liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24: 15-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther.2:205-18 (1995); Alvarez et al., Hum. Gene Ther.5: 597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); and Sterman et al., Hum. Gene Ther. 7: 1083-1089 (1998).

[0360] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include, for example and without limitation, HEK293 cells, and variants thereof, q / 2 cells, and PA317 cells. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. In some embodiments, viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. In certain embodiments, the cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. In certain embodiments, the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. In certain embodiments, contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

[0361] Methods of Use

[0362] Epigenetic Modification

[0363] In some aspects, the present disclosure provides a method for introducing one or more epigenetic modifications (e.g., DNA methylation and / or histone modification) to a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression repressor described herein or a nucleic acid encoding the expression repressor (e.g., an LNP- formulated expression repressor or an LNP-formulated nucleic acid encoding the expression repressor), wherein the expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein (e.g., a DNA methyltransferase; a histone modifying enzyme).

[0364] In some aspects, the present disclosure provides a method for introducing one or more epigenetic modifications (e.g., DNA methylation and / or histone modification) to a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression repressor system described herein comprising at least one expression repressor (e.g., 1, 2, 3, 4, 5 or more expression repressors described herein) or a nucleic acid encoding the at least one expression repressor (e.g., an expression repressor system comprising at least one LNP- formulated expression repressor or an LNP-formulated nucleic acid encoding the at least one expression repressor), wherein the at least one expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein (e.g., a DNA methyltransferase; a histone modifying enzyme).

[0365] In some aspects, the present disclosure provides a method for introducing one or more epigenetic modifications to a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a subject, comprising administering to the subject a dose of an expression repressor described herein or a nucleic acid encoding the expression repressor (e.g., an LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the expression repressor), wherein the expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.

[0366] In some aspects, the present disclosure provides a method of introducing one or more epigenetic modifications to a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a subject, comprising administering to the subject a dose of an expression repressor system described herein comprising at least one expression repressor (e.g., 1, 2, 3, 4, 5 or more expression repressors described herein) or a nucleic acid encoding the at least one expression repressor (e.g., an expression repressor system comprising at least one LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the at least one expression repressor), wherein the at least one expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) an effector domain described herein.

[0367] In some embodiments, epigenetic modification at the site is increased as compared to prior to the contacting or as compared to a control cell or control population of cells not contacted with the expression repressor or expression repressor system. In some embodiments, epigenetic modification at the site is decreased as compared to prior to the contacting or as compared to a control cell or control population of cells not contacted with the expression repressor or expression repressor system. In some embodiments, the increase or decrease of the epigenetic modification at the site is associated with decreased expression of CTNNB1 in the cell or the population of cells. In some embodiments, the epigenetic modification comprises DNA methylation, wherein an increase in DNA methylation at the site is associated with decreased expression of CTNNB1 in the cell or the population of cells. In some embodiments, the epigenetic modification comprises a histone modification, wherein an increase in the histone modification at the site is associated with decreased expression of CTNNB1 in the cell or the population of cells. In some embodiments, the epigenetic modification comprises a histone modification, wherein a decrease in the histone modification at the site is associated with decreased expression of CTNNB1 in the cell or the population of cells. In some embodiments, the histone modification comprises histone acetylation, wherein a decrease in histone acetylation is associated with decreased expression of CTNNB1 in the cell or the population of cells. In some embodiments, the histone modification comprises histone methylation, wherein a decrease in histone methylation is associated with decreased expression of CTNNB1 in the cell or the population of cells.

[0368] In some aspects, the present disclosure provides a method of introducing a histone modification at a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1 ) in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression repressor described herein or a nucleic acid encoding the expression repressor (e.g., an LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the expression repressor), wherein the expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) a transcriptional repressor moiety described herein.

[0369] In some aspects, the present disclosure provides a method of introducing a histone modification at a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression repressor system described herein comprising at least one expression repressor (e.g., 1, 2, 3, 4, 5 or more expression repressors described herein) or a nucleic acid encoding the at least one expression repressor (e.g., an expression repressor system comprising at least one LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the at least one expression repressor), wherein the at least one expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) a transcriptional repressor moiety described herein.

[0370] In some embodiments, the transcriptional repressor moiety is a Kriippel associated box (KRAB) domain or a functional variant or fragment thereof.

[0371] In some embodiments, the transcriptional repressor moiety is a histone modifying enzyme. In some embodiments, the histone modifying enzyme is selected from a histone methyltransferase, a histone deacetylase, and a histone demethylase.

