Compositions and methods for epigenetic regulation of ZNF410 expression

Abstract

This present disclosure relates to compositions and methods for silencing ZNF410, as well as nucleic acids and vectors encoding the same. Also disclosed are cells epigenetically modified by the epigenetic editors.

Description

[0001] COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF ZNF410 EXPRESSION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application no.63 / 661,482, filed June 18, 2024, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF ZNF410 EXPRESSION” and U.S. Provisional Application no.63 / 711,567, filed October 24, 2024, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF ZNF410 EXPRESSION” the disclosure of each of which is hereby incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The content of the electronic sequence listing (C169870051WO00-SEQ-AXW.xml; Size: 1,472,272 bytes; and Date of Creation: June 18, 2025) is herein incorporated by reference in its entirety. BACKGROUND Genome editing has been considered a promising therapeutic approach for the treatment of genetic disease for over a decade. However, manipulation on the DNA level using traditional genetic editors remains risky given the potential for undesired double-strand DNA breaks, heterogenous repair (including large and small insertions and deletions at the intended site), and toxicity. In contrast, targeted epigenetic modification offers the potential to alter gene expression without leading to double-strand break-induced genotoxicity. Hemoglobinopathies, such as beta-hemoglobinopathies, are some of the most common monogenic disorders worldwide. Over 300,000 children are born every year with a beta-hemoglobinopathy, such as beta-thalassemia or sickle cell disease, with high prevalence in Africa, the Mediterranean region, the Middle East, and South Asia. SUMMARY Aspects of the present disclosure relate, at least in part, to a system for repressing transcription of a human ZNF410 gene in a human cell, optionally a human erythroid cell or a precursor cell to a human erythroid cell, comprising

[0002] 1 12541428.1 a) one or more fusion proteins that collectively comprise a DNA methyltransferase (DNMT) domain and / or a domain that recruits a DNMT (recruiter domain), optionally wherein the DNMT domain and / or the recruiter domain comprise a DNMT3A domain and / or a DNMT3L domain, and optionally wherein the recruiter DNMT is a DNMT3L domain and the recruited DNMT is a DNMT3A domain, and a transcriptional repressor domain, each domain being linked to a DNA-binding domain that binds to a target region in the human ZNF410 gene, wherein the target region comprises one or more sequences selected from SEQ ID NOs: 766-1355; or b) one or more nucleic acid molecules encoding the one or more fusion proteins, wherein the system does not generate a DNA break in the ZNF410 gene. In some embodiments, the target region comprises one or more sequences selected from SEQ ID NOs: 806-818. In some embodiments, the DNA-binding domain comprises a catalytically deactivated CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain. In some embodiments, the DNA-binding domain comprises a dCas9 domain and the system further comprises (i) one or more guide RNAs comprising a sequence that binds to any one of SEQ ID NOs: 766-1355, or (ii) nucleic acid molecules coding for the one or more guide RNAs. In some embodiments, the DNA-binding domain comprises a dCas9 domain and the system further comprises (i) one or more guide RNAs comprising a sequence that binds to any one of SEQ ID NOs: 806-818, or (ii) nucleic acid molecules coding for the one or more guide RNAs. Aspects of the present disclosure relate, at least in part, to a system for repressing transcription of a human ZNF410 gene in a human cell, optionally a human erythroid cell or a precursor cell to a human erythroid cell, comprising a) a fusion protein that comprises a DNMT3A domain, a DNMT3L domain, a DNA-binding domain that binds to a target region in the human ZNF410 gene, and a transcriptional repressor domain; or b) a nucleic acid molecule encoding the fusion protein, wherein the system does not generate a DNA break in the ZNF410 gene. In some embodiments, the DNA-binding domain comprises a dCas domain, a ZFP domain, or a TALE domain.

[0003] 2 12541428.1 In some embodiments, the DNA-binding domain comprises a dCas9 domain and the system further comprises (i) one or more guide RNAs comprising a sequence that binds to any one of SEQ ID NOs: 766-1355, or (ii) nucleic acid molecules coding for the one or more guide RNAs. In some embodiments, the one or more guide sequences bind to any one of SEQ ID NOs: 806-818. In some embodiments, the dCas domain comprises a dCas9 sequence, optionally a sequence with at least 90% identity to SEQ ID NO: 15 or 16. In some embodiments, the DNA-binding domain binds to a target sequence selected from in any one of SEQ ID NOs: 766-1355. In some embodiments, the DNA-binding domain binds to a target sequence selected from any one of SEQ ID NOs: 806-818. In some embodiments, the DNMT3A domain comprises a sequence with at least 90% identity to SEQ ID NO: 577 or 578. In some embodiments, the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 581-584. In some embodiments, the DNMT3L domain comprises a sequence with at least 95% identity to a sequence selected from SEQ ID NOs: 581-584. In some embodiments, the DNMT domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 579 and 585. In some embodiments, the transcriptional repressor domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 36-573. In some embodiments, the transcriptional repressor domain comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In some embodiments the KRAB domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 92, 119248, and 258. In some embodiments, the transcriptional repressor domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 574 or 575. In some embodiments, the transcriptional repressor domain is derived from KAP1, MECP2, HP1a / CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2. In some embodiments, the system comprises a) a fusion protein comprising the DNMT3A domain, the DNMT3L domain, the transcriptional repressor domain, and the DNA-binding domain,

[0004] 3 12541428.1 optionally wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and optionally wherein the DNA-binding domain is a catalytically deactivated CRISPR Cas domain, a ZFP domain, or a TELE domain; or b) a nucleic acid molecule encoding the fusion protein. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the DNMT3A domain, a first peptide linker, the DNMT3L domain, a second peptide linker, the DNA-binding domain, a third peptide linker, and the transcriptional repressor domain. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, a first nuclear localization signal (NLS), the DNA-binding domain, a second NLS, the third peptide linker, and the transcriptional repressor domain. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first nuclear localization signal (NLS), the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and a second NLS. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second nuclear localization signals (NLSs), the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and third and fourth NLSs. In some embodiments, the transcriptional repressor domain is a KRAB domain, optionally a human KOX1, ZFP28, ZN627, or ZIM3 KRAB domain. In some embodiments, one or both of the second and third peptide linkers are XTEN linkers, optionally selected from XTEN80 and XTEN16, and further optionally wherein the second peptide linker is XTEN80, and the third peptide linker is XTEN16. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN16 peptide linker, and a human KOX1 KRAB domain. In some embodiments, the fusion protein comprises SEQ ID NO: 734 or a sequence at least 90% identical thereto. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80

[0005] 4 12541428.1 peptide linker, a first NLS, a ZFP or TALE domain, a second NLS, an XTEN16 linker, and a human KOX1 KRAB domain. In some embodiments, the fusion protein comprises SEQ ID NO: 744 or a sequence at least 90% identical thereto. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs. In some embodiments, the fusion protein comprises SEQ ID NO: 1366 or a sequence at least 90% identical thereto. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP or TALE domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs. In some embodiments, the fusion protein comprises SEQ ID NO: 1367 or a sequence at least 90% identical thereto. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP or TALE domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs. In some embodiments, the fusion protein comprises SEQ ID NO: 1368 or a sequence at least 90% identical thereto. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP or TALE domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.

[0006] 5 12541428.1 In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs. In some embodiments, the fusion protein comprises SEQ ID NO: 1369 or a sequence at least 90% identical thereto. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs. In some embodiments, at least one of the NLSs is an SV40 NLS. In some embodiments, the system comprises: a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting the DNMT3A domain, a second fusion protein comprising a second DNA-binding domain and comprising or recruiting the DNMT3L domain, and a third fusion protein comprising a third DNA-binding domain and comprising or recruiting the transcriptional repressor domain; or b) one or more nucleic acid molecules encoding the fusion proteins. Aspects of the present disclosure relate, at least in part, to human cells comprising a system of the present disclosure, or progeny of the cell, optionally wherein the cell is an erythroid cell or a precursor cell to a human erythroid cell. Aspects of the present disclosure relate, at least in part, to human cells modified by a system of the present disclosure, or progeny of the cell, optionally wherein the cell is an erythroid cell or a precursor cell to a human erythroid cell, optionally wherein the cell was modified ex vivo. Aspects of the present disclosure relate, at least in part, to pharmaceutical compositions comprising a system of the disclosure and a pharmaceutically acceptable excipient, optionally wherein the composition comprises lipid nanoparticles (LNPs) comprising the system, and / or the DNA-binding domain is a dCas domain and the LNPs further comprise one or more gRNAs. Aspects of the present disclosure relate, at least in part, to pharmaceutical composition comprising human cells of the present disclosure and a pharmaceutically acceptable excipient.

[0007] 6 12541428.1 Aspects of the present disclosure relate, at least in part, to methods of treating a patient in need thereof, comprising administering the system of the present disclosure, human cells of the present disclosure, or a pharmaceutical composition of the present disclosure to the patient. In some embodiments, the patient has a beta-hemoglobinopathy. In some embodiments, the patient has Beta-thalassemia or sickle cell disease. Aspects of the present disclosure relate, at least in part, to a system of the present disclosure, human cells of the present disclosure, or a pharmaceutical composition of the present disclosure, for use in treating a patient in need thereof, optionally in a method associated with the disclosure. Aspects of the present disclosure relate, at least in part, to use of a system of the disclosure or human cells of the disclosure in the manufacture of a medicament for treating a patient in need thereof, optionally in a method associated with the disclosure. In some embodiments, the system comprises a DNMT3L domain and a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain is a KRAB domain. In some embodiments, the DNA-binding domain comprises a catalytically active Cas domain or a nickase. In some embodiments, the system further comprises (i) one or more guide RNAs comprising a sequence that binds to any one of SEQ ID NOs: 766-1355, (ii) one or more guide RNAs targeting BCL11A, and / or (iii) nucleic acid molecules coding for the one or more guide RNAs of (i) and / or (ii). In some embodiments, the one or more guide RNAs comprises a sequence that binds to any one of SEQ ID NOs: 806-818. BRIEF DESCRIPTION OF THE DRAWINGS FIGs.1A-1C illustrate that reawakening HbF has therapeutic applications. FIG.1A shows a schematic of human chromosome 11 containing genes encoding various globin genes. FIG.1B shows the percentage of globin synthesis attributed to different genes over the course of 15 months post-conception. FIG.1C shows a schematic of the BCL11A network as a target for various potential therapeutic modalities, adapted from Bauer, et al, Blood 2012. Numbers indicate targets points for potential intervention. FIGs.2A-2B show a schematic of a CRISPR-Cas9 screen identifying HbF regulators. FIG.2A shows a workflow for identification of HbF regulators via CRISPR-Cas9 screening.

[0008] 7 12541428.1 FIG.2B is a plot showing regulators of HbF levels. Figures adapted from Vinjamur, et al, Nat Gent.2021 and Lan, et al., Mol Cell.2021. FIG.3 shows GW mapping of ZNF410 binding in hematopoietic cells, adapted from Vinjamur, et al, Nat Gent.2021. CHD indicates Chromodomain-helicase-DNA-binding protein 4. FIGs.4A-4C show in vitro validation experiments demonstrating that loss of ZNF410 leads to γ-globin reactivation, as assessed by flow cytometry (FIG.4A), RT-QPCR (FIG. 4B), and HbF-APC (FIG.4C). Figures adapted from Vinjamur, et al, Nat Gent.2021. FIGs.5A-5B show in vivo transplantation of the edited cells demonstrating that loss of ZNF410 leads to γ-globin reactivation. FIG.5A shows a schematic of the experimental workflow. FIG.5B is a series of graphs showing results of cell characterization in harvested bone marrow. Figures adapted from Vinjamur, et al, Nat Gent.2021. FIG.6 is a schematic of an erythroid cell showing ZNF410 binding to CHD4, leading to inactivation of HbF. LCR, locus control region. Figure adapted from Vinjamur, et al, Nat Gent.2021. FIGs.7A-7B show generation of the ZNF410 reporter cell line (K-562 ZNF410tdTomato). FIG.7A shows a schematic of the human ZNF410 gene containing a 2A:tdTomato cassette inserted in frame with the last coding exon of the gene. Targeting was performed with the CRISPR-Cas9 system. FIG.7B shows a representative flow cytometry dot plot of the K-562 ZNF410tdTomatocells upon their enrichment for tdTomato expression. SSC-A indicates Side Scatter. FIGs.8A-8C show proof-of-principle of epi-silencing of ZNF410 using a panel of selected gRNAs. FIG.8A shows a schematic of the ZNF410 CpG island region showing the sites targeted by the selected gRNAs (black triangles; n=40). The annotated CpG island is represented by a filled rectangle. FIG.8B is a histogram showing the percentage of ZNF410tdTomato-negative cells (% of silenced cells) at day 21 post-nucleofection with plasmids encoding for either the tri-partite ETR (Tri-ETR) or a KRAB-based ETR (KRAB) together with the indicated gRNAs. Experimental conditions were K-562 ZNF410tdTomatoplasmid nucleofection of individual gRNAs (2.5 µg; n=40) and Tri-partite ETR (2.5 µg). Flow cytometry analysis was conducted on day 21. Associated Table 4 reports the sequence, genomic position, and the percent of silencing for each gRNA. FIG.8C shows representative flow cytometry dot plots of Untreated (UT) or K-562 ZNF410tdTomatocells treated as indicated. Data are from the FIG.8B histogram.

[0009] 8 12541428.1 FIG.9 shows the selection of the ZNF410 regions to be targeted by the gRNA library. A schematic of the ZNF410 locus with the regions investigated in the screening experiment highlighted by shading is shown. A total of 550 SpCas9 gRNAs were designed to recognize the promoter region (n=455 gRNA) and a putative regulatory element at the 3’ of the gene (n=95 gRNA). gRNAs predicted not to bind to the human genome (non-targeting gRNAs; n=100) were included in the final library as controls. Control sgRNAs: non-targeting sgRNAs based on Bassik Human CRISPR-Cas9 deletion library. FIG.10 shows a schematic of the experimental flow used for the pooled screening of the ZNF410 gRNAs. A Lentiviral Vector (LV) library expressing the BFP marker together with the 550 gRNAs against ZNF410 or the 100 control guides was prepared and used to transduce, at low Multiplicity of Infection (MOI), the K-562 ZNF410tdTomatocells. LV- positive cells were sorted according to BFP expression (representative flow cytometry dot blot at the bottom) and then transfected with plasmids encoding for (i) the triple ETR combination, (ii) the bi-partite ETR m3ls-dCas9-ZIM3, or (iii) the tri-partite ETR 3A:h3L- dCas9-K. Twenty-one days after transfection, the BFP-positive, tdTomato-negative cells were sorted (see FIG.11) and the complexity of the gRNAs was assessed by targeted deep- sequencing (>300x coverage) followed by bioinformatic analysis (by MAGeCK tool). K-562 ZNF410tdTomatocells transduced with the LV library but not transfected with the ETRs were used to control for input library complexity. FIG.11 shows flow cytometry results of ETR transfection and cell selection. FIG.12 shows identification of enriched gRNAs. Dot plots showing the normalized log10 read counts of each gRNA between ETR-treated (as indicated) and control cells. Red dots indicate non targeting gRNAs while grey dots indicate gRNAs against ZNF410. The number of gRNAs enriched over the control is indicated. Intersection of the enriched gRNAs among treatments showed a sharing of 56 to 66%. FIG.13 shows identification of enriched gRNAs. Histograms showing the fold- change of each gRNA in relation to its target position on the ZNF410 locus (the entire gene) are shown. The y axis is in linear scale. FIG.14 shows identification of enriched gRNAs. Histograms showing the fold- change of each gRNA in relation to its target position on the ZNF410 locus (a zoom on the 5’ of the gene) are shown. The y axis is in linear scale. FIG.15 shows identification of enriched gRNAs. Dot plots showing the log2 fold- change (log2FC) of each ZNF410 gRNA used in the screening are shown. The top 5 gRNAs for each treatment are indicated by colored dots and reported in the tables below. Associated

[0010] 9 12541428.1 Table 5 reports the sequence, genomic position, and log2FC of each gRNA, as well as the mean log2FC of each gRNA across the three treatments. FIG.16 shows validation of the 5 top-performing gRNAs from each screening treatment in K-562 ZNF410tdTomatocells. A histogram showing the percentage of ZNF410tdTomato-negative cells (% of silenced cells) at day 21 post-nucleofection with a non- saturating dose of plasmids encoding for the tri-partite ETR (1.5 µg) and the indicated gRNAs (1.5 µg) is shown. Two gRNAs previously tested in the arrayed screen of FIGs.8A- 8C were also included as controls. FIG.17 shows validation of the 5 top-performing gRNAs from each screening treatment in K-562 ZNF410tdTomatocells. Representative flow cytometry dot plots of K-562 ZNF410tdTomatocells either untreated (control) or transfected with the indicated gRNAs are shown. The bottom right graph shows RT-qPCR results showing the log2FC of ZNF410 mRNA expression in cells treated with the indicated gRNA compared to untreated cells (control). FIGs.18A-18B show validation of selected gRNAs in HUDEP-2 cells. FIG.18A shows a schematic of the experimental flow used to assess the efficacy of ZNF410 epi- silencing in these cells. Cells were co-transfected with in vitro transcribed mRNA encoding for the tri-partite ETR and selected gRNAs alone or in combination from the screening, and the decrease in expression levels of ZNF410 was measured by RT-qPCR at day 21 post- transfection (FIG.18B). Efficient and durable silencing of ZNF410 was observed for both individual (e.g., #177) and gRNA combinations (e.g., #177+#82). FIG.19 shows validation of selected gRNAs in human hematopoietic stem and progenitor cells (HSPCs). A schematic of the experimental flow used to assess the efficacy of ZNF410 epi-silencing in CD34+ cells (namely HSPCs) is shown. FIGs.20A-20C show validation of selected gRNAs in human hematopoietic stem and progenitor cells (HSPCs). Mobilized peripheral blood (mPB) CD34+ cells from a healthy donor were co-transfected with the mRNA encoding for the tri-partite ETR and the indicated gRNAs (also in combination), and either grown in liquid culture for 21 days or plated in semi-solid media for Colony Forming Cell (CFC) assay for 15 days, when the cells were analyzed for the expression of ZNF410 (FIGs.20A and 20B) or beta- and gamma-globin (HBB and HBG, respectively; FIG.20C). Treatment with the tri-partite ETR and ZNF410 gRNAs resulted in ~90% reduction in the expression levels of this gene, both in liquid culture and in differentiated colonies. In erythroid colonies, epi-silencing of ZNF410 was accompanied by a significant increase in fetal HBG expression (>2.5-fold increase) and a

[0011] 10 12541428.1 concomitant reduction in the adult HBB expression, as measured by gene expression analyses. These latter values were comparable to those obtained by disrupting ZNF410 by standard gene editing with CRISPR-Cas9. FIGs.21A-21B show silencing and methylation of ZNF410 in hematopoietic stem and progenitor cells (HSPCs). FIG.21A shows an experimental schematic (top) and line graphs of results (bottom) for % ZNF410 silencing. Bottom left shows results at Day 7 for liquid culture; bottom right shows results at Day 15 for colonies. Results for both bipartite and tripartite ETR architectures are shown. FIG.21B shows a schematic of the ZNF410 CpG island and surrounding regions (top) and both a heat map (bottom left) and bar graph (bottom right) of results of bisulfite sequencing in the region. FIG.22 shows a bar graph of reporter-negative K562 cells achieved with single guide RNAs and pairs of guide RNAs (each paired with guide 1, which corresponds to ZNF410_177). The dotted line throws the threshold of silencing achieved by ZNF410_177 delivered as a single guide with the ETR. Guide pairs exceeding treatment with ZNF410_177 as a single guide are shown in the upper left, and experimental conditions in the upper right. FIG.23A-23B show schematics and results for experiments conducted in HSPCs. FIG.23A shows a schematic (upper left) and a bar graph of RT-qPCR results at day 7 (fold change versus control) for ZNF410 mRNA results of HSPCs treated with ETR and single guide ZNF410_177 (sample 1, on the left) as well as guide pairs (bottom). The lower dotted line indicates the threshold of silencing achieved by ZNF410_177 delivered as a single guide with the ETR. The red arrows indicate guide pairs that considerably outperformed ZNF410_177 as a single guide. The guide pairs are also indicated in the box on the upper right. FIG.23B shows a schematic of three-phase liquid culture for differentiation of erythroid cells (top) and bar graph of RT-qPCR (fold change versus control) results at day 15 of differentiation for ZNF410 mRNA of erythroid cells differentiated in three-phase liquid culture (bottom). The lower dotted line indicates the threshold of silencing achieved by ZNF410_177 delivered as a single guide with the ETR. The upper dotted line indicates the expression levels of ZNF410 in the control sample. FIG.24 shows a bar graph of gamma-globin and beta-globin in the differentiated cells of FIG.23B (left) and a line graph correlating ZNF410 silencing to percent gamma globin (right). The top portion of each bar in the bar graph shows beta globin, and the bottom portion shows gamma globin. The number in each bar shows the percent gamma-globin of total globin.

[0012] 11 12541428.1 FIGs.25A-25B show an experimental setup for xenotransplantation study. FIG.25A shows an experimental workflow. FIG.25B shows a table detailing the experimental groups, including ETR type, RNA doses, sgRNAs used, and number of mice per condition. FIGs.26A-26C show an in vitro assessment of epi-silencing efficiency. FIG.26A shows RT-qPCR quantification of ZNF410 (white bars) and its downstream target CHD4 (gray bars) in mPB CD34⁺ cells maintained in liquid culture for 7 days after nucleofection (Day 9 post-thawing). FIG.26B shows RT-qPCR analysis of ZNF410 and CHD4 expression in erythroid cells derived from edited CD34⁺ HSPCs following in vitro erythroid differentiation using a three-phase medium. In both assays, cells were nucleofected with ETRs or Cas9 two days after thawing. Gene expression is shown as fold change relative to Mock-treated controls. HPRT was used as the normalization control. FIG.26C shows a Colony-Forming Unit (CFU) assay performed on mPB CD34⁺ cells nucleofected two days after thawing and cultured for two additional days before plating in MethoCult. Colonies were scored after 14 days and classified as CFU-GEMM (“A”), BFU-E (“B”), and CFU-GM (“C”). FIGs.27A-27E show bone marrow analysis at 16 weeks post-transplantation. Each dot represents an individual mouse; bars indicate mean values with standard deviation. FIG. 27A shows a gating strategy used for flow cytometric identification of human hematopoietic lineages and erythroid cells. FIG.27B shows human chimerism in the bone marrow, calculated as the percentage of hCD45⁺ cells over total CD45⁺ cells. FIG.27C Lineage distribution within the hCD45⁺ compartment: B lymphocytes (CD19⁺), granulocytes (CD33dim SSChi), and monocytes (CD33bright SSClow). FIG.27D shows frequencies of CD34⁺ hematopoietic stem / progenitor cells (HSPCs) and CD3⁺ T lymphocytes among hCD45⁺CD19⁻CD33⁻ cells. FIG.27E shows erythroid output, expressed as the percentage of hCD235a⁺ cells among hCD45⁻ cells. FIGs.28A-28E shows gene expression and hemoglobin isoform analyses in bone marrow 16 weeks post-transplantation. FIG.28A shows flow cytometry gating strategy used to sort human hematopoietic populations from bone marrow for downstream molecular analyses. FIGs.28B-28C RT-qPCR quantification of ZNF410 (FIG.28B) and its downstream target CHD4 (FIG.28C) in purified human cell populations: B lymphocytes (CD19⁺), myeloid cells (CD33⁺), erythroid cells (CD235a⁺), and HSPCs (CD34⁺). Gene expression was normalized to GAPDH and shown as fold change relative to Mock. FIG.28D show HBG (γ-globin) expression in CD235a⁺ erythroid cells, normalized to HBA, and expressed as fold change relative to Mock. FIG.28E show RT-qPCR quantification of HBG

[0013] 12 12541428.1 and HBB in erythroid cells. Data are shown as percentage of HBG over total β-like globin (HBG + HBB). FIGs.29A-29B show a comparison of ZNF410 silencing efficiency by different ETR platforms. FIG.29A show dose-escalation experiment in K562 ZNF410tdTomato reporter cells nucleofected with increasing doses of Tripartite ETR or CHARM together with sgRNA #177. Silencing efficiency was assessed by flow cytometry 28 days post-nucleofection, measuring the percentage of tdTomato-negative cells. FIG.29B show RT-qPCR quantification of ZNF410 expression in human mPB CD34⁺ cells cultured for 7 days after nucleofection with 1.6 µg of Tripartite ETR or CHARM and sgRNA #177. Expression levels are shown as fold change relative to Mock control. FIGs.30A-30C show transcriptomic analysis of ZNF410-targeting epigenetic editors in K562 cells. K562 ZNF410tdTomatoreporter cells were nucleofected with 400 ng total RNA (200 ng ETR mRNA + 200 ng sgRNA or guide combinations) and cultured for 21 days. At this time point, ZNF410-negative cells were quantified by flow cytometry (FIG.30A) and isolated for total RNA-seq. FIG.30B shows a volcano plot showing differentially expressed genes (DEGs) in ZNF410 KO cells compared to Mock. DEGs were defined as genes with |log₂ fold change| > 1 and FDR < 0.05 (highlighted in red for upregulated and blue for downregulated genes). ZNF410 and CHD4 are indicated. FIG.30C shows number of DEGs identified in ETR-treated samples (Tripartite or CHARM) compared to ZNF410 KO cells, using the same differential expression thresholds (|log₂FC| > 1, FDR < 0.05). Each bar represents a different gRNA or gRNA combination. DETAILED DESCRIPTION The present disclosure provides epigenetic editors for regulating expression of the ZNF410 gene. By altering expression of ZNF410, the systems, compositions and methods described herein may be used for treating conditions, including hemoglobinopathies, such as beta-hemoglobinopathies. Without intending to be bound by theory, silencing ZNF410 reactivates fetal hemoglobin expression (see FIG.2B). Aspects of the present disclosure relate to reactivating fetal hemoglobin expression to relieve one or more symptoms of a hemoglobinopathy, such as a beta-hemoglobinopathy. Unless otherwise stated, “ZNF410,” refers herein to human ZNF410. A human ZNF410 gene sequence can be found at Ensembl with Gene stable ID: ENSG00000119725. The present epigenetic editors have several

[0014] 13 12541428.1 advantages compared to other genome engineering methods, including reversibility, tolerability, decreased risk of translocation, and durable, inheritable silencing. In some embodiments, the region of the human gene targeted for epigenetic regulation is about 4 kb long, and is approximately + / - 500 bps to 2 kb of the ZNF410 transcription start site (TSS). The genomic coordinates and corresponding sequence used for targeting for each gene are shown below. Chr14:73885310-73889650 AACAAAGACAGTTTCAGAAAATGACAGGACTGGGCAAATTAACAAATGTTTGTA AACATGAATGTTCAGGAACTACTGATGTACCTCAAAAGTTTGTTTTATTAATTGT ACTCAACCCTCGCAGAACAGTAAAACTGAAGATTATTGTTTCTGAATGTTTTGGC TTATGATTGTCTACACACTGCCAATATACTTTTGTATGCAACATTTGTAATAAGTA GATTCAAACTGTGTTGGTAATCAGCCTCCCCAGTTTTCGTGAGTGGCCCTTTTTTG TTACTTTTCCTGGACCATTTCATCGCCTGAGGGGGTTTCACTTTTCtctgtatgttgctaactc ccaaatctccatctgtatttctgacctctttcttgagcactggacctttacctccaaGTAAATATCCTCAACTCTGGAT TGCATCTTATACTCTGCttttttcttttcttttcttttttgagatggagtcttgctcagtcgcccggcgtgcagcggcacgatct cggctcactgcaacctccgcctcccgggttcaagcgattctcctgcctcagcctcccgagtagctaggattacaggcgcgtgccacca cgcccggctaatttttgtgtttttagtagagacgggttttcacggcgttacccaggatggtctcgatctcctgacctcgtgatccgcccacc ttagcctcccaaagtgctgggattaaaggcgtgagccacggagtccggccTACTCTGCTTTTCTAAAGTGGAAT TATCTTCCTCTGAAATGTTTCTCCTTTATTTTAAATCACCATTAGTAATTTGTGTAC CCAAACCTGTCATCAAGGCTTATCTTTAACCTTGAAAATGCCTTTTTAAAGAGAA CTCCTTTAATTCCCACGGTTGAGTCTCTCATTCTTCCGCACGCAAATATTTTAAAA TAAGTAATTTCAAGAAGTCAGTCAAACTCACAAAGCTTTAACAAGGTGGCATCC AGCTTGGAAGTCAAGTGTTCAAGCCCGCAGTCGGGACATCCAAGAGCTCTTTACA TCCTAATTACAACACGGAACCCAGACTCACTGCATACCCCGTCCCGTATATAATG CAAAACTTGATTCTGCAAGTTCCTTTCTCGGGCGGTGAAACTTCGAAGGTCCTCT CCATGGAGACCTTTGATAAATGTTTAAGCAGTGATCTTTTCTTTCGCGTTTGCCTC CATTGCAACGCCTCCGCTGATGGGAAAAACTACTCCCTCCGCTACTGCGGGGTGG CAGTGACGTCAACGAATTAGCTGACCTGTTTTTTCGGGCTTCGCGGACATAGGAG TCCCGAAGGCCGAGAGTGAGTGAGTGAGTGGGAGCGGGGCGGCTGCGGCTCCTC CTGTCCGGGGCGGAGCCCTGCGTCGCGGGCCTGGGGAGGCCCCGCCAGCACCCT TGACGTCTGGGTCCCGAGCAGTGCGGCAGCCACACGACACTGAGGCGAGCGAGT GGGTGTGTGCGTGGGGGGTCAGCCAAACTTCCTGTCCCGCGCTCGAGCTGCTTCC GGCGGGAGCCGGAAGACGCTGTGTGTGGACGGAATTCGGGACCGACTGACGGCC GGCCGGCTTCCCGGAACTGGAAGGTGAGCTCCGAGGTGGGCCTGGCCACCCCTC CTCGGTTAGGTAGTACCCGAGAGGGCGGGGGCCAGTACCGGCTACTTGGGGCCT GGGGCCGAGTTCGCCCCGAGAGAGGAAACCGCCCTCCTTGGGCATCCCTCCTGG ATCTGGCGGGCCCGGGAGCGGCCAGTCTGAAGCCCTGCGGCTTTGGCCCTTCCCC TCGCGGGTTTTTAGGGGCGATGTTCTTGCTCCTTTGCTTGGTTTTGGGGGTAGGGA TATAGGAACAGACTGTCCTGAGAAAGCCAGAGTCAGCGAGTCCCCGGGGCGGGG GCTCTTGGACTGCACCCGCAGCCGGGGCCTGCCTACGGACCTTGGGGCCTTATTT CTGAGGCCTTTGTGGTGTGGGGTAGAGGATGCCAGGAGGCCTTGCCCTGAATGTT TCAGTCCAGAGCTACCCTCTCCTTTCATACCACTTACTTTCTGGGTTTGGAAAAGG GACAGCTGCCTACTCGGCTGGGCTGGGTCGCCCGTTTCTCCTTTGCCTGTCAGTTC GGGGGGCAGGAGTGTGTGGATTGTTTACGGCCTTCATACATTTCGTGTCGAATTT ATTTTCTGAAGTTTCTCAAGATAATCCAATTTATTAAAGTTTATCCCACTTGATCA TGAGGCTCAGAAATGCATTTAGGCACCATTTGGAAGTTAAATATTATTAACTTGG GACTTAAGAATTAGTACTTAGGTTGGAACGAAGGTCAACGCAGCATATTTGCAAT AAATAAAAAACATTTGGAAATAAAGTCGGGCATTTTGTTAGGGAGTGAGAAATT

[0015] 14 12541428.1 GCACTTGGTTTTTTCTTagtgtggtgcagagtaaaagacaactaaagtagcagtcagaagttttaagttctagttttttacg ttttgcatctaagtagatgtctaatttttggcaagttgctgaatctctagctctggggagtttttctgtaaacgaaggggttTACCTTTA GGGCTATGACAATTTTTCCGaggcactgaactttgagtagaagtgacataagcatttctttgttttggaaagataacttg aatggctgcataaaggatgtattggaaagaggaattttaaacgcggggtgaccagttagaggcctgttttaatagtatagtagaaagtta ctgagagacctaaacagggcattacagtagggaacagagaacattctgggaacagagaatcacgggaattatttaaaaatggagaat gagagagatttaaagacctagaatttgcagctgagtgggaggaagattgggcgatcattgaaataggtaatacaagaagaacaggttt ggtaaggaagataataatctggattttggacattgttaggtttgagatgctgttggatatacatgtggagatacctagcaagcagttagaa atgaaggtttggagttcagaagagatgactagagaaaaatgtaggtatcatTTGTAGTGTCATTCATTCATTATA CAAATATTTAGTACCTACCAGTTGCTTACTGGGAAGCTTGTATGGAAGCCCTTTA AGCTCATAGTAAACACATTAgaagtgggaagtgaccgttaatgggcacagggtttccttgtgaggtgatgaaatatt gtaaaattgatcgtggttctgcttgtacaactctgaatgaactaaagatattttaattgtactctttaaatgagtcaatagtatggtatgtgaac gatatcttagtgaaactaatttttaaaaaaGATTTACATTGCAGTTTTGGAAATCTGTTGAGTAAGGA ATTGGGAATAGGAATGTTTAATTGTGTATTTGCCGTTACACCTATAGCTAATGTA CATAGTACGTTGCTCTGTTAAAGATGAGATCCCCACAGTACACTTGATAAGATAC TGAGTGCTCTAGTACAAATTCTTTTGAGTTCAGTTGCATATCTTAATGCATCCTGC TATTTTTTATTTTATTCATACCGTACACATACAAATCATAAACTTGGACACAAaaatt gtatggtagtacttagttataatatgaatacccagtgcttgagaaaatgctcgtgttaatttttaaagattattgaagtgaaaacactgaaata aaaagatgtcatagataacATAAAAAGGACAGTAAATAGTTGTCTGGTTCCTCAGAGATTTta caatcctggaatctcaaagttggaggaaccttaaggatgacttaAACAACAAACATCTGATAATTTCTTGGC TTGTTAAGACAAGCTGGAATTGGGGAAGAGGAAttcttgttttttttgagacagcctccatttgttacccag gctggagtgcagtggcgtgatcactgttcagtgcagcctcgatctcctgggctaaagcaatcctcctactcagcctccctagtagctag gaccacaggcatgtgccaccatgtgatgctaattgaaaaatttttttttgtagaaatagagtctcacTGgctgagatcgcgccactgcac tccagcctgcgtgacagagcgagactcagtctcaaaaaaaaaaaaaaaaaaaaaagaaaTAGGGTCTCACTGTGTT GCCTAGGCTGAGAAAAGGAATTCTTAGGCCATGGAGATTCAGGGTCAAACCTAG AGAGATGTAATCACTATTCACGCCAATGATTTCTGCATACTTCTGGTGtgggtgggagtc ctggctttgcaacttaataacttggtaattaagtaatatactgagccttggttccttcctct (SEQ ID NO: 1356) In some embodiments, an epigenetic editor as described herein may comprise one or more fusion proteins, wherein each fusion protein comprises a DNA-binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments, where the DNA-binding domain is a polynucleotide guided DNA-binding domain, the epigenetic editor may further comprise one or more guide polynucleotides. DNA-binding domains, effector domains, and guide polynucleotides of an epigenetic editor as described herein may be selected, e.g., from those described below, in any functional combination. The epigenetic editors described herein may be expressed in a host cell transiently, or may be integrated in a genome of the host cell; such cells and their progeny are also contemplated by the present disclosure. Both transiently expressed and integrated epigenetic editors or components thereof can effect stable epigenetic modifications. For example, after introducing to a host cell an epigenetic editor described herein, the target gene in the host cell may be stably or permanently repressed or silenced. In some embodiments, expression of the target gene is reduced or silenced for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years,

[0016] 15 12541428.1 or for the entire lifetime of the cell or the subject carrying the cell, as compared to the level of expression in the absence of the epigenetic editor. The epigenetic modification may be inherited by the progeny of the host cells into which the epigenetic editor was introduced. The present epigenetic editors may be introduced to a patient in need thereof (e.g., a human patient), e.g., into the patient’s erythroid cells. The epigenetic editors may also be added to any cell that can differentiate into or yield erythroid cell progeny, such as pluripotent stem cells, hematopoietic stem cells, and immortalized human erythroid progenitors (in the singular, "precursor cell to a human erythroid cell"). I. DNA-Binding Domains An epigenetic editor described herein may comprise one or more DNA-binding domains that direct the effector domain(s) of the epigenetic editor to target sequences within or close to the ZNF410 gene locus. A DNA-binding domain as described herein may be, e.g., a polynucleotide guided DNA-binding domain, a zinc finger protein (ZFP) domain, a transcription activator like effector (TALE) domain, a meganuclease DNA-binding domain, and the like. Examples of DNA-binding domains can be found in U.S. Patent 11,162,114, which is incorporated by reference herein in its entirety. In some embodiments, a DNA-binding domain described herein is encoded by its native coding sequence. In other embodiments, the DNA-binding domain is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells. A. Polynucleotide Guided DNA-Binding Domains In some embodiments, a DNA-binding domain herein may be a protein domain directed by a guide nucleic acid sequence (e.g., a guide RNA sequence) to a target site in the ZNF410 gene locus. In certain embodiments, the protein domain may be derived from a CRISPR-associated nuclease, such as a Class I or II CRISPR-associated nuclease. In some embodiments, the protein domain may be derived from a Cas nuclease such as a Type II, Type IIA, Type IIB, Type IIC, Type V, or Type VI Cas nuclease. In certain embodiments, the protein domain may be derived from a Class II Cas nuclease selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas14a, Cas14b, Cas14c, CasX, CasY, CasPhi, C2c4, C2c8, C2c9, C2c10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, and homologues and modified versions thereof. “Derived from” is used to mean that the protein domain comprises the full polypeptide sequence of the parent protein, or comprises a variant