[0372] In some embodiments, the histone modification is deacetylation and the histone modifying enzyme is a histone deacetylase. In some embodiments, the histone deacetylase is selected from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, and a functional variant or fragment thereof. In some embodiments, the histone modification is histone methylation and the histone modifying enzyme is a histone methyltransferase. In some embodiments, the histone methyltransferase is selected from SETDB1, SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, and a functional variant or fragment thereof.

[0373] Methods to measure histone modification are known in the art. As understood by the skilled artisan, methods to detect histone modification of genomic DNA include, but are not limited to, mass spectrometry and genomic approaches based upon chromatin immunoprecipitation (ChIP) in combination with DNA microarray (i.e., ChlP-chip), high- throughput sequencing (i.e., ChlP-seq), or serial analysis of gene expression (ChlP-SAGE). Such methods are described in Kimura, et al (2013) J. Hu. Genetics 58:445.

[0374] In some aspects, the present disclosure provides a method of increasing DNA methylation at a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression repressor described herein or a nucleic acid encoding the expression repressor (e.g., an LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the expression repressor), wherein the expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) a DNA methyltransferase.

[0375] In some aspects, the present disclosure provides a method of increasing DNA methylation at a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a cell or a population of cells, comprising contacting the cell or the population of cells with a dose of an expression repressor system described herein comprising at least one expression repressor (e.g., 1, 2, 3, 4, 5 or more expression repressors described herein) or a nucleic acid encoding the at least one expression repressor (e.g., an expression repressor system comprising at least one LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the at least one expression repressor), wherein the at least one expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) a DNA methyltransferase.

[0376] Methods to measure DNA methylation are known in the art, including, but not limited to, mass spectrometry, methylation-specific PCR, sequencing based-assay such as bisulfite sequencing, the Hpall tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, GLAD-PCR assay, ChlP-on-chip assay, restriction landmark genomic scanning, methylated DNA immunoprecipitation, methyl sensitive southern blotting, high resolution Melt analysis, and methylation sensitive single nucleotide primer extension assay. In some embodiments, the method to measure DNA methylation of a target gene (e.g., CTNNB1) comprises use of a DNA methylation microarray (e.g., an Illumina Methylation Array). Approaches for methylation analysis by microarray are described in Deatherage, et al (2009) Methods Mol Biol 556: 117-139; Schumacher, et al (2006) Nucleic Acids Res 34:528-42; and Willhelm-Benartzi, et al (2013) Br J Cancer 109:1394-1402. In some embodiments, the method comprises a sequencing-based assay, wherein genomic DNA is treated with an agent prior to sequencing that converts cytosine residues to uracil (or another base having distinct hybridization properties from cytosine) but does not affect 5 -methylcytosine residues. Exemplary agents are known in the art and include bisulfite, hydrogen sulfite, disulfite, and combinations thereof. Therefore, DNA treated with bisulfite retains the methylated cytosines, but not unmethylated cytosines. The treated DNA is then subjected to sequencing analysis (see, e.g., Campan et al (2009) Methods Mol Biol 507:325- 37; Adusumalli, et al (2015) Brief Bioinform 16:369-79). Exemplary methods for sequencing analysis are known in the art and include use of next generation sequencing platforms based on sequencing-by-synthesis or sequencing-by-ligation as employed by Illumina, Life Technologies, and Roche; or based on nanopore sequencing or electronic-detection as employed by Ion Torrent technology. In some embodiments, the method to measure DNA methylation comprises enzymatic methyl-seq (EM-seq) (see, e.g., Vaisvila et al (2021) Genome Res 31:1280). In EM- seq, enzymatic reactions (e.g., performed using TET2 and T4-BGT) are used to convert 5- methylcytosine (5mC) and 5 -hydroxy methylcytosine (i.e., the oxidation product of 5mC; also referred to as 5hmC) into products resistant to an enzymatic reaction that deaminates unmodified cytosines by converting them to uracils (e.g., performed using APOBEC3A). The enzymatically processed DNA is then amplified by PCR using EM-seq adaptor primers and subjected to sequencing analysis, e.g., using Illumina sequencing.

[0377] In some embodiments, DNA methylation at the site is increased as compared to prior to the contacting or as compared to a control cell or control population of cells not contacted with the expression repressor or expression repressor system.

[0378] I l l In some embodiments, DNA methylation at the site is increased for an extended duration following the contacting. In some embodiments, DNA methylation at the site is increased for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system.

[0379] In some embodiments, DNA methylation at the site is increased for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is at least about 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days. In some embodiments, the extended duration is at least about 21 days. In some embodiments, the extended duration is at least about 28 days.