[0017] 16 12541428.1 thereof (e.g., with amino acid residue deletions, insertions, and / or substitutions). The variant retains the desired function of the parent protein (e.g., the ability to form a complex with the guide nucleic acid sequence and the target DNA). In some embodiments, the CRISPR-associated protein domain may be a Cas9 domain described herein. Cas9 may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wildtype Cas9 polypeptide described herein. In some embodiments, said wildtype polypeptide is Cas9 from Streptococcus pyogenes (NCBI Ref. No. NC_002737.2 (SEQ ID NO: 1)) and / or UniProt Ref. No. Q99ZW2 (SEQ ID NO: 2). In some embodiments, said wildtype polypeptide is Cas9 from Staphylococcus aureus (SEQ ID NO: 4). In some embodiments, the CRISPR-associated protein domain is a Cpf1 domain or protein, or a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wildtype Cpf1 polypeptide described herein (e.g., Cpf1 from Francisella novicida (UniProt Ref. No. U2UMQ6 or SEQ ID NO: 7). In certain embodiments, the CRISPR-associated protein domain may be a modified form of the wildtype protein comprising one or more amino acid residue changes such as a deletion, an insertion, or a substitution; a fusion or chimera; or any combination thereof. Cas9 sequences and structures of variant Cas9 orthologs have been described for various organisms. Exemplary organisms from which a Cas9 domain herein can be derived include, but are not limited to, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans,

[0018] 17 12541428.1 Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina. Cas9 sequences also include those from the organisms and loci disclosed in Chylinski et al., RNA Biol. (2013) 10(5):726-37. In some embodiments, the Cas9 domain is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 domain is from Staphylococcus aureus (SaCas9). Other Cas domains are also contemplated for use in the epigenetic editors herein. These include, for example, those from CasX (Cas12E) (e.g., SEQ ID NO: 8), CasY (Cas12d) (e.g., SEQ ID NO: 9), Casφ (CasPhi) (e.g., SEQ ID NO: 10), Cas12f1 (Cas14a) (e.g., SEQ ID NO: 11), Cas12f2 (Cas14b) (e.g., SEQ ID NO: 12), Cas12f3 (Cas14c) (e.g., SEQ ID NO: 13), and C2c8 (e.g., SEQ ID NO: 14). For epigenetic editing, the nuclease-derived protein domain (e.g., a Cas9 or Cpf1 domain) may have reduced or no nuclease activity through mutations such that the protein domain does not cleave DNA or has reduced DNA-cleaving activity while retaining the ability to complex with the guide nucleic acid sequence (e.g., guide RNA) and the target DNA. For example, the nuclease activity may be reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the wildtype domain. In some embodiments, a CRISPR-associated protein domain described herein is catalytically active. In some embodiments, a CRISPR-associated protein domain described herein is catalytically inactive (“dead”). In some embodiments, a CRISPR-associated protein domain described herein is partially catalytically inactivated (“nickase”). Examples of such domains include, for example, dCas9 (“dead” Cas9), dCpf1, ddCpf1, dCasPhi, ddCas12a, dLbCpf1, and dFnCpf1. A dCas9 protein domain, for example, may comprise one, two, or more mutations as compared to wildtype Cas9 that abrogate its nuclease activity. The DNA cleavage domain of Cas9 is known to include two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A (in RuvC1) and H840A (in HNH) completely inactivate the nuclease activity of SpCas9. SaCas9, similarly, may be inactivated by the mutations D10A and

[0019] 18 12541428.1 N580A. In some embodiments, the dCas9 comprises at least one mutation in the HNH subdomain and / or the RuvC1 subdomain that reduces or abrogates nuclease activity. In some embodiments, the dCas9 only comprises a RuvC1 subdomain, or only comprises an HNR subdomain. It is to be understood that any mutation that inactivates the RuvC1 and / or the HNH domain may be included in a dCas9 herein, e.g., insertion, deletion, or single or multiple amino acid substitution in the RuvC1 domain and / or the HNH domain. In some embodiments, a dCas9 protein herein comprises a mutation at position(s) corresponding to position D10 (e.g., D10A), H840 (e.g., H840A), or both, of a wildtype SpCas9 sequence as numbered in the sequence provided at UniProt Accession No. Q99ZW2 (SEQ ID NO: 2). In particular embodiments, the dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 15). In some embodiments, a dCas9 protein as described herein comprises a mutation at position(s) corresponding to position D10 (e.g., D10A), N580 (e.g., N580A), or both, of a wildtype SaCas9 sequence (e.g., SEQ ID NO: 4). In particular embodiments, the dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO: 16). Additional suitable mutations that inactivate Cas9 will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure. Such mutations may include, but are not limited to, D839A, N863A, and / or K603R in SpCas9. The present disclosure contemplates any mutations that reduce or abrogate the nuclease activity of any Cas9 described herein (e.g., mutations corresponding to any of the Cas9 mutations described herein). A dCpf1 protein domain may comprise one, two, or more mutations as compared to wildtype Cpf1 that reduce or abrogate its nuclease activity. The Cpf1 protein has a RuvC- like endonuclease domain that is similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. In some embodiments, the dCpf1 comprises one or more mutations corresponding to position D917A, E1006A, or D1255A as numbered in the sequence of the Francisella novicida Cpf1 protein (FnCpf1; SEQ ID NO: 7). In certain embodiments, the dCpf1 protein comprises mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A, or corresponding mutation(s) in any of the Cpf1 amino acid sequences described herein. In some embodiments, the dCpf1 comprises a D917A mutation. In particular embodiments, the dCpf1 comprises the amino acid sequence of dFnCpf1 (SEQ ID NO: 17).

[0020] 19 12541428.1 Further nuclease inactive CRISPR-associated protein domains contemplated herein include those from, for example, dNmeCas9 (e.g., SEQ ID NO: 18), dCjCas9 (e.g., SEQ ID NO: 19), dSt1Cas9 (e.g., SEQ ID NO: 20), dSt3Cas9 (e.g., SEQ ID NO: 21), dLbCpf1 (e.g., SEQ ID NO: 22), dAsCpf1 (e.g., SEQ ID NO: 23), denAsCpf1 (e.g., SEQ ID NO: 24), dHFAsCpf1 (e.g., SEQ ID NO: 25), dRVRAsCpf1 (e.g., SEQ ID NO: 26), dRRAsCpf1 (e.g., SEQ ID NO: 27), dCasX (e.g., SEQ ID NO: 28), and dCasPhi (e.g., SEQ ID NO: 29). In some embodiments, a Cas9 domain described herein may be a high-fidelity Cas9 domain, e.g., comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA to confer increased target binding specificity. In certain embodiments, the high fidelity Cas9 domain may be nuclease inactive as described herein. A CRISPR-associated protein domain described herein may recognize a protospacer adjacent motif (PAM) sequence in a target gene. A “PAM” sequence is typically a 2 to 6 bp DNA sequence immediately following the sequence targeted by the CRISPR-associated protein domain. The PAM sequence is required for CRISPR protein binding and cleavage but is not part of the target sequence. The CRISPR-associated protein domain may either recognize a naturally occurring or canonical PAM sequence or may have altered PAM specificity. CRISPR-associated protein domains that bind to non-canonical PAM sequences have been described in the art. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver et al., Nature (2015) 523(7561):481-5 and Kleinstiver et al., Nat Biotechnol. (2015) 33:1293-8. Such Cas9 domains may include, for example, those from “VRER” SpCas9, “EQR” SpCas9, “VQR” SpCas9, “SpG Cas9,” “SpRYCas9,” and “KKH” SaCas9. Nuclease inactive versions of these Cas9 domains are also contemplated, such as nuclease inactive VRER SpCas9 (e.g., SEQ ID NO: 30), nuclease inactive EQR SpCas9 (e.g., SEQ ID NO: 31), nuclease inactive VQR SpCas9 (e.g., SEQ ID NO: 32), nuclease inactive SpG Cas9 (e.g., SEQ ID NO: 33), nuclease inactive SpRY Cas9 (e.g., SEQ ID NO: 34), and nuclease inactive KKH SaCas9 (e.g., SEQ ID NO: 35). Another example is the Cas9 of Francisella novicida engineered to recognize 5’-YG-3’ (where “Y” is a pyrimidine). Additional suitable CRISPR-associated proteins, orthologs, and variants, including nuclease inactive variants and sequences, will be apparent to those of skill in the art based on this disclosure. Guide RNAs that can be used in conjunction with the CRISPR-associated protein domains herein are further described in Section II below.

[0021] 20 12541428.1 B. Zinc Finger Protein Domains In some embodiments, the DNA-binding domain of an epigenetic editor described herein comprises a zinc finger protein (ZFP) domain (or “ZF domain” as used herein). ZFPs are proteins having at least one zinc finger, and bind to DNA in a sequence-specific manner. A “zinc finger” (ZF) or “zinc finger motif” (ZF motif) refers to a polypeptide domain comprising a beta-beta-alpha (ββα)-protein fold stabilized by a zinc ion. A ZF binds from two to four base pairs of nucleotides, typically three or four base pairs (contiguous or noncontiguous). Each ZF typically comprises approximately 30 amino acids. ZFP domains may contain multiple ZFs that make tandem contacts with their target nucleic acid sequence. A tandem array of ZFs may be engineered to generate artificial ZFPs that bind desired nucleic acid targets. ZFPs may be rationally designed by using databases comprising triplet (or quadruplet) nucleotide sequences and individual ZF amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of ZFs that bind the particular triplet or quadruplet sequence. See, e.g., U.S. Patents 6,453,242, 6,534,261, and 8,772,453. ZFPs are widespread in eukaryotic cells, and may belong to, e.g., C2H2 class, CCHC class, PHD class, or RING class. An exemplary motif characterizing one class of these proteins (C2H2 class) is -Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His- (SEQ ID NO: 742), where X is any independently chosen amino acid. In some embodiments, a ZFP domain herein may comprise a ZF array comprising sequential C2H2-ZFs each contacting three or more sequential nucleotides. A ZFP domain of an epigenetic editor described herein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more ZFs. The ZFP domain may include an array of two-finger or three- finger units, e.g., 3, 4, 5, 6, 7, 8, 9 or 10 or more units, wherein each unit binds a subsite in the target sequence. In some embodiments, a ZFP domain comprising at least three ZFs recognizes a target DNA sequence of 9 or 10 nucleotides. In some embodiments, a ZFP domain comprising at least four ZFs recognizes a target DNA sequence of 12 to 14 nucleotides. In some embodiments, a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18 to 21 nucleotides. In some embodiments, ZFs in a ZFP domain described herein are connected via peptide linkers. The peptide linkers may be, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In some embodiments, a linker comprises 5 or more amino acids. In some embodiments, a linker comprises 7-17 amino acids. The linker may be flexible or rigid.

[0022] 21 12541428.1 In some embodiments a zinc finger array may have the sequence: SRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH[lin ker]FQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH[linker ]PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTHLRGS (SEQ ID NO: 739), or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, where “XXXXXXX” represents the amino acids of the ZF recognition helix, which confers DNA-binding specificity upon the zinc finger; each X may be independently chosen. In the above sequence, “XX” in italics may be TR, LR or LK, and “[linker]” represents a linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 740); this linker may be used when sub-sites targeted by the ZFs are adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO: 741); this linker may be used when there is a base between the sub-sites targeted by the zinc fingers. The two indicated linkers may be the same or different. ZFP domains herein may contain arrays of two or more adjacent ZFs that are directly adjacent to one another (e.g., separated by a short (canonical) linker sequence), or are separated by longer, flexible or structured polypeptide sequences. In some embodiments, directly adjacent fingers bind to contiguous nucleic acid sequences, i.e., to adjacent trinucleotides / triplets. In some embodiments, adjacent fingers cross-bind between each other’s respective target triplets, which may help to strengthen or enhance the recognition of the target sequence, and leads to the binding of overlapping sequences. In some embodiments, distant ZFs within the ZFP domain may recognize (or bind to) non-contiguous nucleotide sequences. C. TALEs In some embodiments, the DNA-binding domain of an epigenetic editor described herein comprises a transcription activator-like effector (TALE) domain. The DNA-binding domain of a TALE comprises a highly conserved sequence of about 33-34 amino acids, with a repeat variable di-residue (RVD) at positions 12 and 13 that is central to the recognition of specific nucleotides. TALEs can be engineered to bind practically any desired DNA sequence. Methods for programming TALEs are known in the art. For example, such methods are described in Carroll et al., Genet Soc Amer. (2011) 188(4):773-82; Miller et al., Nat Biotechnol. (2007) 25(7):778-85; Christian et al., Genetics (2008) 186(2):757-61; Li et al., Nucl Acids Res. (2010) 39(1):359-72; and Moscou et al., Science (2009) 326(5959):1501.

[0023] 22 12541428.1 D. Other DNA-Binding Domains Other DNA-binding domains are contemplated for the epigenetic editors described herein. In some embodiments, the DNA-binding domain comprises an argonaute protein domain, e.g., from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease that is guided to its target site by 5' phosphorylated ssDNA (gDNA), where it produces double-strand breaks. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Thus, using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described, e.g., in Gao et al., Nat Biotechnol. (2016) 34(7):768-73; Swarts et al., Nature (2014) 507(7491):258-61; and Swarts et al., Nucl Acids Res. (2015) 43(10):5120-9. In some embodiments, the DNA-binding domain comprises an inactivated nuclease, for example, an inactivated meganuclease. Additional non-limiting examples of DNA- binding domains include tetracycline-controlled repressor (tetR) DNA-binding domains, leucine zippers, helix-loop-helix (HLH) domains, helix-turn-helix domains, β-sheet motifs, steroid receptor motifs, bZIP domains homeodomains, and AT-hooks. II. Guide Polynucleotides Epigenetic editors described herein that comprise a polynucleotide guided DNA- binding domain may also include a guide polynucleotide that is capable of forming a complex with the DNA-binding domain. The guide polynucleotide may comprise RNA, DNA, or a mixture of both. For example, where the polynucleotide guided DNA-binding domain is a CRISPR-associated protein domain, the guide polynucleotide may be a guide RNA (gRNA). A “guide RNA” or “gRNA” refers to a nucleic acid that is able to hybridize to a target sequence and direct binding of the CRISPR-Cas complex to the target sequence. Methods of using guide polynucleotide sequences with programmable DNA-binding proteins (e.g., CRISPR-associated protein domains) for site-specific DNA targeting (e.g., to modify a genome) are known in the art. A guide polynucleotide sequence (e.g., a gRNA sequence) may comprises two parts: 1) a nucleotide sequence (“targeting sequence”) that is complementary to a target nucleic acid sequence; and 2) a nucleotide sequence that binds a polynucleotide guided DNA-binding domain (e.g., a CRISPR-Cas protein domain). The nucleotide sequence in 1) may comprise a spacer sequence that hybridizes to a target sequence, and may be, e.g., crispr RNA, or crRNA. The nucleotide sequence in 2) may be referred to as a scaffold sequence of a guide nucleic acid, e.g., a tracrRNA, or an activating region of a guide nucleic acid, and may

[0024] 23 12541428.1 comprise a stem-loop structure. Parts 1) and 2) as described above may be fused to form one single guide (e.g., a single guide RNA, or sgRNA), or may be on two separate nucleic acid molecules. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a linker. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a non-nucleic acid linker, for example, a peptide linker or a chemical linker. Part 2 (the scaffold sequence) of a guide polynucleotide as described herein may be, for example, as described in Jinek et al., Science (2012) 337:816-21; U.S. Patent Publication 2016 / 0208288; or U.S. Patent Publication 2016 / 0200779. Variants of part 2) are also contemplated by the present disclosure. For example, the tetraloop and stem loop of a gRNA scaffold (tracrRNA) sequence may be modified to include RNA aptamers, which can be bound by specific protein domains. In some embodiments, such modified gRNAs can be used to facilitate the recruitment of repressive or activating domains fused to the protein- interacting RNA aptamers. Methods for designing, selecting, and validating gRNAs are described herein and known in the art. Software tools can be used to optimize the gRNAs corresponding to a target DNA sequence, e.g., to minimize total off-target activity across the genome. For example, DNA sequence searching algorithms can be used to identify a target sequence in crRNAs of a gRNA for use with Cas9. Exemplary gRNA design tools include the ones described in Bae et al., Bioinformatics (2014) 30:1473-5. Guide polynucleotides (e.g., gRNAs) described herein may be of various lengths. In some embodiments, the length of the spacer or targeting sequence depends on the CRISPR- associated protein component of the epigenetic editor system used. For example, Cas proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the spacer sequence may comprise, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides in length. In some embodiments, the spacer comprises 10-24, 11-20, 11-16, 18-24, 19-21, or 20 nucleotides in length. In some embodiments, a guide polynucleotide (e.g., gRNA) is from 15-100 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length and comprises a spacer sequence of at least 10 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) contiguous nucleotides complementary to the target sequence. In some embodiments, the spacer sequence for targeting ZNF410 is between 10 and 18 nucleotides complementary to the target

[0025] 24 12541428.1 sequence (inclusive). In some embodiments, the spacer sequence for targeting ZNF410 contains one or more mismatches in the complementary sequence. In some embodiments, a guide polynucleotide described herein may be truncated, e.g., by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides. In certain embodiments, the 3’ end of the ZNF410 target sequence is immediately adjacent to a PAM sequence (e.g., a canonical PAM sequence such as NGG for SpCas9). The degree of complementarity between the targeting sequence of the guide polynucleotide (e.g., the spacer sequence of a gRNA) and the target sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In particular embodiments, the targeting and the target sequence may be 100% complementary. In other embodiments, the targeting sequence and the target sequence may contain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. A guide polynucleotide (e.g., gRNA) may be modified with, for example, chemical alterations and synthetic modifications. A modified gRNA, for instance, can include an alteration or replacement of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage, an alteration of the ribose sugar (e.g., of the 2’ hydroxyl on the ribose sugar), an alteration of the phosphate moiety, modification or replacement of a naturally occurring nucleobase, modification or replacement of the ribose-phosphate backbone, modification of the 3’ end and / or 5’ end of the oligonucleotide, replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker, or any combination thereof. In some embodiments, one or more ribose groups of the gRNA may be modified. Examples of chemical modifications to the ribose group include, but are not limited to, 2’-O- methyl (2’-OMe), 2’-fluoro (2’-F), 2’-deoxy, 2’-O-(2-methoxyethyl) (2’-MOE), 2’-NH2, 2’- O-allyl, 2’-O-ethylamine, 2’-O-cyanoethyl, 2’-O-acetalester, or a bicyclic nucleotide such as locked nucleic acid (LNA), 2’-(5-constrained ethyl (S-cEt)), constrained MOE, or 2’-0,4’-C- aminomethylene bridged nucleic acid (2’,4’-BNANC). 2’-O-methyl modification and / or 2’- fluoro modification may increase binding affinity and / or nuclease stability of the gRNA oligonucleotides. In some embodiments, one or more phosphate groups of the gRNA may be chemically modified. Examples of chemical modifications to a phosphate group include, but are not limited to, a phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification. In some embodiments, a guide polynucleotide described herein may comprise one, two, three, or more