[0380] In some embodiments, DNA methylation at the site is increased for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is about 10 to about 100 days, about 20 days to about 90 days, about 20 days to about 80 days, about 20 days to about 70 days, about 20 days to about 60 days, about 25 days to about 75 days, about 25 days to about 65 days, about 30 days to about 100 days, about 30 days to about 90 days, about 30 days to about 80 days, or about 30 days to about 70 days. In some embodiments, the extended duration is about 21 days to about 100 days. In some embodiments, the extended duration is about 21 days to about 200 days. In some embodiments, the extended duration is about 28 days to about 100 days. In some embodiments, the extended duration is about 28 days to about 200 days.

[0381] In some embodiments, DNA methylation at the site is increased for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments, the extended duration is at least about 3 weeks. In some embodiments, the extended duration is at least about 4 weeks.

[0382] In some embodiments, DNA methylation at the site is increased for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is about 1 week to about 48 weeks, about 1 week to about 36 weeks, about 1 week to about 24 weeks, about 1 week to about 12 weeks, about 2 weeks to about 48 weeks, about 2 weeks to about 36 weeks, about 2 weeks to about 24 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 48 weeks, about 3 weeks to about 36 weeks, about 3 weeks to about 24 weeks, about 3 weeks to about 12 weeks, about 4 weeks to about 48 weeks, about 4 weeks to about 36 weeks, about 4 weeks to about 24 weeks, or about 4 weeks to about 12 weeks. In some embodiments, the extended duration is at least about 3 weeks to about 12 weeks. In some embodiments, the extended duration is at least about 3 weeks to about 24 weeks. In some embodiments, the extended duration is at least about 3 weeks to about 48 weeks. In some embodiments, the extended duration is at least about 4 weeks to about 12 weeks. In some embodiments, the extended duration is at least about 4 weeks to about 24 weeks. In some embodiments, the extended duration is at least about 4 weeks to about 48 weeks.

[0383] In some embodiments, DNA methylation at the site is increased for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is at least 21 days.

[0384] In some embodiments, increased DNA methylation is maintained at the site for an extended duration following the contacting. In some embodiments, increased DNA methylation is maintained at the site for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system.

[0385] In some embodiments, increased DNA methylation is maintained at the site for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is at least about 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days. In some embodiments, the extended duration is about 10 to about 100 days, about 20 days to about 90 days, about 20 days to about 80 days, about 20 days to about 70 days, about 20 days to about 60 days, about 25 days to about 75 days, about 25 days to about 65 days, about 30 days to about 100 days, about 30 days to about 90 days, about 30 days to about 80 days, or about 30 days to about 70 days.

[0386] In some embodiments, increased DNA methylation is maintained at the site for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks.

[0387] In some embodiments, increased DNA methylation is maintained at the site for an extended duration following the contacting and prior to contacting the cell or the population of cells with a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is about 1 week to about 48 weeks, about 1 week to about 36 weeks, about 1 week to about 24 weeks, about 1 week to about 12 weeks, about 2 weeks to about 48 weeks, about 2 weeks to about 36 weeks, about 2 weeks to about 24 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 48 weeks, about 3 weeks to about 36 weeks, about 3 weeks to about 24 weeks, about 3 weeks to about 12 weeks, about 4 weeks to about 48 weeks, about 4 weeks to about 36 weeks, about 4 weeks to about 24 weeks, or about 4 weeks to about 12 weeks.

[0388] In some aspects, the disclosure provides a method of increasing DNA methylation at a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a subject, comprising administering to the subject a dose of an expression repressor described herein or a nucleic acid encoding the expression repressor (e.g., an LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the expression repressor), wherein the expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) a DNA methyltransferase.

[0389] In some aspects, the disclosure provides a method of increasing DNA methylation at a site in a region of the genome comprising CTNNB1 (e.g., human CTNNB1) in a subject, comprising administering to the subject a dose of an expression repressor system described herein comprising at least one expression repressor (e.g., 1, 2, 3, 4, 5 or more expression repressors described herein) or a nucleic acid encoding the at least one expression repressor (e.g., an expression repressor system comprising at least one LNP-formulated expression repressor or an LNP-formulated nucleic acid encoding the at least one expression repressor), wherein the at least one expression repressor comprises (i) a DNA targeting moiety (e.g., a TALE, a ZFN, a dCas9 / gRNA) that binds a target sequence described herein, and (ii) a DNA methyltransferase.

[0390] In some embodiments, DNA methylation at the site is increased as compared to prior to the administering or as compared to a control subject.

[0391] In some embodiments, DNA methylation at the site is increased for an extended duration following the administering. In some embodiments, DNA methylation at the site is increased for an extended duration following the administering and prior to administering to the subject a subsequent dose of the expression repressor or the expression repressor system.