[0026] 25 12541428.1 PS linkages at or near the 5’ end and / or the 3’ end; the PS linkages may be contiguous or noncontiguous. In some embodiments, the gRNA herein comprises a mixture of ribonucleotides and deoxyribonucleotides and / or one or more PS linkages. In some embodiments, one or more nucleobases of the gRNA may be chemically modified. Examples of chemically modified nucleobases include, but are not limited to, 2- thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups. Chemical modifications can be made in the spacer region, the tracr RNA region, the stem loop, or any combination thereof. Any tracr sequence known in the art is contemplated for a gRNA described herein. In some embodiments, a gRNA described herein has a tracr sequence shown in Table 1 below, or a tracr sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown below. Table 1. Exemplary TRACR Sequences In some embodiments, the gRNA herein is provided to the cell directly (e.g., through an RNP complex together with the CRISPR-associated protein domain). In some embodiments, the gRNA is provided to the cell through an expression vector (e.g., a plasmid vector or a viral vector) introduced into the cell, where the cell then expresses the gRNA from the expression vector. Methods of introducing gRNAs and expression vectors into cells are well known in the art. III. Effector Domains Epigenetic editors described herein include one or more effector protein domains (also “epigenetic effector domains,” or “effector domains,” as used herein) that effect

[0027] 26 12541428.1 epigenetic modification of a target gene. An epigenetic editor with one or more effector domains may modulate the expression of a target gene without altering its nucleobase sequence. In some embodiments, an effector domain described herein may provide repression or silencing of expression of a target gene by repressing transcription or by modifying or remodeling chromatin. Such effector domains are also referred to herein as “repression domains” or “epigenetic repression domains.” Non-limiting examples of chemical modifications that may be mediated by effector domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation and / or ubiquitination of DNA or histone residues. In some embodiments, an effector domain of an epigenetic editor described herein may make histone tail modifications, e.g., by adding or removing active marks on histone tails. In some embodiments, an effector domain of an epigenetic editor described herein may comprise or recruit a transcription-related protein, e.g., a transcription repressor. The transcription-related protein may be endogenous or exogenous. In some embodiments, an effector domain of an epigenetic editor described herein may, for example, comprise a protein that directly or indirectly blocks access of a transcription factor to the gene of interest harboring the target sequence. An effector domain may be a full-length protein or a fragment thereof that retains the epigenetic effector function (a “functional domain”). Functional domains that are capable of modulating (e.g., repressing) gene expression can be derived from a larger protein. For example, functional domains that can reduce target gene expression may be identified based on sequences of repressor proteins. Amino acid sequences of gene expression-modulating proteins may be obtained from available genome browsers, such as the UCSD genome browser or Ensembl genome browser. Protein annotation databases such as UniProt or Pfam can be used to identify functional domains within the full protein sequence. As a starting point, the largest sequence, encompassing all regions identified by different databases, may be tested for gene expression modulation activity. Various truncations then may be tested to identify the minimal functional unit. Variants of effector domains described herein are also contemplated by the present disclosure. A variant may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wildtype effector domain described herein. In particular

[0028] 27 12541428.1 embodiments, the variant retains at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the epigenetic effector function of the wildtype effector domain. In some embodiments, an effector domain described herein may comprise a fusion of two or more effector domains (e.g., KOX1 KRAB and ZIM3). The effector domain may, for example, comprise a fusion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector domains, such as effector domains described herein. In certain embodiments, an effector domain comprises a fusion of a truncated form of an effector domain and a second effector domain. In certain embodiments, an effector domain comprises a fusion of the truncated forms of two effector domains (e.g., fusions of the N- and C-terminal portions of the two effector domains). In some embodiments, an epigenetic editor described herein may comprise 1 effector domain, 2 effector domains, 3 effector domains, 4 effector domains, 5 effector domains, 6 effector domains, 7 effector domains, 8 effector domains, 9 effector domains, 10 effector domains, or more. In certain embodiments, the epigenetic editor comprises one or more fusion proteins (e.g., one, two, or three fusion proteins), each with one or more effector domains (e.g., one, two, or three effector domains) linked to a DNA-binding domain. In some embodiments, the effector domains may induce a combination of epigenetic modifications, e.g., transcription repression and DNA methylation, DNA methylation and histone deacetylation, DNA methylation and histone demethylation, DNA methylation and histone methylation, DNA methylation and histone phosphorylation, DNA methylation and histone ubiquitylation, DNA methylation, and histone SUMOylation. In certain embodiments, an effector domain described herein (e.g., DNMT3A and / or DNMT3L) is encoded by a nucleotide sequence as found in the native genome (e.g., human or murine) for that effector domain. In other embodiments, an effector domain described herein is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells. Effector domains described herein may include, for example, transcriptional repressors, DNA methyltransferases, and / or histone modifiers, as further detailed below. A. Transcriptional Repressors In some embodiments, an epigenetic effector domain described herein mediates repression of a target gene’s expression (e.g., transcription). The effector domain may comprise, e.g., a Krüppel-associated box (KRAB) repression domain, a Repressor Element Silencing Transcription Factor (REST) repression domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, a FKHR (forkhead in rhabdosarcoma gene) repressor domain, an EGR-1 (early growth response gene product-1) repressor domain, an ets2

[0029] 28 12541428.1 repressor factor repressor domain (ERD), a MAD smSIN3 interaction domain (SID), a WRPW motif of the hairy-related basic helix-loop-helix (bHLH) repressor proteins, an HP1 alpha chromo-shadow repression domain, an HP1 beta repression domain, or any combination thereof. The effector domain may recruit one or more protein domains that repress expression of the target gene, e.g., through a scaffold protein. In some embodiments, the effector domain may recruit or interact with a scaffold protein domain that recruits a PRMT protein, a HDAC protein, a SETDB1 protein, or a NuRD protein domain. In some embodiments, the effector domain comprises a functional domain derived from a zinc finger repressor protein, such as a KRAB domain. KRAB domains are found in approximately 400 human ZFP-based transcription factors. Descriptions of KRAB domains may be found, for example, in Ecco et al., Development (2017) 144(15):2719-29 and Lambert et al., Cell (2018) 172:650-65. In certain embodiments, the effector domain comprises a repression domain (e.g., KRAB) derived from KOX1 / ZNF10, KOX8 / ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain comprises a repression domain (e.g., KRAB) derived from ZIM3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF680, ZNF41, ZNF189, ZNF528, ZNF543, ZNF554, ZNF140, ZNF610, ZNF264, ZNF350, ZNF8, ZNF582, ZNF30, ZNF324, ZNF98, ZNF669, ZNF677, ZNF596, ZNF214, ZNF37, ZNF34, ZNF250, ZNF547, ZNF273, ZNF354, ZFP82, ZNF224, ZNF33, ZNF45, ZNF175, ZNF595, ZNF184, ZNF419, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF566, ZNF729, ZIM2, ZNF254, ZNF764, ZNF785, KOX1, or any combination thereof. For example, the repression domain may be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In particular embodiments, the repression domain is a ZIM3 KRAB domain. In further embodiments, the effector domain is derived from a human protein, e.g., a human ZIM3, a human KOX1, a human ZFP28, or a human ZN627. Exemplary effector domains that may reduce or silence target gene expression are provided in Table 2 below, with their corresponding sequences found in the "Sequences" listing after the Examples. Further examples of repressors and transcriptional repressor domains can be found, e.g., in PCT Patent Publication WO 2021 / 226077 and Tycko et al., Cell (2020) 183(7):2020-35, each of which is incorporated herein by reference in its entirety.

[0030] 29 12541428.1 Table 2. Exemplary Effector Domains That May Reduce or Silence Gene Expression

[0031] 30 12541428.1

[0032] 31 12541428.1

[0033] 32 12541428.1

[0034] 33 12541428.1

[0035] 34 12541428.1

[0036] 35 12541428.1

[0037] 36 12541428.1 A functional analog of any one of the above-listed proteins, i.e., a molecule having the same or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more) of the protein’s transcription factor function) is encompassed by the present disclosure. For example, the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and / or containing mutations relative to the above protein. In some embodiments, the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences of the domains listed in Table 2. In certain embodiments, an epigenetic editor described herein comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, and / or an effector domain derived from KAP1, MECP2, HP1a, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, optionally wherein the parental protein is a human protein. In particular embodiments, an epigenetic editor described herein comprises a domain derived from KOX1, ZIM3, ZFP28, and / or ZN627, optionally wherein the parental protein is a human protein. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from KOX1, e.g., a human KOX1. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZIM3, e.g., a human ZIM3. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZFP28, e.g., a human ZFP28. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZN627, e.g., a human ZN627. In certain embodiments, an epigenetic editor described herein may comprise a CDYL2, e.g., a human CDYL2, and / or a TOX

[0038] 37 12541428.1 domain (e.g., a human TOX domain) in combination with a KOX1 KRAB domain (e.g., a human KOX1 KRAB domain). In certain embodiments, an epigenetic effector described herein comprises a repression domain derived from ZNF10. For example, the repression domain may comprise the sequence provided in the "Sequences" table for ZNF10, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence provided in the "Sequences" table for ZNF10. B. Transcriptional Activators In some embodiments, an epigenetic effector domain described herein mediates activation of a target gene’s expression (e.g., transcription). In some embodiments, an epigenetic editor for activation of a target as described herein may recruit a domain that is associated with changes in DNA methylation. A non-limiting example of a domain that is associated with changes in DNA methylation is the ten eleven translocation (Tet) enzyme family. In some embodiments, Tet enzymes oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation. Non-limiting examples of Tet enzymes are human Tet1 (UniProt ID: Q8NFU7) and human Tet2 (UniProt ID: Q6N021). The Tet1 protein has been characterized in the art and has at least one catalytic domain. In some embodiments of the present disclosure, fusion proteins comprise the catalytic domain (CD) of Tet1. Some exemplary and non-limiting activator effector (transcriptional activation domains) proteins, domains, truncations, trimmings, or combinations of effector domains that may activate target gene expression through various mechanisms that are contemplated by the present disclosure are OCT4 (POU5F1), GATA4, PU.1 (SPI1), EOMES, PAX6 (FVH1), FOXA1 (HNF3A), FOXA2 (HNF3B), FOXD3 (HFH2), CEBPalpha, CEBPbeta, Satb1, KLF4, HNF4 alpha, HNF1 alpha, GATA2, GATA6, PBX1, EBF1, HOXB2, FOXN1, FOXR2, KLF14, SOX7, SPDYE4, CSRNP1, ATF6, CITED2-TAD, CITED1-TAD, C3orf62- TAD, CBX-C (CBX2), FAM22F-23, KLF6-1, SPDYE4-3, Med21, Med25, ZN473_SND, FOXO3_SND, FOXO1_SND, MYBA_SND, MYB_SND, NCOA2_SND, SMCA2_SND, NCOA3_SND, ZN597_SND, APBB1_SND, ANM2_SND, CXXC1_SND, CRTC2_SND, NOTC2_SND, p53 (gene: TP53), PPARgamma, BZLF1, BanP, GCN5_HAT, KDM4A_JMJN_C, KDM4D_FL, KDM6A_JMJC, KDM6B_JMJC, MLL3_SET, MLL4_SET, MLL4_SET2, Dpy-30, ASH1L_SET, ASH2L, Brd9, ZNF473_SND (trimmed), Vp64, P65-RTA (PR), HSF1, P300, SS18, VPH, FOXP3, Vp64, GADD45a, GADD45b, GADD45g, SOX2, MYOD1 (MYF3), GATA3, PAX3, PAX7 (HUP1), ASCL1, FOXO1

[0039] 38 12541428.1 (FKHR), Rap1, Reb1, Cbf1, HNF4gamma, NEUROD1, NEUROD2, ILS1, KLF15, KLF6, ATMIN, CHOP (DDIT3), C3orf62, KLF7-TAD, ATF6-TAD, KIBRA_SND, c-Myc, BRD2, Med6, KDM4A_JMJC, KDM4B_JMJC, KDM4C_JMJC, KDM4D_JMJC, SMYD5_H316L_C318A, BRG1_Trunc. Alternative names used in the art, potential amino acid changes, trimmings, and truncations are indicated. C. DNA Methyltransferases In some embodiments, an effector domain of an epigenetic editor described herein alters target gene expression through DNA modification, such as methylation. Highly methylated areas of DNA tend to be less transcriptionally active than less methylated areas. DNA methylation occurs primarily at CpG sites (shorthand for “C-phosphate-G-” or “cytosine-phosphate-guanine” sites). Many mammalian genes have promoter regions near or including CpG islands (nucleic acid regions with a high frequency of CpG dinucleotides). An effector domain described herein may be, e.g., a DNA methyltransferase (DNMT) or a catalytic domain thereof, or may be capable of recruiting a DNA methyltransferase. DNMTs are enzymes that catalyze the transfer of a methyl group to a DNA nucleotide, thereby repressing expression of the target gene through the recruitment of repressive regulatory proteins. In some embodiments, the methylation is at a CG (or CpG) dinucleotide sequence. In some embodiments, the methylation is at a CHG or CHH sequence, where H is any one of A, T, or C. DNMTs in the epigenetic editors may include, e.g., DNMT1, DNMT3A, DNMT3B, and / or Dnmt3c. In some embodiments, the DNMT is a mammalian (e.g., human or murine) DNMT. In particular embodiments, the DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, an epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 577, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 577. In certain embodiments, an epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 578, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 578. In some embodiments, the DNMT3A domain may have, e.g., a mutation at position H739 (such as H739A or H739E), R771 (such as R771L) and / or R836 (such as R836A or R836Q), or any combination thereof (numbering according to SEQ ID NO: 577). In some embodiments, an effector domain described herein may be a DNMT-like domain. As used herein a “DNMT-like domain” is a regulatory factor of DNA methyltransferase that may activate or recruit other DNMT domains, but does not itself possess methylation activity. In some embodiments, the DNMT-like domain is a mammalian

[0040] 39 12541428.1 (e.g., human or mouse) DNMT-like domain. In certain embodiments, the DNMT-like domain is DNMT3L, which may be, for example, human DNMT3L or mouse DNMT3L. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 581, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 581. In certain embodiments, an epigenetic editor herein comprises a DNMT3L domain comprising SEQ ID NO: 582, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 582. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 583, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 583. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 584, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 584. In some embodiments, the DNMT3L domain may have, e.g., a mutation corresponding to that at position D226 (such as D226V), Q268 (such as Q268K), or both (numbering according to SEQ ID NO: 581). In certain embodiments, an epigenetic editor herein may comprise both DNMT and DNMT-like effector domains. For example, the epigenetic editor may comprise a DNMT3A- 3L domain, wherein DNMT3A and DNMT3L may be covalently linked. In other embodiments, an epigenetic editor described herein may comprise an effector domain that comprises only a DNMT3A domain (e.g., human DNMT3A), or only a DNMT-like domain (e.g., DNMT3L, which may be human or mouse DNMT3L). In some embodiments, the DNMT3A of the epigenetic editor is a binding partner for DNMT3A that recruits endogenous DNMT3A, e.g., an affinity domain that binds DNMT3A. In some embodiments, the affinity domain is a histone tail. Table 3 below provides exemplary methyltransferases from which an effector domain of an epigenetic editor described herein may be derived. Their corresponding sequences can be found in the "Sequences" listing after the Examples. Table 3. Exemplary Methyltransferase Sequences

[0041] 40 12541428.1

[0042] 41 12541428.1 A functional analog of any one of the above-listed proteins, i.e., a molecule having the same or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more) of the protein’s DNA methylation function or recruiting function) is encompassed by the present disclosure. For example, the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and / or containing mutations relative to the above protein. In some embodiments, the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences of the domains listed in Table 3. In some embodiments, the effector domain herein comprises only the functional domain (or functional analog thereof), e.g., the catalytical domain or recruiting domain, of the above-listed proteins. As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is identical to the parental protein (e.g., a human or murine DNMT3A or DNMT3L) or a functional analog thereof (e.g., having a functional fragment, such as a catalytic fragment or recruiting fragment, of the parental protein; and / or having mutations that improve the activity of the DNMT protein). An epigenetic editor herein may effect methylation at, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more CpG dinucleotide sequences in the target gene or chromosome. The CpG dinucleotide sequences may be located within or near the target gene in CpG islands, or may be located in a region that is not a CpG island. A CpG island generally refers to a nucleic acid sequence or chromosome region that comprises a high frequency of CpG dinucleotides. For example, a CpG island may comprise at least 50% GC content. The CpG island may have a high observed-to-expected CpG ratio, for example, an observed-to-expected CpG ratio of at least 60%. As used herein, an observed-to-expected CpG ratio is determined by Number of CpG * (sequence length) / (Number of C * Number of G). In some embodiments, the CpG island has an observed-to-expected CpG ratio of at least 60%, 70%, 80%, 90% or more. A CpG island may be a sequence or region of, e.g., at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, only 1, or less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 CpG dinucleotides are methylated by the epigenetic editor. In some embodiments, an epigenetic editor herein effects methylation at a hypomethylated nucleic acid sequence, i.e., a sequence that may lack methyl groups on the 5- methyl cytosine nucleotides (e.g., in CpG) as compared to a standard control.