[0392] In some embodiments, DNA methylation at the site is increased for an extended duration following the administering and prior to administering to the subject a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is at least about 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days. In some embodiments, the extended duration is about 10 to about 100 days, about 20 days to about 90 days, about 20 days to about 80 days, about 20 days to about 70 days, about 20 days to about 60 days, about 25 days to about 75 days, about 25 days to about 65 days, about 30 days to about 100 days, about 30 days to about 90 days, about 30 days to about 80 days, or about 30 days to about

[0393] 70 days.

[0394] In some embodiments, DNA methylation at the site is increased for an extended duration following the administering and prior to administering to the subject a subsequent dose of the expression repressor or the expression repressor system, wherein the extended duration is at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about ...

Claims

CLAIMS1. An expression repressor targeting a gene encoding P-catenin (CTNNB1) comprising(i) a DNA targeting moiety that binds to a target sequence, wherein the target sequence is about 15-20 nucleotides of a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chromosome 3 (chr3); and(ii) an effector domain.

2. The expression repressor of claim 1, wherein the region spans position 41,240,170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3.

3. The expression repressor of claim 1 or 2, wherein the region spans position 41,240,553 to position 41,240,770, according to the hgl9 reference genome for chr3.

4. The expression repressor of claim 1, wherein the region spans position 41,240,170 to position 41,240,300; position 41,240,250 to position 41,240,350; position 41,240,300 to position 41,240,400; position 41,240,350 to position 41,240,450; position 41,240,400 to position 41,240,500; position 41,240,450 to position 41,240,550; position 41,240,500 to position 41,240,600; position 41,240,550 to position 41,240,650; position 41,240,600 to position 41,240,700; position 41,240,650 to position 41,240,750; position 41,240,700 to position 41,240,800; position 41,240,750 to position 41,240,850; position 41,240,800 to position 41,240,900; position 41,240,850 to position 41,240,950; position 41,240,900 to position 41,241,000; position 41,240,950 to position 41,241,050; position 41,241,000 to position 41,241,100; position 41,241,050 to position 41,241,150; position 41,241,100 to position 41,241,200; position 41,241,150 to position 41,241,250; position 41,241,200 to position 41,241,300; position 41,241,250 to position 41,241,350; position 41,241,300 to position 41,241,400; position 41,241,350 to position 41,241,450; position 41,241,400 to position 41,241,523; position 41,241,450 to position 41,241,550; or position 41,241,500 to position 41,241,623, each according to the hgl9 reference genome for chr3.

5. The expression repressor of any one of claims 1-4, wherein the CTNBB1 target sequence is 15, 16, 17, 18, 19, or 20 nucleotides in length.

6. An expression repressor targeting CTNNB1 comprising(i) a DNA targeting moiety that binds to a target sequence, wherein the target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20; and(ii) an effector domain.

7. The expression repressor of claim 6, wherein the target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20.

8. The expression repressor of claim 6, wherein the target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20.

9. The expression repressor of one of claims 1-8, wherein the DNA-targeting moiety comprises a zinc finger (ZF) domain.

10. The expression repressor of one of claims 1-8, wherein the DNA-targeting moiety comprises a transcription activator-like effector (TALE) domain.

11. The expression repressor of one of claims 1-8, wherein the DNA-targeting moiety comprises a nuclease inactive Cas polypeptide (dCas) and a gRNA comprising a sequence complementary to the target sequence.

12. An expression repressor targeting CTNNB1 comprising(i) a DNA targeting moiety that binds to a target sequence, wherein the DNA targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 13; and(ii) an effector domain.

13. The expression repressor of claim 12, wherein the DNA targeting moiety comprises a SEQ ID NO: 13.

14. An expression repressor targeting CTNNB1 comprising(i) a DNA targeting moiety that binds to a target sequence of about 15-20 nucleotides in an insulated genomic domain (IGD) comprising CTNNB1, wherein the DNA targeting moiety comprises a ZF domain or a TALE domain; and(ii) an effector domain.

15. The expression repressor of claim 14, wherein the target sequence is in a region of about 500 bases to about 5,000 bases comprising a CpG island.

16. The expression repressor of claim 14 or 15, wherein the target sequence is (i) in a CpG island; or (ii) up to 200 bases upstream or downstream of a CpG island.

17. The expression repressor of any one of claims 14-16, wherein the target sequence is in or near a promoter region of CTNNB1.

18. The expression repressor of any one of claims 14-17, wherein the target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20.

19. The expression repressor of any one of claims 14-18, wherein the target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20.

20. The expression repressor of any one of claims 14-19, wherein the target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20.

21. The expression repressor of any one of claims 14-20, wherein the DNA targeting moiety comprises a ZF domain.

22. The expression repressor of any one of claims 14-20, wherein the DNA targeting moiety comprises a TALE domain.

23. The expression repressor of claim 22, wherein the DNA targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 13.

24. The expression repressor of claim 22 or 23, wherein the DNA targeting moiety comprises SEQ ID NO: 13.

25. The expression repressor of any one of claims 1-24, wherein the effector domain comprises a transcriptional repressor moiety.