[0043] 42 12541428.1 Hypomethylation may occur, for example, in aging cells or in cancer (e.g., early stages of neoplasia) relative to a younger cell or non-cancer cell, respectively. In some embodiments, an epigenetic editor described herein induces methylation at a hypermethylated nucleic acid sequence. In some embodiments, methylation may be introduced by the epigenetic editor at a site other than a CpG dinucleotide. For example, the target gene sequence may be methylated at the C nucleotide of CpA, CpT, or CpC sequences. In some embodiments, an epigenetic editor comprises a DNMT3A domain and effects methylation at CpG, CpA, CpT, CpC sequences, or any combination thereof. In some embodiments, an epigenetic editor comprises a DNMT3A domain that lacks a regulatory subdomain and only maintains a catalytic domain. In some embodiments, the epigenetic editor comprising a DNMT3A catalytic domain effects methylation exclusively at CpG sequences. In some embodiments, an epigenetic editor comprising a DNMT3A domain that comprises a mutation, e.g. a R836A or R836Q mutation (numbering according to SEQ ID NO: 577), has higher methylation activity at CpA, CpC, and / or CpT sequences as compared to an epigenetic editor comprising a wildtype DNMT3A domain. D. Histone Modifiers In some embodiments, an effector domain of an epigenetic editor herein mediates histone modification. Histone modifications play a structural and biochemical role in gene transcription, such as by formation or disruption of the nucleosome structure that binds to the histone and prevents gene transcription. Histone modifications may include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, SUMOylation and the like, e.g., at their N-terminal ends (“histone tails”). These modifications maintain or specifically convert chromatin structure, thereby controlling responses such as gene expression, DNA replication, DNA repair, and the like, which occur on chromosomal DNA. Post-translational modification of histones is an epigenetic regulatory mechanism and is considered essential for the genetic regulation of eukaryotic cells. Recent studies have revealed that chromatin remodeling factors such as SWI / SNF, RSC, NURF, NRD, and the like, which facilitate transcription factor access to DNA by modifying the nucleosome structure; histone acetyltransferases (HATs) that regulate the acetylation state of histones; and histone deacetylases (HDACs), act as important regulators. In particular, the unstructured N-termini of histones may be modified by acetylation, deacetylation, methylation, ubiquitylation, phosphorylation, SUMOylation, ribosylation, citrullination O-GlcNAcylation, crotonylation, or any combination thereof. For example,

[0044] 43 12541428.1 histone acetyltransferases (HATs) utilize acetyl-CoA as a cofactor and catalyze the transfer of an acetyl group to the epsilon amino group of the lysine side chains. This neutralizes the lysine’s positive charge and weakens the interactions between histones and DNA, thus opening the chromosomes for transcription factors to bind and initiate transcription. Acetylation of K14 and K9 lysines of histone H3 by histone acetyltransferase enzymes may be linked to transcriptional competence in humans. Lysine acetylation may directly or indirectly create binding sites for chromatin-modifying enzymes that regulate transcriptional activation. On the other hand, histone methylation of lysine 9 of histone H3 may be associated with heterochromatin, or transcriptionally silent chromatin. In certain embodiments, an effector domain of an epigenetic editor described herein comprises a histone methyltransferase domain. The effector domain may comprise, for example, a DOT1L domain, a SET domain, a SUV39H1 domain, a G9a / EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof. In particular embodiments, the effector domain comprises a histone-lysine-N- methyltransferase SETDB1 domain. In some embodiments, the effector domain comprises a histone deacetylase protein domain. In certain embodiments, the effector domain comprises a HDAC family protein domain, for example, a HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In particular embodiments, the effector domain comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones. E. Other Effector Domains In some embodiments, the effector domain comprises a tripartite motif containing protein (TRIM28, TIF1-beta, or KAP1). In certain embodiments, the effector domain comprises one or more KAP1 proteins. A KAP1 protein in an epigenetic editor herein may form a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in modulation of gene expression in a cellular environment. For example, KAP1 may be recruited by a KRAB domain of a transcriptional repressor. A KAP1 protein domain may interact with or recruit one or more protein complexes that reduces or silences gene expression. In some embodiments, KAP1 interacts with or recruits a histone deacetylase protein, a histone-lysine methyltransferase protein, a chromatin remodeling protein, and / or a heterochromatin protein. For example, a KAP1 protein domain may interact with or recruit a heterochromatin protein 1 (HP1) protein, a SETDB1 protein, an HDAC protein, and / or a NuRD protein complex component. In some embodiments, a KAP1 protein

[0045] 44 12541428.1 domain interacts with or recruits a ZFP90 protein (e.g., isoform 2 of ZFP90), and / or a FOXP3 protein. An exemplary KAP1 amino acid sequence is shown in SEQ ID NO: 611. In some embodiments, the effector domain comprises a protein domain that interacts with or is recruited by one or more DNA epigenetic marks. For example, the effector domain may comprise a methyl CpG binding protein 2 (MECP2) protein that interacts with methylated DNA nucleotides in the target gene (which may or may not be at a CpG island of the target gene). An MECP2 protein domain in an epigenetic editor described herein may induce condensed chromatin structure, thereby reducing or silencing expression of the target gene. In some embodiments, an MECP2 protein domain in an epigenetic editor described herein may interact with a histone deacetylase (e.g. HDAC), thereby repressing or silencing expression of the target gene. In some embodiments, an MECP2 protein domain in an epigenetic editor described herein may block access of a transcription factor or transcriptional activator to the target sequence, thereby repressing or silencing expression of the target gene. An exemplary MECP2 amino acid sequence is shown in SEQ ID NO: 612. Also contemplated as effector domains for the epigenetic editors described herein are, e.g., a chromoshadow domain, a ubiquitin-2 like Rad60 SUMO-like (Rad60-SLD / SUMO) domain, a chromatin organization modifier domain (Chromo) domain, a Yaf2 / RYBP C- terminal binding motif domain (YAF2_RYBP), a CBX family C-terminal motif domain (CBX7_C), a zinc finger C3HC4 type (RING finger) domain (ZF-C3HC4_2), a cytochrome b5 domain (Cyt-b5), a helix-loop-helix domain (HLH), a helix-hairpin-helix motif domain (e.g., HHH_3), a high mobility group box domain (HMG-box), a basic leucine zipper domain (e.g., bZIP_1 or bZIP_2), a Myb_DNA-binding domain, a homeodomain, a MYM-type Zinc finger with FCS sequence domain (ZF-FCS), an interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), an SSX repression domain (SSXRD), a B-box-type zinc finger domain (ZF-B_box), a CXXC zinc finger domain (ZF-CXXC), a regulator of chromosome condensation 1 domain (RCC1), an SRC homology 3 domain (SH3_9), a sterile alpha motif domain (SAM_1), a sterile alpha motif domain (SAM_2), a sterile alpha motif / Pointed domain (SAM_PNT), a Vestigial / Tondu family domain (Vg_Tdu), a LIM domain, an RNA recognition motif domain (RRM_1), a paired amphipathic helix domain (PAH), a proteasomal ATPase OB C-terminal domain (Prot_ATP_ID_OB), a nervy homology 2 domain (NHR2), a hinge domain of cleavage stimulation factor subunit 2 (CSTF2_hinge), a PPAR gamma N-terminal region domain (PPARgamma_N), a CDC48 N- terminal domain (CDC48_2), a WD40 repeat domain (WD40), a Fip1 motif domain (Fip1), a PDZ domain (PDZ_6), a Von Willebrand factor type C domain (VWC), a NAB conserved

[0046] 45 12541428.1 region 1 domain (NCD1), an S1 RNA-binding domain (S1), an HNF3 C-terminal domain (HNF_C), a Tudor domain (Tudor_2), a histone-like transcription factor (CBF / NF-Y) and archaeal histone domain (CBFD_NFYB_HMF), a zinc finger protein domain (DUF3669), an EGF-like domain (cEGF), a GATA zinc finger domain (GATA), a TEA / ATTS domain (TEA), a phorbol esters / diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), a transactivation domain of FOXO protein family (FOXO-TAD), a homeobox KN domain (Homeobox_KN), a BED zinc finger domain (ZF-BED), a zinc finger of C3HC4-type RING domain (ZF-C3HC4_4), a RAD51 interacting motif domain (RAD51_interact), a p55-binding region of a methyl-CpG-binding domain protein MBD (MBDa), a Notch domain, a Raf-like Ras-binding domain (RBD), a Spin / Ssty family domain (Spin-Ssty), a PHD finger domain (PHD_3), a Low-density lipoprotein receptor domain class A (Ldl_recept_a), a CS domain, a DM DNA-binding domain, and a QLQ domain. In some embodiments, the effector domain is a protein domain comprising a YAF2_RYBP domain or homeodomain or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain. In particular embodiments, the YAF2_RYBP domain may comprise a 32 amino acid Yaf2 / RYBP C-terminal binding motif domain (32 aa RYBP). In some embodiments, the effector domain comprises a protein domain selected from a group consisting of SUMO3 domain, Chromo domain from M phase phosphoprotein 8 (MPP8), chromoshadow domain from Chromobox 1 (CBX1), and SAM_1 / SPM domain from Scm Polycomb Group Protein Homolog 1 (SCMH1). In some embodiments, the effector domain comprises an HNF3 C-terminal domain (HNF_C). The HNF_C domain may be from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif. In some embodiments, the effector domain may comprise an interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), a Cyt-b5 domain from DNA repair factor HERC2 E3 ligase, a variant SH3 domain (SH3_9) from Bridging Integrator 1 (BIN1), an HMG-box domain from transcription factor TOX or ZF-C3HC4_2 RING finger domain from the polycomb component PCGF2, a Chromodomain-helicase-DNA binding protein 3 (CHD3) domain, or a ZNF783 domain. In some embodiments, the effector domain comprises a D3A methyltransferase recruiting domain. In some embodiments, the D3A methyltransferase recruiting domain is a CHARM (coupled histone tail for autoinhibition release of methyltransferase) domain. In some embodiments, a CHARM effector comprises both a DNA-binding domain and a

[0047] 46 12541428.1 CHARM domain. Such CHARM effectors may be named after the DNA-binding domain, for example, CRISPRcharm, ZFcharm, or a TALEcharm. CHARM domains are described in WO 2024 / 173896 (PCT / US2024 / 016308), which is incorporated herein by reference, in particular for its disclosure of CHARM structures (e.g., histone H3 tail-Dnmt3l fusion) and uses thereof. CHARM is also described in Neumann et al. Science.2024 Jun 28;384(6703):ado7082. doi: 10.1126 / science.ado7082, which is incorporated herein by reference. IV. Epigenetic Editors The present disclosure provides epigenetic editors (also referred to herein as epigenetic editing systems) for repressing or activating expression of a gene of interest, e.g., by directing epigenetic modification(s) to a target sequence in the gene of interest. The DNA-binding domain (in concert with a guide polynucleotide such as one described herein, where the DNA-binding domain is a polynucleotide guided DNA-binding domain) directs the effector domain to epigenetically modify the target sequence, resulting in gene repression or silencing that may be durable and inheritable across cell generations. In some aspects, the epigenetic editors described herein can repress or silence genes reversibly or irreversibly in cells. In particular embodiments, an epigenetic editor described herein comprises one or more fusion proteins, each comprising (1) DNA-binding domain(s) and (2) effector domain(s). The effector domains may be on one or more fusion proteins comprised by the epigenetic editor. For example, a single fusion protein may comprise all of the effector domains with a DNA-binding domain. Alternatively, the effector domains or subsets thereof may be on separate fusion proteins, each with a DNA-binding domain (which may be the same or different). A fusion protein described herein may further comprise one or more linkers (e.g., peptide linkers), detectable tags, nuclear localization signals (NLSs), or any combination thereof. As used herein, a “fusion protein” refers to a chimeric protein in which two or more coding sequences (e.g., for DNA-binding domain(s) and / or effector domain(s)) are covalently or non-covalently joined, directly or indirectly. In some embodiments, an epigenetic editor described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more effector (e.g., repression) domains, which may be identical or different. In certain embodiments, two or more of said effector domains function synergistically. Combinations of effector domains may comprise DNA methylation domains, histone deacetylation domains, histone methylation domains, and / or scaffold domains that recruit any of the above. For example, an epigenetic editor described herein may comprise one or more

[0048] 47 12541428.1 transcriptional repressor domains (e.g., a KRAB domain such as KOX1, ZIM3, ZFP28, or ZN627 KRAB) in combination with one or more DNA methylation domains (e.g., a DNMT domain) and / or recruiter domain (e.g., a DNMT3L domain). Such an epigenetic editor may comprise, for instance, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, the epigenetic editor further comprises an additional effector domain (e.g., a KAP1, MECP2, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, RBBP4, RCOR1, or SCML2 domain). In some embodiments, the additional effector domain is a CDYL2, TOX, TOX3, TOX4, or HP1a domain. For example, an epigenetic editor described herein may comprise a CDYL2 and / or a TOX domain in combination with a KRAB domain (e.g., a KOX1 KRAB domain). In some embodiments of the present disclosure, the epigenetic editor comprises a first fusion protein comprising a nuclease-dead (e.g., catalytically deactivated) RNA-guided nuclease fused to an epigenetic effector domain and a first guide RNA as well as a second fusion protein, comprising a nuclease-dead RNA-guided nuclease fused to an epigenetic effector domain and a second guide RNA. In some embodiments, the first nuclease-dead RNA-guided nuclease is different from the second nuclease-dead RNA-guided nuclease. "Different" can mean taken or derived from a different species or of a different enzymatic class. "Different" also means heterologous or functioning in an orthogonal manner. In some embodiments, the first and second nuclease-dead RNA-guided nucleases are derived from the same species. In some embodiments, the first and second nuclease-dead RNA-guided nucleases are derived from different species. In some embodiments, the first guide RNA forms a complex with the first nuclease- dead (e.g., catalytically deactivated) RNA-guided nuclease. In some embodiments, the second guide RNA forms a complex with the second nuclease-dead RNA-guided nuclease. In some embodiments, the second nuclease-dead RNA-guided nuclease does not form a complex with the first guide RNA. In some embodiments, the first nuclease-dead RNA- guided nuclease does not form a complex with the second guide RNA. In some embodiments, a guide RNA directs a nuclease-dead RNA to a target site in the genome of a cell. In some embodiments, the present disclosure includes an epigenetic editor comprising fusion proteins with a nuclease-dead (e.g., catalytically deactivated) RNA-guided nuclease and a fusion protein comprising a ZF domain. In some embodiments, the present disclosure includes an epigenetic editor comprising both an epigenetic repressor and an epigenetic activator. In some embodiments, the epigenetic editor includes a single epigenetic repressor and multiple epigenetic activators. In

[0049] 48 12541428.1 some embodiments, the epigenetic editor includes a single epigenetic activator and multiple epigenetic repressors. In some embodiments, the epigenetic repressors and epigenetic activators are linked to orthogonal species, for example, the repressor linked to a dSpCas9 and the epigenetic activator linked to a dSaCas9. In some embodiments, the present disclosure includes an epigenetic editor capable of "polyfunctional" editing: the editor can accomplish both epigenetic editing and gene editing (i.e. gene disruption and / or targeted transgene integration). In some embodiments, the polyfunctional epigenetic editor comprises a guide polynucleotide with a spacer sequence of 20 nucleotides. In some of these embodiments, the epigenetic editor cleaves both DNA strand or a single one proximal to the 20-nucleotide spacer sequence. In some embodiments, the polyfunctional epigenetic editor comprises a guide polynucleotide with a spacer sequence of between 10 and 18 nucleotides, inclusive. In some of these embodiments, the epigenetic editor epigenetically edits DNA proximal to the between 10 and 18-nucleotide spacer sequence. A. Linkers A fusion protein as described herein may comprise one or more linkers that connect components of the epigenetic editor. A linker may be a peptide or non-peptide linker. In some embodiments, one or more linkers utilized in an epigenetic editor provided herein is a peptide linker, i.e., a linker comprising a peptide moiety. A peptide linker can be any length applicable to the epigenetic editor fusion proteins described herein. In some embodiments, the linker can comprise a peptide between 1 and 200 (e.g., between 1 and 80) amino acids. In some embodiments, the linker comprises from 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 6050 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker is 4, 16, 24, 32, 40, 64, 92, or 104 amino acids in length. The peptide linker may be a flexible or rigid linker. In particular embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 722-730, 738, 740-741, 749-750, 1357-1362, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0050] 49 12541428.1 In certain embodiments, the peptide linker is an XTEN linker. The term “XTEN” as used herein refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. XTEN linkers typically are unstructured and comprise a limited set of natural amino acids. Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the fusion protein. These XTEN fusion proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways. The XTEN linker may be, for example, 24, 40, 64, or 92 amino acids in length, and in particular embodiments may comprise the amino acid sequence of any one of SEQ ID NOs: 727-731 and 738 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the XTEN linker may be XTEN10, XTEN16, XTEN20, or XTEN80. In some embodiments, one or more linkers utilized in an epigenetic editor provided herein is a non-peptide linker. For example, the linker may be a carbon bond, a disulfide bond, or carbon-heteroatom bond. In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker. In some embodiments, one or more linkers utilized in an epigenetic editor provided herein is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker may comprise, for example, a monomer, dimer, or polymer of aminoalkanoic acid; an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.); a monomer, dimer, or polymer of aminohexanoic acid (Ahx); or a polyethylene glycol moiety (PEG); or an aryl or heteroaryl moiety. In certain embodiments, the linker may be based on a carbocyclic moiety (e.g., cyclopentane or cyclohexane) or a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates. Various linker lengths and flexibilities can be employed between any two components of an epigenetic editor (e.g., between an effector domain (e.g., a repression domain) and a DNA-binding domain (e.g., a Cas9 domain), between a first effector domain and a second effector domain, etc.). The linkers may range from very flexible linkers of the form (GGGGS)n (SEQ ID NO: 1357), (e.g., (GGGGS)4(SEQ ID NO: 1358)), and (G)n (SEQ ID NO: 1359) to more rigid linkers of the form (EAAAK)n (SEQ ID NO: 1360), (SGGS)n (SEQ

[0051] 50 12541428.1 ID NO: 1361), and (XP)n (SEQ ID NO: 1362) in order to achieve the optimal length for effector domain activity for the specific application. In some embodiments, n is any integer between 3 and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n (SEQ ID NO: 1363) motif, wherein n is 1, 3, or 7. In some embodiments, a linker in an epigenetic editor described herein comprises a nuclear localization signal, for example, with the amino acid sequence of any one of SEQ ID NOs: 732-737. In some embodiments, a linker in an epigenetic editor described herein comprises a cleavable peptide, e.g., a T2A peptide, a p2A peptide, or a furin / p2A peptide. In some embodiments, a linker in an epigenetic editor described herein comprises an expression tag, e.g., a detectable tag such as a green fluorescence protein. B. Nuclear Localization Signals A fusion protein described herein may comprise one or more nuclear localization signals, and in certain embodiments, may comprise two or more nuclear localization signals. For example, the fusion protein may comprise 1, 2, 3, 4, or 5 nuclear localization signals. As used herein, a “nuclear localization signal” (NLS) is an amino acid sequence that directs proteins to the nucleus. In certain embodiments, the NLS may be an SV40 NLS. The fusion protein may comprise an NLS at its N-terminus, C-terminus, or both, and / or an NLS may be embedded in the middle of the fusion protein (e.g., at the N- or C- terminus of a DNA- binding domain or an effector domain). In certain embodiments, an NLS comprises the amino acid sequence of any one of SEQ ID NOs: 732-737. , or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the selected sequence. Additional NLSs are known in the art. C. Tags Epigenetic editors provided herein may comprise one or more additional sequences (“tags”) for tracking, detection, and localization of the editors. In some embodiments, the epigenetic editor comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more detectable tags. Each of the detectable tags may be the same or different. For example, an epigenetic editor fusion protein may comprise cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG- tags, hemagglutinin (HA)-tags, poly-histidine tags (also referred to as histidine tags or His-