26. The expression repressor of claim 25, wherein the transcriptional repressor moiety comprises a Kruppel associated box (KRAB) domain or a functional variant or fragment thereof.

27. The expression repressor of claim 25, wherein the transcriptional repressor moiety comprises a histone modifying enzyme selected from a histone methyltransferase, a histone deacetylase, and a histone demethylase.

28. The expression repressor of claim 27, wherein the histone modifying enzyme is a histone deacetylase.

29. The expression repressor of claim 28, wherein the histone deacetylase is HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC1 1, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment thereof.

30. The expression repressor of claim 27, wherein the histone modifying enzyme is a histone methyltransferase.

31. The expression repressor of claim 30, wherein the histone methyltransferase is SETDB 1 , SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof.

32. The expression repressor of claim 25, wherein the transcriptional repressor moiety comprises a DNA methyltransferase.

33. The expression repressor of claim 32, wherein the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof.

34. The expression repressor of claim 33, wherein the DNA methyltransferase is MQ1, or a functional variant or fragment thereof.

35. A nucleic acid comprising a nucleotide sequence encoding the expression repressor of any one of claims 1-33.

36. A recombinant expression vector comprising the nucleic acid of claim 35.

37. A messenger RNA (mRNA) encoding the expression repressor of any one of claims 1-34.

38. A lipid nanoparticle (LNP) comprising the expression repressor of any one of claims 1- 34, the nucleic acid of claim 35, the recombinant expression vector of claim 36, or the mRNA of claim 37.

39. A pharmaceutical composition comprising the expression repressor of any one of claims 1-34, the nucleic acid of claim 35, the recombinant expression vector of claim 36, the mRNA of claim 37, or the LNP of claim 38, and a pharmaceutically acceptable carrier.

40. A system for modulating expression of human CTNNB1 comprising(i) the expression repressor according to any one of claims 1 -34, and(ii) a second expression repressor comprising: a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain.

41. The system of claim 40, wherein the expression repressor and the second expression repressor are in the same composition.

42. The system of claim 40, wherein the expression repressor and the second expression repressor are in different compositions.

43. The system of claim 40, comprising a first nucleic acid encoding the expression repressor and a second nucleic acid encoding the second expression repressor.

44. The system of claim 43, wherein the first nucleic acid and the second nucleic acid are in the same composition.

45. The system of claim 43, wherein the first nucleic acid and the second nucleic acid are in different compositions.

46. The system of claim 43, wherein the first nucleic acid and the second nucleic acid are formulated in the same LNP.

47. The system of claim 43, wherein the first nucleic acid and the second nucleic acid are formulated in different LNPs.

48. The system of claim 43, comprising a first recombinant expression vector comprising the first nucleic acid and a second recombinant expression vector comprising the second nucleic acid.

49. The system of claim 48, wherein the first recombinant expression vector and the second recombinant expression vector are formulated in the same LNP.

50. The system of claim 48, wherein the first recombinant expression vector and the second recombinant expression vector are formulated in different LNPs.

51. The system of claim 43, comprising a recombinant expression vector comprising the first nucleic acid and the second nucleic acid.

52. The system of claim 51 , wherein the recombinant expression vector is formulated in an LNP.

53. A nucleic acid comprising a first nucleotide sequence encoding the expression repressor according to any one of claims 1-34, and a second nucleotide sequence encoding a second expression repressor comprising a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain.

54. A recombinant expression vector comprising the nucleic acid of claim 53.

55. An mRNA that encodes: the expression repressor according to any one of claims 1-34; and a second expression repressor comprising: a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain.

56. The mRNA of claim 55, wherein the ORF comprises a ribosome skipping sequence between the first nucleotide sequence and the second nucleotide sequence.

57. An LNP comprising the nucleic acid of claim 53, the recombinant expression vector of claim 54, or the mRNA of claim 55 or 56.

58. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of any one of claims 40-57, wherein the second target sequence is about 15-20 nucleotides in an insulated genomic domain (IGD) comprising CTNNB1.

59. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 58, wherein the second target sequence is in a region spanning position 41,240,170 to position 41,241,623, according to the hgl9 reference genome for chr3.

60. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 59, wherein the region spans position 41,240,170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3.

61. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 59, wherein the region spans position 41,240,170 to position 41,240,300; position 41,240,250 to position 41,240,350; position 41,240,300 to position 41,240,400; position 41,240,350 to position 41,240,450; position 41,240,400 to position 41,240,500; position 41,240,450 to position 41,240,550; position 41,240,500 to position 41,240,600; position 41,240,550 to position 41,240,650; position 41,240,600 to position 41,240,700; position 41,240,650 to position 41,240,750; position 41,240,700 to position 41,240,800; position 41,240,750 to position 41,240,850; position 41,240,800 to position 41,240,900; position 41,240,850 to position 41,240,950; position 41,240,900 to position 41,241,000; position 41,240,950 to position 41,241,050; position 41,241,000 to position 41,241,100; position 41,241,050 to position 41,241,150; position 41,241,100 to position 41,241,200; position 41,241,150 to position 41,241,250; position 41,241,200 to position 41,241,300; position 41,241,250 to position41,241,350; position 41,241,300 to position 41,241,400; position 41,241,350 to position41,241,450; position 41,241,400 to position 41,241,523; position 41,241,450 to position 41,241,550; or position 41,241,500 to position 41,241,623, each according to the hgl9 reference genome for chr362. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of any one of claims 40-61 , wherein the second target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20.

63. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 62, wherein the second target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20.

64. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 62 or63, wherein the second target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20.

65. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of any one of claims 40-64, wherein the second DNA-targeting moiety of the second fusion protein comprises a zinc finger (ZF) domain.

66. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of any one of claims 40-64, wherein the second DNA-targeting moiety of the second fusion protein comprises a TALE domain.

67. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 66, wherein the second DNA targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 13.

68. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 66 or 67, wherein the second DNA targeting moiety comprise SEQ ID NO: 13.

69. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of any one of claims 40-64, wherein the second DNA-targeting moiety of the second fusion protein comprises a dCas and a gRNA comprising a sequence complementary to the second target sequence.

70. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of any one of claims 40-69, wherein the second effector domain comprises a second transcriptional repressor moiety.

71. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 70, wherein the second transcriptional repressor moiety comprises a Kruppel associated box (KRAB) domain or a functional variant or fragment thereof.

72. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 70, wherein the second transcriptional repressor moiety comprises a histone modifying enzyme selected from a histone methyltransferase, a histone deacetylase, and a histone demethylase.

73. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 72, wherein the histone modifying enzyme is a histone deacetylase.

74. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 73, wherein the histone deacetylase is 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 thereof.

75. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 72, wherein the histone modifying enzyme is a histone methyltransferase.

76. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 75, wherein the histone methyltransferase is SETDB1, SETDB2, EHMT2, EHMT1, SUV39H1,EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof.

77. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 70, wherein the second transcriptional repressor moiety comprises a DNA methyltransferase.

78. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 77, wherein the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A1, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof.

79. The system, nucleic acid, recombinant expression vector, mRNA, or LNP of claim 77, wherein the DNA methyltransferase is MQ1, or a functional variant or fragment thereof.

80. A pharmaceutical composition comprising the system of any one of claims 40-52 and 58- 79, the nucleic acid of any one of claims 53 and 58-79, the recombinant expression vector of any one of claims 54 and 58-79, the mRNA of any one of claims 55-56 and 58-79, or the LNP of any one of claims 57-79, and a pharmaceutically acceptable carrier.

81. A cell comprising the expression repressor of any one of claims 1 -34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80.

82. A method of altering expression of CTNNB1 in a cell, comprising contacting the cell with the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80.

83. The method of claim 82, wherein expression of CTNNB1 is decreased.

84. A method of introducing one or more epigenetic modifications to CTNNB1 in a cell, comprising contacting the cell with the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80.

85. The method of claim 84, wherein the epigenetic modification is DNA methylation or histone methylation.

86. A method of treating a condition associated with CTNNB1 expression in a subject, comprising administering to the subject the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80.

87. The method of claim 86, wherein the condition is associated with a mutation in CTNNB1.

88. The method of claim 86 or 87, wherein the conditions is associated with overexpression of CTNNB1.

89. The method of any one of claims 86-88, wherein the condition is cancer.

90. A method of treating cancer in a subject comprising administering to the subject the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of anyone of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80.

91. A method of reducing tumor burden in a subject having cancer comprising administering to the subject the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80.

92. The method of claim 90 or 91 , wherein the cancer is associated with a mutation in CTNNB1.

93. The method of any one of claims 90-92, wherein the cancer is selected from endometrial cancer, hepatobiliary cancer, melanoma, colorectal cancer, bladder cancer, skin cancer, prostate cancer, non-small cell lung cancer, and pancreatic cancer.

94. A kit comprising a container comprising a pharmaceutical composition comprising the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, or the LNP of any one of claims 38 and 57-79, and a pharmaceutically acceptable carrier, and instructions for use in treating a condition associated with CTNNB1 expression in a subject.

95. The kit of claim 94, wherein the condition is associated with a mutation in CTNNB1.

96. The kit of claim 94 or 95, wherein the conditions is associated with overexpression of CTNNB1.

97. The kit of any one of claims 94-96, wherein the condition is cancer.

98. A kit comprising a container comprising a pharmaceutical composition comprising the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, or the LNP of any one of claims 38 and 57-79, and a pharmaceutically acceptable carrier, and instructions for use in treating cancer in a subject.

99. A kit comprising a container comprising a pharmaceutical composition comprising the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, or the LNP of any one of claims 38 and 57-79, and a pharmaceutically acceptable carrier, and instructions for use in reducing tumor burden in a subject having cancer.

100. The kit of claim 98 or 99, wherein the cancer is associated with a mutation in CTNNB1.

101. A method of treating a condition associated with CTNNB1 expression in a subject, comprising administering to the subject (i) the expression repressor according to any one of claims 1-34, and (ii) a second expression repressor comprising: a second DNA targeting moiety that binds to a second target sequence that is different from the target sequence, and a second effector domain, wherein the second effector domain is the same as or different from the effector domain.

102. The method of claim 101, wherein the condition is associated with a mutation in CTNNB1.

103. The method of claim 101 or 102, wherein the conditions is associated with overexpression of CTNNB1.

104. The method of any one of claims 101-103, wherein the condition is cancer.

105. The method of any one of claims 101-104, comprising administering the expression repressor and the second expression repressor in the same composition or in different compositions.

106. The method of any one of claims 101-104, comprising administering a first nucleic acid encoding the expression repressor and a second nucleic acid encoding the second repression repressor.

107. The method of claim 106, wherein the first nucleic acid is an mRNA encoding the expression repressor.

108. The method of claim 106 or 107, wherein the second nucleic acid is an mRNA encoding the second expression repressor.

109. The method of any one of claims 106-108, comprising administering the first nucleic acid and the second nucleic acid in the same composition or in different compositions.

110. The method of any one of claims 106-108, comprising administering the first nucleic acid and the second nucleic acid formulated in the same LNP or in separate LNPs.

111. The method of claim 106, comprising administering a first recombinant expression vector comprising the first nucleic acid and a second recombinant expression vector comprising the second nucleic acid.

112. The method of claim 111, wherein the first recombinant expression vector and the second recombinant expression vector are formulated in the same LNP or in separate LNPs.

113. The method of claim 106, comprising administering a recombinant expression vector comprising the first nucleic acid and the second nucleic acid.

114. The method of claim 113, wherein the recombinant expression vector is formulated in an LNP.

115. The method of any one of claims 101-114, wherein the second target sequence is about 15-20 nucleotides in an IGD comprising CTNNB1.

116. The method of claim 115, wherein the second target sequence is in a region spanning position 41,240,270 to position 41,241,523 according to the hgl9 reference genome for chr3.

117. The method of claim 116, wherein the region spans position 41 ,240, 170 to position 41,240,387, position 41,240,553 to position 41,240,770, or position 41,241,406 to position 41,241,623, each according to the hgl9 reference genome for chr3.

118. The method of claim 116, wherein the region spans position 41,240,170 to position 41,240,300; position 41,240,250 to position 41,240,350; position 41,240,300 to position 41,240,400; position 41,240,350 to position 41,240,450; position 41,240,400 to position 41,240,500; position 41,240,450 to position 41,240,550; position 41,240,500 to position 41,240,600; position 41,240,550 to position 41,240,650; position 41,240,600 to position 41,240,700; position 41,240,650 to position 41,240,750; position 41,240,700 to position 41,240,800; position 41,240,750 to position 41,240,850; position 41,240,800 to position 41,240,900; position 41,240,850 to position 41,240,950; position 41,240,900 to position 41,241,000; position 41,240,950 to position 41,241,050; position 41,241,000 to position 41,241,100; position 41,241,050 to position 41,241,150; position 41,241,100 to position 41,241,200; position 41,241,150 to position 41,241,250; position 41,241,200 to position 41,241,300; position 41,241,250 to position 41,241,350; position 41,241,300 to position 41,241,400; position 41,241,350 to position 41,241,450; position 41,241,400 to position 41,241,523; position 41,241,450 to position 41,241,550; or position 41,241,500 to position 41,241,623, each according to the hgl9 reference genome for chr3.

119. The method of any one of claims 115-118, wherein the second target sequence comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 8, 14, and 20.

120. The method of claim 119, wherein the second target sequence comprises a sequence selected from SEQ ID NOs: 8, 14, and 20.

121. The method of claim 119 or 120, wherein the second target sequence consists of a sequence selected from SEQ ID NOs: 8, 14, and 20.