[0052] 51 12541428.1 tags), maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1 or Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. D. Fusion Protein Configurations A fusion protein of an epigenetic editor described herein may have its components structured in different configurations. For example, the DNA-binding domain may be at the C-terminus, the N-terminus, or in between two or more epigenetic effector domains or additional domains. In some embodiments, the DNA-binding domain is at the C-terminus of the epigenetic editor. In some embodiments, the DNA-binding domain is at the N-terminus of the epigenetic editor. In some embodiments, the DNA-binding domain is linked to one or more nuclear localization signals. In some embodiments, the DNA-binding domain is flanked by an epigenetic effector domain and / or an additional domain on both sides. In some embodiments, where “DBD” indicates DNA-binding domain and “ED” indicates effector domain, the epigenetic editor comprises the configuration of: - N’]-[ED1]-[DBD]-[ED2]-[C’ - N’]-[ED1]-[DBD]-[ED2]-[ED3]-[C’ - N’]-[ED1]-[ED2]-[DBD]-[ED3]-[C’ or - N’]-[ED1]-[ED2]-DBD]-[ED3]-[ED4]-[C’. In some embodiments, an epigenetic editor comprises a DNA-binding domain (DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repressor (“repressor”) domain that represses or silences expression of a target gene. The DBD, DNMT, and transcriptional repressor domains may be any as described herein, in any combination. In some embodiments, the epigenetic editor comprises a fusion protein with the configuration of: N’]-[DNA methyltransferase domain]-[DBD]-[repressor domain]-[C’ N’]-[repressor domain]-[DBD]-[DNA methyltransferase domain]-[C’ N’]-[DNA methyltransferase domain]-[repressor domain]- [DBD]-[C’ or N’]-[repressor domain]-[DNA methyltransferase domain]- [DBD]-[C’. In some embodiments, a connecting structure “]-[“in any one of the epigenetic editor structures is a linker, e.g., a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and / or a promoter or regulatory sequence. In an epigenetic editor

[0053] 52 12541428.1 structure, the multiple connecting structures “]-[“ may be the same or may each be a different linker, tag, NLS, or peptide bond. In particular embodiments, the DNA methyltransferase domain comprises DNMT3A, DNMT3L, or both. In particular embodiments, the DBD is a catalytically inactive polynucleotide guided DNA-binding domain (e.g., a dCas9) or a ZFP domain. In particular embodiments, the repressor domain is a KRAB domain. In some embodiments, the epigenetic editor comprises a configuration selected from N’]-[DNMT3A-DNMT3L]-[DBD]-[KRAB]-[C’ N’]-[KRAB]-[DBD]-[DNMT3A-DNMT3L]-[C’ N’]-[KRAB]-[DBD]-[DNMT3A]-[C’ N’]-[DNMT3A]-[DBD]-[KRAB]-[C’ N’]-[KRAB]-[DBD]-[DNMT3A]-[DNMT3L]-[C’ N’]-[DNMT3A]- [DNMT3L]- [DBD]-[KRAB]-[C’ N’]-[DNMT3A]-[DBD]-[C’ N’]-[DBD]-[DNMT3A]-[C’ N’]-[DNMT3L]-[DBD]-[C’ N’]-[DBD]-[DNMT3L]-[C’ wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure ]-[ is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, KRAB, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. In particular embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain; the KRAB domain is derived from KOX1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure. In some embodiments, the epigenetic editor comprises a configuration selected from N’]-[DNMT3A]-[DBD]-[SETDB1]-[C’ N’]-[DNMT3A]- [DNMT3L]- [DBD]-[SETDB1]-[C’ N’]- [DNMT3A-DNMT3L]- [DBD]- [SETDB1]- [C’ N’]-[SETDB1]-[DBD]-[DNMT3A]-[DNMT3L]-[C’ N’]-[SETDB1]-[DBD]-[DNMT3A]-[C’

[0054] 53 12541428.1 wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure ]-[ is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, SETDB1, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. In particular embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain; the SETDB1 domain is derived from human SETDB1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure. Particular constructs contemplated herein include: DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1 KRAB (Configuration 1), and DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1 KRAB (Configuration 2). In particular embodiments, the DNMT3L and DNMT3A are both derived from human parental proteins. In particular embodiments, the DNMT3L and DNMT3A are derived from human and mouse parental proteins, respectively. In particular embodiments, the DNMT3L and DNMT3A are derived from mouse and human parental proteins, respectively. In particular embodiments, the DNMT3L and DNMT3A are both derived from mouse parental proteins. In some embodiments, the dCas9 is dSpCas9. In some embodiments, the KOX1 is human KOX1. In particular embodiments, a fusion construct described herein may have Configuration 1 and comprise SEQ ID NO: 743, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 743 below, the XTEN linkers are underlined, the NLS sequences are bolded, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the dCas9 domain is bolded and italicized, and the KOX1 KRAB domain is underlined and bolded: MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITV GMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEF YRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWG NLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDI LWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNAN SRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGS GGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQE SQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPL

[0055] 54 12541428.1 TPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPPSGGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEPKKKRKVYMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAI LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLD NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLL DTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP (SEQ ID NO: 743) In particular embodiments, a fusion construct described herein may have Configuration 2 and comprise SEQ ID NO: 744, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 744 below, the XTEN linkers are underlined, the NLS sequences are bolded and underlined, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the ZFP domain is bolded, and the KOX1 KRAB domain is underlined and bolded. Variable amino acids represented by Xs are the amino acids of the DNA-recognition helix of the zinc finger and XX in italics may be either TR, LR or LK. MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITV GMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEF YRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWG NLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDI LWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNAN SRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGS GGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQE SQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPL TPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPPSGGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEPKKKRKVYSRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXX

[0056] 55 12541428.1 XXXHXXTH[linker]PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXX XHXXTH[linker]PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXH XXTHLRGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVY RNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP (SEQ ID NO: 744) [linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 740). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 741). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 740 and the other linker sequence may have the amino acid sequence of SEQ ID NO: 741. In some embodiments, a fusion construct described herein may have Configuration 7 and comprise SEQ ID NO: 1366, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, a fusion construct described herein may have Configuration 9 and comprise SEQ ID NO: 1367, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, a fusion construct described herein may have Configuration 11 and comprise SEQ ID NO: 1368, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, a fusion construct described herein may have Configuration 13 and comprise SEQ ID NO: 1369, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, a fusion construct described herein is within an expression construct that comprises a WPRE sequence, a polyadenylation site, or both. In certain embodiments, the WPRE sequence is in a 3’ noncoding region. In certain embodiments, the WPRE sequence is upstream from a poly-adenylation site. In particular embodiments, the expression construct comprises the fusion construct and a WPRE sequence in a 3’ noncoding region upstream from a polyadenylation site. Multiple epigenetic editors may be used to effect activation or repression of a target gene or multiple target genes. For example, an epigenetic editor fusion protein comprising a DNA-binding domain (e.g., a dCas9 domain) and an effector domain may be co-delivered with two or more guide polynucleotides (e.g., gRNAs), each targeting a different target DNA sequence. The target sites for two of the DNA-binding domains may be the same or in the vicinity of each other, or separated by, for example, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, or about 600 or more base pairs. In addition, when targeting double-strand DNA, such as an endogenous gene

[0057] 56 12541428.1 locus, the guide polynucleotides may target the same or different strands (one or more to the positive strand and / or one or more to the negative strand). V. Target Sequences An epigenetic editor herein may be directed to a target sequence in ZNF410 to effect epigenetic modification of the ZNF410 gene. As used herein, a “target sequence,” a “target site,” or a “target region” is a nucleic acid sequence present in a gene of interest; in some instances, the target sequence may be outside but in the vicinity of the gene of interest wherein methylation or binding by a repressor of the target sequence represses expression of the gene. In some embodiments, the target sequence may be a hypomethylated or hypermethylated nucleic acid sequence. The target sequence may be in any part of a target gene. In some embodiments, the target sequence is part of or near a noncoding sequence of the gene. In some embodiments, the target sequence is part of an exon of the gene. In some embodiments, the target sequence is part of or near a transcriptional regulatory sequence of the gene, such as a promoter or an enhancer. In some embodiments, the target sequence is adjacent to, overlaps with, or encompasses a CpG island. In certain embodiments, the target sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking a TSS. In certain embodiments, the target sequence is within 500 bp flanking the ZNF410 TSS. In certain embodiments, the target sequence is within 1000 bp flanking the ZNF410 TSS. In some embodiments, the target sequence may hybridize to a guide polynucleotide sequence (e.g., gRNA) complexed with a fusion protein comprising a polynucleotide guided DNA-binding domain (e.g., a CRISPR protein such as dCas9) and effector domain(s). The guide polynucleotide sequence may be designed to have complementarity to the target sequence, or identity to the opposing strand of the target sequence. In some embodiments, the guide polynucleotide comprises a spacer sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a protospacer sequence in the target sequence. In particular embodiments, the guide polynucleotide comprises a spacer sequence that is 100% identical to a protospacer sequence in the target sequence. In some embodiments, where the DNA-binding domain of an epigenetic editor described herein is a zinc finger array, the target sequence may be recognized by said zinc finger array.

[0058] 57 12541428.1 In some embodiments, where the DNA-binding domain of an epigenetic editor described herein is a TALE, the target sequence may be recognized by said TALE. A target sequence described herein may be specific to one copy of a target gene, or may be specific to one allele of a target gene. Accordingly, the epigenetic modification and modulation of expression thereof may be specific to one copy or one allele of the target gene. For example, an epigenetic editor may repress expression of a specific copy harboring a target sequence recognized by the DNA-binding domain (e.g., a copy associated with a disease or condition, or that harbors a mutation associated with a disease or condition). VI. Epigenetic Modifications An epigenetic editor described herein may perform sequence-specific epigenetic modification(s) (e.g., alteration of chemical modification(s)) of a target gene that harbors the target sequence. Such epigenetic modulation may be safer and more easily reversible than modulation due to gene editing, e.g., with generation of DNA double-strand breaks. In some embodiments, the epigenetic modulation may reduce or silence the target gene. In some embodiments, the modification is at a specific site of the target sequence. In some embodiments, the modification is at a specific allele of the target gene. Accordingly, the epigenetic modification may result in modulated (e.g., reduced) expression of one copy of a target gene harboring a specific allele, and not the other copy of the target gene. In some embodiments, the specific allele is associated with a disease, condition, or disorder. In some embodiments, the epigenetic modification reduces or abolishes transcription of the target gene harboring the target sequence. In some embodiments, the epigenetic modification reduces or abolishes transcription of a copy of the target gene harboring a specific allele recognized by the epigenetic editor. In some embodiments, the epigenetic editor reduces the level of or eliminates expression of a protein encoded by the target gene. In some embodiments, the epigenetic editor reduces the level of or eliminates expression of a protein encoded by a copy of the target gene harboring a specific allele recognized by the epigenetic editor. The target ZNF410 gene may be epigenetically modified in vitro, ex vivo, or in vivo. The effector domain of an epigenetic editor described herein may alter (e.g., deposit or remove) a chemical modification at a nucleotide of the target gene or at a histone associated with the target gene. The chemical modification may be altered at a single nucleotide or a single histone, or may be altered at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0059] 58 12541428.1 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or more nucleotides. In some embodiments, an effector domain of an epigenetic editor described herein may alter a CpG dinucleotide within the target gene. In some embodiments, all CpG dinucleotides within 2000, 1500, 1000, 500, or 200 bps flanking a target sequence (e.g., in an alteration site as described herein) are altered according to a modification type described herein, as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more of the CpG dinucleotides are altered as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the CpG dinucleotides are altered as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, one single CpG dinucleotide is altered, as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. An effector domain of an epigenetic editor described herein may alter a histone modification state of a histone associated with or bound to the target gene. For example, an effector domain may deposit a modification on one or more lysine residues of histone tails of histones associated with the target gene. In some embodiments, the effector domain may result in deacetylation of one or more histone tails of histones associated with the target gene, thereby reducing or silencing expression of the target gene. In some embodiments, the histone modification state is a methylation state. For example, the effector domain may result in a H3K9, H3K27 or H4K20 methylation (e.g. one or more of a H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3 methylation) at one or more histone tails associated with the target gene, thereby reducing or silencing expression of the target gene. In some embodiments, all histone tails of histones bound to DNA nucleotides within 2000, 1500, 1000, 500, or 200 bps flanking the target sequence are altered according to a modification type as described herein, as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more histone tails of the bound histones are altered as compared to the original state of the chromosome or the

[0060] 59 12541428.1 chromosome in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. For example, one single histone tail of the bound histones may be altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. As another example, one single bound histone octamer may be altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. The chemical modification deposited at target gene DNA nucleotides or histone residues may be at or in close proximity to a target sequence in the target gene. In some embodiments, an effector domain of an epigenetic editor described herein alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide 100-200, 200-300, 300-400, 400-55, 500-600, 600-700, or 700-800 nucleotides 5’ or 3’ to the target sequence in the target gene. In some embodiments, an effector domain alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides flanking the target sequence. As used herein, “flanking” refers to nucleotide positions 5’ to the 5’ end of and 3’ to the 3’ end of a particular sequence, e.g. a target sequence. In some embodiments, an effector domain mediates or induces a chemical modification change of a nucleotide or a histone tail bound to a nucleotide distant from a target sequence. Such modification may be initiated near the target sequence, and may subsequently spread to one or more nucleotides in the target gene distant from the target sequence. For example, an effector domain may initiate alteration of a chemical modification state of one or more nucleotides or one or more histone residues bound to one or more nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nucleotides flanking the target sequence, and the chemical modification state alteration may spread to one or more nucleotides at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or more nucleotides from the target sequence in the target gene, either upstream or downstream of the target sequence. In certain embodiments, the chemical modification may be initiated at less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides in the target gene and spread to at least 100, 200, 300, 400,

[0061] 60 12541428.1 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides in the target gene. In some embodiments, the chemical modification spreads to nucleotides in the entire target gene. Additional proteins or transcription factors, for example, transcription repressors, methyltransferases, or transcription regulation scaffold proteins, may be involved in the spreading of the chemical modification. Alternatively, the epigenetic editor alone may be involved. In some embodiments, an epigenetic editor described herein reduces expression of a target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the target gene in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject (e.g., in the absence of the epigenetic editor). In some embodiments, the epigenetic editors described herein reduces expression of a copy of target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the copy of the target gene in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject. In certain embodiments, the copy of the target gene harbors a specific sequence or allele recognized by the epigenetic editor. In particular embodiments, the epigenetically modified copy encodes a functional protein, and accordingly an epigenetic editor disclosed herein may reduce or abolish expression and / or function of the protein. For example, an epigenetic editor described herein may reduce expression and / or function of a protein encoded by the target gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100 fold in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject. Modulation of target gene expression can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in transcription or activity of reporter genes such as, for example, luciferase, CAT, beta-galactosidase, or GFP; changes in signal transduction; changes in phosphorylation and dephosphorylation; changes in

[0062] 61 12541428.1 receptor-ligand interactions; changes in concentrations of second messengers such as, for example, cGMP, cAMP, IP3, and Ca2+; changes in cell growth; changes in neovascularization; and / or changes in any functional effect of gene expression. Measurements can be made in vitro, in vivo, and / or ex vivo, and can be made by conventional methods, e.g., measurement of RNA or protein levels, measurement of RNA stability, and / or identification of downstream or reporter gene expression. Readout can be by way of, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3), changes in intracellular calcium levels; cytokine release, and the like. Methods for determining the expression level of a gene, for example the target of an epigenetic editor, may include, e.g., determining the transcript level of a gene by reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blot, RNA sequencing, DNA sequencing (e.g., sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA); next generation (Next-Gen) sequencing, nanopore sequencing, pyrosequencing, or Nanostring sequencing. Levels of protein expressed from a gene may be determined, e.g., by Western blotting, enzyme linked immuno-absorbance assays, mass- spectrometry, immunohistochemistry, or flow cytometry analysis. Gene expression product levels may be normalized to an internal standard such as total messenger ribonucleic acid (mRNA) or the expression level of a particular gene, e.g., a housekeeping gene. In some embodiments, the effect of an epigenetic editor in modulating target gene expression may be examined using a reporter system. For example, an epigenetic editor may be designed to target a reporter gene encoding a reporter protein, such as a fluorescent protein. Expression of the reporter gene in such a model system may be monitored by, e.g., flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a population of cells may be transfected with a vector that harbors a reporter gene. The vector may be constructed such that the reporter gene is expressed when the vector transfects a cell. Suitable reporter genes include genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins. The population of cells carrying the reporter system may be transfected with DNA, mRNA, or vectors encoding the epigenetic editor targeting the reporter gene. VII. Pharmaceutical Compositions Another aspect of the present disclosure is a pharmaceutical composition comprising

[0063] 62 12541428.1 as an active ingredient (or as the sole active ingredient) one or more epigenetic editors described herein or component(s) (e.g., fusion proteins and / or guide polynucleotides) thereof, or nucleic acid molecule(s) encoding said epigenetic editors or component(s) thereof. For example, a pharmaceutical composition may comprise nucleic acid molecule(s) encoding the fusion protein(s) (and guide polynucleotides, where applicable) of an epigenetic editor described herein. In some embodiments, separate pharmaceutical compositions comprise the fusion protein(s) and the guide polynucleotide(s). A pharmaceutical composition may also comprise cells that have undergone epigenetic modification(s) mediated or induced by an epigenetic editor provided herein. Generally, the epigenetic editors described herein or component(s) thereof, or nucleic acid molecule(s) encoding said epigenetic editors or component(s) thereof, of the present disclosure are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s), e.g., as described below. The term “excipient” is used herein to describe any ingredient other than the compound(s) of the present disclosure. The choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody. Formulations of a pharmaceutical composition suitable for parenteral administration typically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. In some embodiments, the epigenetic editor or its component(s) are introduced to target cells in the form of nucleic acid molecule(s) encoding the epigenetic editor or its component(s); accordingly, the pharmaceutical compositions herein comprise the nucleic acid molecule(s). Such nucleic acid molecule(s) may be, for example, DNA, RNA or mRNA, and / or modified

[0064] 63 12541428.1 nucleic acid sequence(s) (e.g., with chemical modifications, a 5’ cap, or one or more 3’ modifications). In some embodiments, the nucleic acid molecule(s) may be delivered as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by target cells. In some embodiments, the nucleic acid molecule(s) may be in nucleic acid expression vector(s), which may include expression control sequences such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art. A vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein. Examples of vectors include, but are not limited to, plasmid vectors; viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, or spleen necrosis virus, vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and other recombinant vectors. In certain embodiments, the vector is a plasmid or a viral vector. Viral particles may also be used to deliver nucleic acid molecule(s) encoding epigenetic editors or component(s) thereof as described herein. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles may also be engineered to incorporate targeting ligands to alter target tissue specificity. In certain embodiments, an epigenetic editor as described herein or component(s) thereof are encoded by nucleic acid sequence(s) present in one or more viral vectors, or a suitable capsid protein of any viral vector. Examples of viral vectors include adeno- associated viral vectors (e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and / or variants thereof); retroviral vectors (e.g., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., HIV and FIV-based vectors), and herpesvirus vectors (e.g., HSV-2). In some embodiments, delivery involves an adeno-associated virus (AAV) vector. AAV vector delivery may be particularly useful where the DNA-binding domain of an epigenetic editor fusion protein is a zinc finger array. Without wishing to be bound by any theory, the smaller size of zinc finger arrays compared to larger DNA-binding domains such

[0065] 64 12541428.1 as Cas protein domains may allow such a fusion protein to be conveniently packed in viral vectors such as an AAV vector. Any AAV serotype, e.g., human AAV serotype, can be used for an AAV vector as described herein, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), and AAV serotype 11 (AAV11), as well as variants thereof. In some embodiments, an AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a wildtype AAV. In certain embodiments, the AAV variant may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans. In some instances, one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate a chimeric variant. For example, a chimeric AAV may comprise inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV can have a different antigenic reactivity or recognition compared to its parental serotypes. In some embodiments, a chimeric variant of an AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes. Non-viral systems are also contemplated for delivery as described herein. Non-viral systems include, but are not limited to, nucleic acid transfection methods including electroporation, sonoporation, calcium phosphate transfection, microinjection, DNA biolistics, lipid-mediated transfection, transfection through heat shock, compacted DNA- mediated transfection, lipofection, cationic agent-mediated transfection, and transfection with liposomes, immunoliposomes, or cationic facial amphiphiles (CFAs). In certain embodiments, one or more mRNAs encoding epigenetic editor fusion proteins as described herein may be co-electroporated with one or more guide polynucleotides (e.g., gRNAs) as described herein. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold). For instance, organic (e.g. lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. In some embodiments, delivery is accomplished using a lipid nanoparticle (LNP). LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. In some embodiments, a LNP refers to any particle that has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.

[0066] 65 12541428.1 Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. An LNP as described herein may be made from cationic, anionic, or neutral lipids. In some embodiments, an LNP may comprise neutral lipids, such as the fusogenic phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as helper lipids to enhance transfection activity and nanoparticle stability. In some embodiments, an LNP may comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. The lipids may be combined in any molar ratios to produce the LNP. In some embodiments, the LNP is a liver-targeting (e.g., preferentially or specifically targeting the liver) LNP. LNP formulations and methods of LNP delivery that can be used will be apparent to those skilled in the art from the present disclosure and the state of the art. Non-limiting exemplary compositions and methods can be found in Shah, R., Eldridge, D., Palombo, E., and Harding, I., Lipid Nanoparticles: Production, Characterization and Stability, Springer, 2015, ISBN-13978-3319107103; Mitchell, M.J., Billingsley, M.M., Haley, R.M. et al. Engineering precision nanoparticles for drug delivery, Nat Rev Drug Discov 20, 101–124 (2021); Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078–1094 (2021); Lipid-Nanoparticle-Based Delivery of CRISPR / Cas9 Genome- Editing Components, Pardis Kazemian, Si-Yue Yu, Sarah B. Thomson, Alexandra Birkenshaw, Blair R. Leavitt, and Colin J. D. Ross. Molecular Pharmaceutics 202219 (6), 1669-1686; Cullis PR, Hope MJ. Lipid Nanoparticle Systems for Enabling Gene Therapies, Mol Ther.2017 Jul 5;25(7):1467-1475; Hatit, M.Z.C., Lokugamage, M.P., Dobrowolski, C.N. et al. Species-dependent in vivo mRNA delivery and cellular responses to nanoparticles, Nat. Nanotechnol.17, 310–318 (2022); Lam, K., Schreiner, P., Leung, A., Stainton, P., Reid, S., Yaworski, E., Lutwyche, P. and Heyes, J. (2023), Optimizing Lipid Nanoparticles for Delivery in Primates, Adv. Mater; Dilliard, S.A., Siegwart, D.J. Passive, active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs, Nat Rev Mater (2023); Kasiewicz, L.N., et.al., Lipid nanoparticles incorporating a GalNAc ligand enable in vivo liver ANGPTL3 editing in wild-type and somatic LDLR knockout non-human primates, bioRxiv 2021.11.08.467731, doi: https: / / doi.org / 10.1101 / 2021.11.08.467731; Tombácz, I., et.al., Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs, Molecular Therapy, Volume 29, Issue 11, 2021, 3293-3304;

[0067] 66 12541428.1 Cheng, Q., Wei, T., Farbiak, L. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing, Nat. Nanotechnol.15, 313–320 (2020); Zhang, Y., et.al., Lipids and Lipid Derivatives for RNA Delivery, Chemical Reviews 2021121 (20); Lam, K., et.al, Unsaturated, Trialkyl Ionizable Lipids are Versatile Lipid-Nanoparticle Components for Therapeutic and Vaccine Applications, Adv. Mater.2023, 35; Han, X., Zhang, H., Butowska, K. et al. An ionizable lipid toolbox for RNA delivery, Nat Commun 12, 7233 (2021); US Patent No.9,364,435; US Patent No.8,058,069; US Patent No.8,822,66; US Patent No.8,492,359; US Patent No.11,141,378; US Patent No. 9,518,272; US Patent No.9,404,127; US Patent No.9,006,417; US Patent No.7,901,708; US Patent No.9,005,654; US Patent No.9,878,042; US Patent No.9,682,139; US Patent No. 8,642,076; US Patent No.9,593,077; US Patent No.9,415,109; US Patent No.9,701,623; US Patent No.10,369,226; US Patent No.9,999,673; US Patent No.9,301,923; US Patent No. 10,342,761; US Patent No.10,137,201; International Publication No. WO2015199952A1; International Publication No. WO2017075531A1; International Publication No. WO2018081480A1; International Publication No. WO2016081029A1; European Application No. EP3852911A2; each of which are incorporated herein by reference in their entirety. Other methods of delivery to target cells will be known to those skilled in the art and can be used with the compositions of the present disclosure. Any type of cell may be targeted for delivery of an epigenetic editor or component(s) thereof as described herein. For example, the cells may be eukaryotic or prokaryotic. In some embodiments, the cells are mammalian (e.g., human) cells. Human cells may include, for example, hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells. In some embodiments of the present disclosure, the hematopoietic cells of bone marrow are targeted for delivery. In some embodiments, targeting of the bone marrow is accomplished using a viral delivery system. In other embodiments, targeting of the bone marrow is accomplished using an LNP. In some embodiments, an epigenetic editor described herein, or component(s) thereof, are delivered to a host cell for transient expression, e.g., via a transient expression vector. Transient expression of the epigenetic editor or its component(s) may result in prolonged or permanent epigenetic modification of the target gene. For example, the epigenetic modification may be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.11, or 12 weeks or more; or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, after introduction of the epigenetic editor into the host cell. The epigenetic modification may be maintained after one or more mitotic

[0068] 67 12541428.1 and / or meiotic events of the host cell. In particular embodiments, the epigenetic modification is maintained across generations in offspring generated or derived from the host cell. VIII. Therapeutic Uses of Epigenetic Editors The present disclosure also provides methods for treating or preventing a condition in a subject, comprising administering to the subject an epigenetic editor or pharmaceutical composition as described herein. The epigenetic editor may effect an epigenetic modification of a target polynucleotide sequence in a target gene associated with a disease, condition, or disorder in the subject, thereby modulating expression of the target gene to treat or prevent the disease, condition, or disorder. In some embodiments, the epigenetic editor reduces the expression of the target gene to an extent sufficient to achieve a desired effect, e.g., a therapeutically relevant effect such as the prevention or treatment of the disease, condition, or disorder. In some embodiments, a subject is administered one or more systems for modulating (e.g., repressing or activating) expression of ZNF410, wherein the system comprises (1) the fusion protein(s) and, where relevant, guide polynucleotide(s) of an epigenetic editor as described herein, or (2) nucleic acid molecules encoding said fusion protein(s) and, where relevant, guide polynucleotide(s). “Treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a biological disorder and / or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and / or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment. In some embodiments, as compared with an equivalent untreated control, alleviating a symptom may involve reduction of the symptom by at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% as measured by any standard technique. In some embodiments, the subject may be a mammal, e.g., a human. In some embodiments, the subject is selected from a non-human primate such as chimpanzee, cynomolgus monkey, or macaque, and other apes and monkey species. In some embodiments, the human patient has a condition selected from a hemoglobinopathy, a beta-hemoglobinopathy, beta-thalassemia, and sickle cell disease. In some embodiments, the human patient has one or more sequences that differ from the canonical sequence of ZNF410 targeted by the guide polynucleotide.

[0069] 68 12541428.1 In some embodiments, a patient to be treated with an epigenetic editor of the present disclosure has received prior treatment for the condition to be treated (e.g., hemoglobinopathy, a beta-hemoglobinopathy, beta-thalassemia, sickle cell disease). In other embodiments, the patient has not received such prior treatment. In some embodiments, the patient has failed on a prior treatment for the condition (e.g., a prior beta-thalassemia treatment). An epigenetic editor of the present disclosure may be administered in a therapeutically effective amount to a patient with a condition described herein. “Therapeutically effective amount,” as used herein, refers to an amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated, and / or result in clinical endpoint(s) desired by healthcare professionals. An effective amount for therapy may be measured by its ability to stabilize disease progression and / or ameliorate symptoms in a patient, and preferably to reverse disease progression. The ability of an epigenetic editor of the present disclosure to reduce or silence ZNF410 expression, may be evaluated by in vitro assays, e.g., as described herein, as well as in suitable animal models that are predictive of the efficacy in humans. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case. An epigenetic editor of the present disclosure may be administered without additional therapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with an epigenetic editor of the present disclosure may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the additional therapeutic agent is any known in the art to treat a hemoglobinopathy (e.g., beta-thalassemia). In some embodiments, the additional therapeutic agent genetically disrupts the erythroid- specific enhancer of BCL11A. The epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure may be administered by any method accepted in the art (e.g., parenterally, intravenously, intradermally, or intramuscularly). XII. Definitions The term “nucleic acid” as used herein refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single- or

[0070] 69 12541428.1 double-strand form, and includes DNA and RNA. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, and are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which include natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modified versions which place new reactive groups such as amines, alcohols, thiols, carboxylates, alkylhalides, etc. Nucleic acids may contain known nucleotide analogs and / or modified backbone residues or linkages, which may be synthetic, naturally occurring, and non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are well known in the art, and may provide a nucleic acid molecule with enhanced cellular uptake, reduced immunogenicity, and / or increased stability in the presence of nucleases. As used herein, an “isolated” or “purified” nucleic acid molecule is a nucleic acid molecule that exists apart from its native environment. For example, an “isolated” or “purified” nucleic acid molecule (1) has been separated away from the nucleic acids of the genomic DNA or cellular RNA of its source of origin; and / or (2) does not occur in nature. In some embodiments, an “isolated” or “purified” nucleic acid molecule is a recombinant nucleic acid molecule. It will be understood that in addition to the specific proteins and nucleic acid molecules mentioned herein, the present disclosure also contemplates the use of variants, derivatives, homologs, and fragments thereof. A variant of any given sequence may have the specific sequence of residues (whether amino acid or nucleic acid residues) modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring sequence (in some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues). For specific proteins described herein (e.g., KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), the present disclosure also contemplates any of the protein’s naturally occurring forms, or variants or homologs that retain at least one of its endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98%, or 99% of its function as compared to the specific protein described). As used herein, a homologue of any polypeptide or nucleic acid sequence contemplated herein includes sequences having a certain homology with the wildtype amino acid and nucleic sequence. A homologous sequence may include a sequence, e.g. an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%< 93%,

[0071] 70 12541428.1 94%, 95%, 96%, 97%, 98%, or 99% identical to the subject sequence. The term “percent identical” in the context of amino acid or nucleotide sequences refers to the percent of residues in two sequences that are the same when aligned for maximum correspondence. In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence. Sequence identity may be measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence. The percent identity of two nucleotide or polypeptide sequences is determined by, e.g., BLAST® using default parameters (available at the U.S. National Library of Medicine’s National Center for Biotechnology Information website). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%) of the reference sequence. It will be understood that the numbering of the specific positions or residues in polypeptide sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. The term “modulate” or “alter” refers to a change in the quantity, degree, or extent of a function. For example, an epigenetic editor as described herein may modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or suppressing transcription of a gene operatively linked to the promoter sequence. As other examples, an epigenetic editor as described herein may block RNA polymerase from transcribing a gene, or may inhibit translation of an mRNA transcript. The terms “inhibit,” “repress,” “suppress,” “silence” and the like, when used in reference to an epigenetic editor or a component thereof as described herein, refers to decreasing or preventing the activity (e.g., transcription) of a nucleic acid sequence (e.g., a target gene) or

[0072] 71 12541428.1 protein relative to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or component thereof. The term may include partially or totally blocking activity, or preventing or delaying activity. The inhibited activity may be, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% less than that of a control, or may be, e.g., at least 1.5-fold, 2-fold, 3-fold, 4- fold, 5-fold, or 10-fold less than that of a control. The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” should be assumed to mean an acceptable error range for the particular value. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The recitation of a listing of elements herein includes any of the elements singly or

[0073] 72 12541428.1 in any combination. The recitation of an embodiment herein includes that embodiment as a single embodiment, or in combination with any other embodiment(s) herein. All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety. To the extent that references incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. In order that the present disclosure may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present disclosure in any manner. EXAMPLES Example 1: Identification of ZNF410 as a target Reactivation of HbF at the beta globin locus is a potentially valuable therapeutic approach (FIG.1A-1C). A screen identified ZNF410 amongst targets potentially useful for regulating HbF (FIG.2A-2B). GW mapping of ZNF410 binding in hematopoeitic has been performed (FIG.3). Experiments knocking out potential targets in CD34+ cells differentiated into erythroid cells and transplanted into mice established that loss of ZNF410 leads to reactivation of gamma globin, a component of HbF, likely through its specific interaction with CHD4, and that loss of ZNF410 both does not significantly disrupt differentiation into erythroid cells and upregulates HbF with no severe phenotypes (FIGs. 4A-5B). A model of ZNF410 modulation of the beta globin locus is shown in FIG.6. Example 2: Fusion Protein Design and Synthesis A number of bipartite and tripartite fusion proteins have been designed to be used singly or in combination, including: • dCas9-K, • dCas-3A • dCas-3L • m3ls-dCas9-ZIM3 • 3A:m3Ls-dCas9-ZIM3 • 3A:h3L-dCas9-K

[0074] 73 12541428.1 Example 3: Developing an engineered cell line that reports for ZNF410 expression and to perform the primary gRNA screens The human erythroleukemia cell line K-562 was engineered to express the tdTomato transgene from the ZNF410 locus (hereafter referred as to the K-562 ZNF410tdTomato cell line; FIG.7A). Genomic DNA from bulk-sorted, tdTomato-positive cells (FIG.7B) was analyzed to confirm on-targeted insertion of the transgene. This line was used for the arrayed screens (results shown in Table 4 below; target sites shown in FIG.8A and results in FIG. 8B-8C). The most-specific - according to in silico prediction - spCas9 gRNAs were tested for targeting the CGI of ZFN410 and an upstream CpG-reach region (n=150). A Lentiviral Vector (LV) library expressing the BFP marker together with the 550 gRNAs against ZNF410 or the 100 control guides (FIG.9) was prepared and used to transduce, at low Multiplicity of Infection (MOI), the K-562 ZNF410tdTomatocells. LV-positive cells were sorted according to BFP expression (FIG.10, representative flow cytometry dot blot at the bottom) and then transfected with plasmids encoding for (i) the triple ETR combination, (ii) the bi-partite ETR m3ls-dCas9-ZIM3, or (iii) the tri-partite ETR 3A:h3L-dCas9-K. Twenty one days after transfection, the BFP-positive, tdTomato-negative cells were sorted (see FIG. 11) and the complexity of the gRNAs was assessed by targeted deep-sequencing (>300x coverage) followed by bioinformatic analysis (by MAGeCK tool). K-562 ZNF410tdTomatocells transduced with the LV library but not transfected with the ETRs were used to control for input library complexity. Further validation of the top 5 gRNAs from each screen was performed in the K-562 ZNF410tdTomato cell line. Results are shown in Table 5 below and in FIGs.11-17. Table 4. Results of Arrayed Screen.

[0075] 74 12541428.1 Table 5. Results of Validation Screen. _

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[0096] 95 12541428.1 _ Example 4: gRNA validation in the HUDEP-2 cell line. The human immortalized erythroid progenitor cell line HUDEP-2 was used to validate candidate gRNAs, as this line: (i) expresses ZNF410, (ii) can be pushed towards terminal erythroid differentiation, and (iii) can reactivate HbF expression when ZNF410 or other gamma-globin regulators are genetically disabled. B2M epi-silencing reagents were used to optimize RNA delivery of the fusion protein components in this cell line, comparing nucleofection, LNPs and other lipid-based delivery approaches. An overview of the validation protocol is shown in FIG.18A. Top performing gRNAs were identified and transfected, individually or in combination, with mRNAs encoding for the fusion proteins in the HUDEP-2. Two weeks later, the cells were analyzed by flow cytometry and ddPCR to identify the gRNA(s) and / or combination(s) able to induce robust and durable reactivation of gamma-globin expression and ZNF410 silencing, respectively. Results are shown in FIG. 18B. Similar experiments are performed upon erythroid differentiation of the treated HUDEP-2. Genetic inactivation of ZNF410 or the BCL11A enhancer are used as positive controls in these experiments. Example 5: Validation of candidate gRNAs in primary human HSPCs. A schematic of the experimental flow used to assess the efficacy of ZNF410 epi- silencing in CD34+ cells (namely HSPCs) is shown in FIG.19. Mobilized peripheral blood (mPB) CD34+ cells from a healthy donor were co-transfected with the mRNA encoding for the tri-partite ETR and the indicated gRNAs (also in combination), and either grown in liquid

[0097] 96 12541428.1 culture for 21 days or plated in semi-solid media for Colony Forming Cell (CFC) assay for 15 days, when the cells were analyzed for the expression of ZNF410 (FIGs.20A and 20B) or beta- and gamma-globin (HBB and HBG, respectively; FIG.20C). Treatment with the tri- partite ETR and ZNF410 gRNAs resulted in ~90% reduction in the expression levels of this gene, both in liquid culture and in differentiated colonies. In colonies (>90% of cells are of erythroid origin), epi-silencing of ZNF410 was accompanied by a significant increase in fetal HBG expression (>2.5-fold increase) and a concomitant reduction in the adult HBB expression. These latter values were comparable to those obtained by disrupting ZNF410 by standard gene editing with CRISPR-Cas9. Successful candidates are used for epigenetic-silencing of ZNF410 in HSPCs for Beta- Thalassemia (Beta-Tal) patients. Example 6: In vivo validation of ZNF410 epigenetic silencing for fetal hemoglobin reactivation. Epigenetically silenced human HSPCs are transplanted into immunodeficient mice (NBSGW), and the composition of the human graft are monitored for up to 16 weeks by multiparametric flow cytometry analyses. Secondary transplants are then performed to assess the durability of epigenetic silencing (up 12 weeks follow up). Genetic disruption of ZNF410 is used as a positive control. Human cells are analyzed for ZNF410 expression and, for the erythroid lineages, also for the expression of the globin genes. Example 7: In vivo validation of ZNF410 epigenetic silencing for fetal hemoglobin reactivation. To further increase efficacy of gamma-globin reactivation, poly-functional editing is tested in CD34+ cells. ZNF410 is epigenetically silenced and the erythroid specific enhancer of BCL11A is genetically disrupted. In one experiment, catalytically active dCas9 is utilized for the polyfunctional editing. For targeting of BCL11A, guide RNAs with 20 nucleotides of complementary spacer sequence are used. For targeting of ZNF410, guide RNAs of 10 to 18 nucleotides of complementary spacer sequence (with and without mismatches) are used. Example 8: ZNF410 silencing and DNA methylation in HSPCs A dose escalation experiment targeting ZNF410 was performed in CD34+ cells from three different donors, both in liquid culture and in colonies. Both the tripartite and bipartite ETR architectures were compared, and ZNF410_177 was used as the guide RNA. Results