122. The method of any one of claims 101-121, wherein the second DNA-targeting moiety of the second fusion protein comprises a zinc finger (ZF) domain.

123. The method of any one of claims 101-121, wherein the second DNA-targeting moiety of the second fusion protein comprises a TALE domain.

124. The method of claim 123, wherein the second DNA targeting moiety comprises a sequence having at least 90% identity to SEQ ID NO: 13.

125. The method of claim 123 or 124, wherein the second DNA targeting moiety comprises SEQ ID NO: 13.

126. The method of any one of claims 101-121, wherein the second DNA-targeting moiety of the second fusion protein comprises a dCas9 and a gRNA comprising a sequence complementary to the second target sequence.

127. The method of any one of claims 101-126, wherein the second effector domain comprises a second transcriptional repressor moiety.

128. The method of claim 127, wherein the second transcriptional repressor moiety comprises a Kruppel associated box (KRAB) domain or a functional variant or fragment thereof.

129. The method of claim 127, wherein the second transcriptional repressor moiety comprises a histone modifying enzyme selected from a histone methyltransferase, a histone deacetylase, and a histone demethylase.

130. The method of claim 129, wherein the histone modifying enzyme is a histone deacetylase.

131. The method of claim 130, wherein the histone deacetylase is 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 thereof.

132. The method of claim 129, wherein the histone modifying enzyme is a histone methyltransferase.

133. The method of claim 132, wherein the histone methyltransferase is SETDB 1, SETDB2, EHMT2, EHMT1, SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment thereof.

134. The method of claim 127, wherein the second transcriptional repressor moiety comprises a DNA methyltransferase.

135. The method of claim 134, wherein the DNA methyltransferase is MQ1, DNMT1, DNMT3A1, DNMT3A1, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment thereof.

136. The method of claim 134, wherein the DNA methyltransferase is MQ1, or a functional variant or fragment thereof.

137. A method of decreasing expression of CTNNB1 in a cell, comprising contacting the cell with the expression repressor of any one of claims 1-34, the system of any one of claims 40-52and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80, thereby decreasing expression of CTNNB1 by at least about 15%.

138. The method of claim 137, wherein expression of CTNNB1 is decreased as compared to a control cell not contacted with the expression repressor, system, nucleic acid, recombinant expression vector, mRNA, LNP, or pharmaceutical composition.

139. The method of claim 137 or 138, wherein decreased expression of CTNNB1 is measured as a decrease in the level of an RNA transcript of CTNNB1 and / or P-catenin in the cell.

140. The method of any one of claims 137-139, wherein expression of CTNNB1 is decreased by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

141. The method of any one of claims 137-139, wherein expression of CTNNB1 is decreased by about 1.5-fold, about 2-fold, about 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as compared to a control cell not contacted with the expression repressor, system, nucleic acid, recombinant expression vector, mRNA, LNP, or pharmaceutical composition.

142. Use of the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80 for treating a condition associated with CTNNB1 expression in a subject, comprising administering the expression repressor, the system, the nucleic acid, the recombinant expression vector, the mRNA, the LNP, or the pharmaceutical composition to the subject.

143. Use of the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80 for treating cancer in a subject, comprising administering the expression repressor, the system, the nucleic acid, the recombinant expression vector, the mRNA, the LNP, or the pharmaceutical composition to the subject.

144. Use of the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80 for reducing tumor burden in a subject, comprising administering the expression repressor, the system, the nucleic acid, the recombinant expression vector, the mRNA, the LNP, or the pharmaceutical composition to the subject.

145. Use of the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80 in the manufacture of a medicament for treating a condition associated with CTNNB1 expression in a subject, comprising administering the medicament o the subject.

146. Use of the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80 in the manufacture of a medicament for treating cancer in a subject, comprising administering the medicament to the subject.

147. Use of the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80 in the manufacture of a medicament for reducing tumor burden in a subject, comprising administering the medicament to the subject.

148. A method of reducing cell viability in a population of cells, comprising contacting the population of cells with the expression repressor of any one of claims 1-34, the system of any one of claims 40-52 and 58-79, the nucleic acid of any one of claims 35, 53, and 58-79, the recombinant expression vector of any one of claims 36, 54, and 58-79, the mRNA of any one of claims 37, 55-56, and 58-79, the LNP of any one of claims 38 and 57-79, or the pharmaceutical composition of claim 39 or 80, thereby reducing cell viability in the population of cells.

149. The method of claim 148, wherein cell viability is reduced as compared to a population of control cells not contacted with the expression repressor, system, nucleic acid, recombinant expression vector, mRNA, LNP, or pharmaceutical composition.

150. The method of claim 148 or 149, wherein reduced cell viability is measured as a decrease in cell proliferation. 1

Images