[0098] 97 12541428.1 were assayed by RT-qPCR. Targeted bisulfite sequencing of a 1kb region surrounding the CpG island in ZNF410 in was also performed. The three donors responded efficiently to ZNF410 epi-silencing, with the tripartite ETR consistently outperforming the bipartite ETR at all tested doses. Saturation of silencing was achieved around 1.6 µg of total RNA. Results are shown in FIG.21A. Methylation levels (shown in FIG.21B) correlate with silencing efficiency: the tripartite ETR at the highest dose showed the maximum methylation at the CpG island, which aligns with the robust silencing effect observed in that sample. Example 9: Testing gRNA combinations in K-562 reporter cell line 12 modified* gRNAs were used to conduct a first experiment in the K-562 reporter cell line with the tripartite ETR at non-saturating doses (600 ng of RNA: 300 ng tripartite ETR RNA and 300 ng gRNA). Results are shown in FIG.22. ZNF410_177 again performing strongly, along with a few other gRNAs that also show high efficiency. Notably, a few combinations—such as ZNF410_177 with ZNF410_48, ZNF410_177 with ZNF410_18 and ZNF410_177 with ZNF410_196—drastically increase silencing efficiency, reaching up to 80% ZNF410-negative cells with a very low dose of gRNA and the tripartite ETR. Adding a third gRNA did not lead to any further improvements, indicating that dual sgRNA combinations may be the optimal approach for enhancing silencing efficiency. *An example of the modification pattern in the modified gRNAs: Chemically synthesized sgRNA was obtained from IDT with the following chemical modifications, in which - RNA (ribose) is expressed as 'r_', for example rA, rU - 2' O-methyl RNA bases are expressed as 'm_' - Phosphorothioated 2'-O-methyl RNA bases are expressed as 'm *': Example 10: Testing gRNA combinations in HSPCs: semisolid media and three-phase liquid culture After the results in K-562 cells, validation was performed in primary human HSPCs grown in StemSpan + cytokines and small molecules used to expand the cells and keep them undifferentiated. Cells were treated with a non-saturating dose of tripartite ETR mRNA and

[0099] 98 12541428.1 pairs of gRNAs (600ng RNA: 300 ng tripartite RNA, 300 ng gRNA). RT-qPCR was used to measure ZNF410 expression in undifferentiated ETR-treated cells 7 days after treatment. An experimental schematic and results are shown in FIG.23A. The same gRNA combinations that worked well in K-562 cells produced robust silencing in HSPCs, confirming that these combinations can achieve high levels of ZNF410 epi-silencing in primary cells. Silencing was tested in a three-phase liquid culture (to determine persistence of silencing at full erythroid differentiation). Three-phase liquid culture for erythroid differentiation is known in the art, and the nucleofection and phases with timing are shown at the top of FIG.23B. Five days after ETR treatment (600ng RNA: 300 ng tripartite RNA, 300 ng gRNA), differentiation was inducted, and after 15 days of differentiation, RT-qPCR was performed to measure ZNF410 expression. Results are shown in the bar graph of FIG.23B. The data confirmed that epi-silencing was maintained at levels similar to the input undifferentiated cells, indicating that the silencing effect persists even through the complex process of erythroid differentiation. Example 11: Measuring expression of the γ-globin gene in differentiated cells with ZNF410 silenced with gRNA pairs The expression of the γ-globin gene was measured in the differentiated cells of Example 10. Results are shown in FIG.24. There was an upregulation of γ-globin in the treated cells compared to controls. Furthermore, the degree of γ-globin upregulation correlates with the level of epi-silencing achieved, providing strong evidence that ZNF410 silencing leads to γ-globin reactivation post-differentiation. Example 12: ZNF410 efficacy in vivo In order to assess the durability of ZNF410 epigenetic silencing in vivo and its effects on the engraftment and differentiation potential of long-term hematopoietic stem cells (HSCs), a xenotransplantation experiment was performed in immunodeficient NBSGW mice, as outlined in FIGs.25A-25B. Mobilized peripheral blood (mPB) CD34⁺ cells were nucleofected with mRNA encoding the ETR and chemically modified sgRNAs two days after thawing. Three days post-nucleofection (Day 5), cells were split into two arms: 1) In vivo: 500,000 cells were transplanted per single busulfan-conditioned NBSGW mouse. Mice were monitored for 16 weeks prior to bone marrow harvest and analysis.2) In vitro: Remaining cells were cultured under liquid conditions until either Day 9 (Liquid culture) or Day 23 (erythroid differentiation), to evaluate silencing efficiency. The sgRNA used for ZNF410

[0100] 99 12541428.1 knockout (Bauer2) corresponded to “sg_2” from Vinjamur et al., Nat Genet 2021 (doi.org / 10.1038 / s41588-021-00843-w), and was included as a gene editing control. In addition to xenotransplantation, a fraction of the treated CD34⁺ cells was cultured ex vivo to measure the efficiency of ZNF410 silencing and test clonogenic potential through colony-forming assays as outlined in FIGs.26A-26C. Mobilized peripheral blood (mPB) CD34+ cells were nucleofected with mRNA encoding the Tripartite ETR targeting ZNF410, or with Cas9 and a knockout sgRNA (according to the scheme in FIG.25A). Cells were cultured in either liquid conditions (Day 9), erythroid differentiation media (Day 23), or plated in methylcellulose for colony-forming unit (CFU) assays. Overall, the results shown in FIGs.26A-26C demonstrate that the Tripartite ETR effectively silences ZNF410 and downregulates its downstream target CHD4, while preserving the clonogenic potential of human HSPCs. To assess the impact of ZNF410 epigenetic silencing on human hematopoietic engraftment and lineage differentiation, the bone marrow of transplanted NBSGW mice was analyzed 16 weeks post-infusion. mPB CD34⁺ HSPCs from a single donor were edited and transplanted into NBSGW mice as outlined in FIGs.25A-25B. Sixteen weeks post- transplantation, bone marrow was harvested to evaluate human engraftment and lineage output. As shown in FIGs.27A-27E, these data show that ZNF410 epi-silencing did not affect the composition of the human graft, supporting the tolerability of the treatment. To evaluate the long-term stability of ZNF410 epigenetic silencing and its functional consequences in vivo, we analyzed gene expression and globin isoform production in distinct human hematopoietic populations sorted from the bone marrow 16 weeks after transplantation. CD34⁺ HSPCs edited with ZNF410-targeting ETRs or Cas9 were transplanted into NBSGW mice. After 16 weeks, human bone marrow cells were sorted into distinct populations and analyzed by RT-qPCR. As shown in FIGs.28A-28E, these data show that: (1) ZNF410 epi-silencing occurred in the more primitive HSC compartment and that it fully resisted to hematopoietic differentiation (>90% ZNF410 reduction in all hematopoietic lineages analyzed); (2) ZNF410 epi-silencing resulted in down regulation of CHD4, predominantly in erythroid cells (CD235a+ cells); (3) ZNF410 epi-silencing resulted in significant reactivation of γ-globin in erythroid cells. To identify the most effective and specific configuration of architectures and gRNAs for ZNF410 epigenetic silencing, the best-performing gRNA combinations were systematically compared in combination with two distinct ETR platforms: the Tripartite-ETR and CHARM architectures.

[0101] 100 12541428.1 Silencing efficiency of the two platforms were first compared in both K562 reporter cells and primary CD34⁺ HSPCs (FIGs.29A-29B). Overall, these data indicate that Tripartite ETR and CHARM platforms achieve comparable efficiency in epi-silencing of ZNF410. RNA-seq was then performed on sorted K562 ZNF410-silenced cells to evaluate the transcriptome-wide specificity of the tested configurations (FIGs.30A-30C). These findings demonstrate that both ETR designs enable potent and highly specific repression of ZNF410, with minimal off-target transcriptional activity.

[0102] 101 12541428.1 SEQUENCES The SEQ ID NOs (SEQ) of nucleotide (nt) and amino acid (aa) sequences described in the present disclosure are listed below.

[0103] 102 12541428.1

[0104] 103 12541428.1

[0105] 104 12541428.1

[0106] 105 12541428.1

[0107] 106 12541428.1

[0108] 107 12541428.1

[0109] 108 12541428.1

[0110] 109 12541428.1

[0111] 110 12541428.1

[0112] 111 12541428.1

[0113] 112 12541428.1

[0114] 113 12541428.1

[0115] 114 12541428.1

[0116] 115 12541428.1

[0117] 116 12541428.1

[0118] 117 12541428.1

[0119] 118 12541428.1

[0120] 119 12541428.1

[0121] 120 12541428.1

[0122] 121 12541428.1

[0123] 122 12541428.1

[0124] 123 12541428.1

[0125] 124 12541428.1

[0126] 125 12541428.1

[0127] 126 12541428.1

[0128] 127 12541428.1

[0129] 128 12541428.1

[0130] 129 12541428.1

[0131] 130 12541428.1

[0132] 131 12541428.1

[0133] 132 12541428.1

[0134] 133 12541428.1

[0135] 134 12541428.1

[0136] 135 12541428.1

[0137] 136 12541428.1

[0138] 137 12541428.1

[0139] 138 12541428.1

[0140] 139 12541428.1

[0141] 140 12541428.1

[0142] 141 12541428.1

[0143] 142 12541428.1

[0144] 143 12541428.1

[0145] 144 12541428.1

[0146] 145 12541428.1

[0147] 146 12541428.1

[0148] 147 12541428.1

[0149] 148 12541428.1

[0150] 149 12541428.1

[0151] 150 12541428.1

[0152] 151 12541428.1

[0153] 152 12541428.1

[0154] 153 12541428.1

[0155] 154 12541428.1

[0156] 155 12541428.1

[0157] 156 12541428.1

[0158] 157 12541428.1

[0159] 158 12541428.1

[0160] 159 12541428.1

[0161] 160 12541428.1

[0162] 161 12541428.1

[0163] 162 12541428.1

[0164] 163 12541428.1

[0165] 164 12541428.1

[0166] 165 12541428.1

[0167] 166 12541428.1

[0168] 167 12541428.1

[0169] 168 12541428.1

[0170] 169 12541428.1

[0171] 170 12541428.1

[0172] 171 12541428.1

[0173] 172 12541428.1

[0174] 173 12541428.1

[0175] 174 12541428.1

[0176] 175 12541428.1

[0177] 176 12541428.1

[0178] 177 12541428.1

[0179] 178 12541428.1

[0180] 179 12541428.1

[0181] 180 12541428.1

[0182] 181 12541428.1

[0183] 182 12541428.1

[0184] 183 12541428.1

[0185] 184 12541428.1

[0186] 185 12541428.1

[0187] 186 12541428.1

[0188] 187 12541428.1

[0189] 188 12541428.1

[0190] 189 12541428.1

[0191] 190 12541428.1

[0192] 191 12541428.1

[0193] 192 12541428.1

[0194] 193 12541428.1

Claims

CLAIMS What is claimed is:

1. A system for repressing transcription of a human ZNF410 gene in a human cell, optionally a human erythroid cell or a precursor cell to a human erythroid cell, comprising a) one or more fusion proteins that collectively comprise a DNA methyltransferase (DNMT) domain and / or a domain that recruits a DNMT (recruiter domain), optionally wherein the DNMT domain and / or the recruiter domain comprise a DNMT3A domain and / or a DNMT3L domain, and optionally wherein the recruiter DNMT is a DNMT3L domain and the recruited DNMT is a DNMT3A domain, and a transcriptional repressor domain, each domain being linked to a DNA-binding domain that binds to a target region in the human ZNF410 gene, wherein the target region comprises one or more sequences selected from SEQ ID NOs: 766-1355; or b) one or more nucleic acid molecules encoding the one or more fusion proteins, wherein the system does not generate a DNA break in the ZNF410 gene.

2. The system of claim 1, wherein the DNA-binding domain comprises a catalytically deactivated CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain.

3. The system of claim 2, wherein the DNA-binding domain comprises a dCas9 domain and the system further comprises (i) one or more guide RNAs comprising a sequence that binds to any one of SEQ ID NOs: 766-1355, or (ii) nucleic acid molecules coding for the one or more guide RNAs.

4. A system for repressing transcription of a human ZNF410 gene in a human cell, optionally a human erythroid cell or a precursor cell to a human erythroid cell, comprising a) a fusion protein that comprises a DNMT3A domain, a DNMT3L domain, a DNA-binding domain that binds to a target region in the human ZNF410 gene, and a transcriptional repressor domain; or b) a nucleic acid molecule encoding the fusion protein,194 12541428.1wherein the system does not generate a DNA break in the ZNF410 gene.

5. The system of claim 4, wherein the DNA-binding domain comprises a dCas domain, a ZFP domain, or a TALE domain.

6. The system of claim 5, wherein the DNA-binding domain comprises a dCas9 domain and the system further comprises (i) one or more guide RNAs comprising a sequence that binds to any one of SEQ ID NOs: 766-1355, or (ii) nucleic acid molecules coding for the one or more guide RNAs.

7. The system of any one of claims 2, 3, 5, and 6, wherein the dCas domain comprises a dCas9 sequence, optionally a sequence with at least 90% identity to SEQ ID NO: 15 or 16.

8. The system of any one of claims 1-7, wherein the DNA-binding domain binds to a target sequence selected from any one of SEQ ID NO: 766-1355.

9. The system of any one of claims 1-8, wherein the DNMT3A domain comprises a sequence with at least 90% identity to SEQ ID NO: 577 or 578.

10. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 581-584.

11. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence with at least 95% identity to a sequence selected from SEQ ID NOs: 581-584.

12. The system of any one of claims 1-3 and 5-8, wherein the DNMT domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 579 and 585.

13. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 36-573.

14. The system of any one of claims 1-13, wherein the transcriptional repressor domain comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627.195 12541428.

115. The system of claim 14, wherein the KRAB domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 92, 119, 248, and 258 16. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 574 or 575.

17. The system of any one of claims 1-12, wherein the transcriptional repressor domain is derived from KAP1, MECP2, HP1a / CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.

18. The system of any one of claims 1-17, wherein the system comprises a) a fusion protein comprising the DNMT3A domain, the DNMT3L domain, the transcriptional repressor domain, and the DNA-binding domain, optionally wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and optionally wherein the DNA-binding domain is a catalytically deactivated CRISPR Cas domain, a ZFP domain, or a TELE domain; or b) a nucleic acid molecule encoding the fusion protein.

19. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, the DNMT3A domain, a first peptide linker, the DNMT3L domain, a second peptide linker, the DNA-binding domain, a third peptide linker, and the transcriptional repressor domain.

20. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, a first nuclear localization signal (NLS), the DNA-binding domain, a second NLS, the third peptide linker, and the transcriptional repressor domain.

21. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, a first nuclear localization signal (NLS), the DNMT3A domain, the first peptide196 12541428.1linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and a second NLS.

22. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second nuclear localization signals (NLSs), the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and third and fourth NLSs.

23. The system of any one of claims 18-22, wherein the transcriptional repressor domain is a KRAB domain, optionally a human KOX1, ZFP28, ZN627, or ZIM3 KRAB domain.

24. The system of any one of claims 19-23, wherein one or both of the second and third peptide linkers are XTEN linkers, optionally selected from XTEN80 and XTEN16, and further optionally wherein the second peptide linker is XTEN80, and the third peptide linker is XTEN16.

25. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN16 peptide linker, and a human KOX1 KRAB domain.

26. The system of claim 25, wherein the fusion protein comprises SEQ ID NO: 734 or a sequence at least 90% identical thereto.

27. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a ZFP or TALE domain, a second NLS, an XTEN16 linker, and a human KOX1 KRAB domain.

28. The system of claim 27, wherein the fusion protein comprises SEQ ID NO: 735 or a sequence at least 90% identical thereto.197 12541428.

129. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.

30. The system of claim 29, wherein the fusion protein comprises SEQ ID NO: 1366 or a sequence at least 90% identical thereto.

31. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP or TALE domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.

32. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.

33. The system of claim 32, wherein the fusion protein comprises SEQ ID NO: 1367 or a sequence at least 90% identical thereto.

34. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP or TALE domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.

35. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.

36. The system of claim 35, wherein the fusion protein comprises SEQ ID NO: 1368 or a sequence at least 90% identical thereto.198 12541428.

137. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP or TALE domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.

38. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.

39. The system of claim 38, wherein the fusion protein comprises SEQ ID NO: 1369 or a sequence at least 90% identical thereto.

40. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C- terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.

41. The system of any one of claims 20-40, wherein at least one of the NLSs is an SV40 NLS.

42. The system of any one of claims 1-3 and 7-17, wherein the system comprises: a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting the DNMT3A domain, a second fusion protein comprising a second DNA-binding domain and comprising or recruiting the DNMT3L domain, and a third fusion protein comprising a third DNA-binding domain and comprising or recruiting the transcriptional repressor domain; or b) one or more nucleic acid molecules encoding the fusion proteins.

43. A human cell comprising the system of any one of claims 1-42, or progeny of the cell, optionally wherein the cell is an erythroid cell or a precursor cell to a human erythroid cell.199 12541428.

144. A human cell modified by the system of any one of claims 1-42, or progeny of the cell, optionally wherein the cell is an erythroid cell or a precursor cell to a human erythroid cell, optionally wherein the cell was modified ex vivo.

45. A pharmaceutical composition comprising the system of any one of claims 1-42 and a pharmaceutically acceptable excipient, optionally wherein the composition comprises lipid nanoparticles (LNPs) comprising the system, and / or the DNA-binding domain is a dCas domain and the LNPs further comprise one or more gRNAs.

46. A pharmaceutical composition comprising human cells of claim 43 or 44 and a pharmaceutically acceptable excipient.

47. A method of treating a patient in need thereof, comprising administering the system of any one of claims 1-42, human cells of claim 43 or 44, or the pharmaceutical composition of claim 45 or 46 to the patient.

48. The method of claim 47, wherein the patient has a beta-hemoglobinopathy.

49. The method of claim 48, wherein the patient has Beta-thalassemia or sickle cell disease.

50. The system of any one of claims 1-42, human cells of claim 43 or 44, or the pharmaceutical composition of claim 45 or 46, for use in treating a patient in need thereof, optionally in the method of any one of claims 47-49.

51. Use of the system of any one of claims 1-42 or the human cells of claim 43 or 44 in the manufacture of a medicament for treating a patient in need thereof, optionally in the method of claim 47-49.

52. The system of claim 1, wherein the system comprises a DNMT3L domain and a transcriptional repressor domain.200 12541428.

153. The system of claim 53, wherein the transcriptional repressor domain is a KRAB domain.

54. The system of claim 1, wherein the DNA-binding domain comprises a catalytically active Cas domain or a nickase.

55. The system of claim 54, wherein the system further comprises (i) one or more guide RNAs comprising a sequence that binds to any one of SEQ ID NOs: 766-1355, (ii) one or more guide RNAs targeting BCL11A, and / or (iii) nucleic acid molecules coding for the one or more guide RNAs of (i) and / or (ii).201 12541428.1

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