Compositions and Methods for Epigenetic Regulation of PCSK9 Expression

Abstract

This application relates to compositions and methods comprising epigenetic editors for the epigenetic modification of PCSK9, as well as nucleic acids and vectors encoding the same. Also disclosed are cells epigenetically modified by an epigenetic editor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 119(e) to U.S. Provisional Patent Application No. 63 / 337,164, entitled "COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF PCSK9 EXPRESSION," filed May 1, 2022; U.S. Provisional Patent Application No. 63 / 337,167, entitled "COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF PCSK9 EXPRESSION," filed May 1, 2022; and U.S. Provisional Patent Application No. 63 / 355,083, entitled "COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF PCSK9 EXPRESSION," filed June 23, 2022, the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (C169870034WO00-SEQ-AXW.xml, size: 1,831,551 bytes, and creation date: May 1, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Genome editing has been considered a promising therapeutic approach for the treatment of genetic diseases for over a decade. However, manipulation at the DNA level using conventional gene editors remains risky given the potential for unwanted double-strand DNA breaks, uneven repair (including large and small insertions and deletions at the intended site), and toxicity. In contrast, targeted epigenetic modifications offer the ability to alter gene expression without double-strand break-induced genotoxicity.

[0004] One promising candidate for epigenetic silencing is the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene. PCSK9 is an important target in the treatment of cardiovascular disease, a leading cause of death worldwide (Berberich et al., Nature Rev Cardiol. (2019) 16(1):9-20). The human PCSK9 gene, located on chromosome 1, shares approximately 94% and 80% homology with its cynomolgus monkey and mouse counterparts, respectively. The gene contains a CpG island in the promoter region, making it distal to other genes and cis-regulatory features. PCSK9 protein is primarily produced by the liver.

[0005] In humans, PCSK9 plays an important role in regulating circulating levels of low-density lipoprotein (LDL) particles as a result of binding to the LDL receptor (LDLR). LDLR reduces circulating LDL particle concentrations by mediating their endocytosis and degradation in cells. In the absence of PCSK9 or when the interaction between PCSK9 and LDLR is blocked, the recycling rate of LDLR to the cell surface increases, and the recycled LDLR protein continues to remove LDL particles from the extracellular fluid (Tombling et al., Atherosclerosis (2021) 330:52-60). In contrast, when endocytosed LDLR is bound to PCSK9, the LDLR is degraded along with its passenger LDL particles. Clinical and genetic studies have established that circulating LDL contributes to atherosclerotic cardiovascular disease (Ference et al., Eur Heart J (2017) 38:2459-72). In addition, loss-of-function mutations in PCSK9 are associated with low LDL levels (Zhao et al., Am J Hum Genet. (2006) 79(3):514-23). ​​Genetic or pharmacological reduction of PCSK9 reduces cardiovascular events (Ference et al., N Engl J Med. (2016) 375(22):2144-53, Sabatine et al., N Engl J Med. (2017) 376(18):1713-22). Reducing PCSK9 expression may help increase LDLR recycling, resulting in lower blood LDL particle concentrations. Summary of the Invention [Problem to be solved by the invention]

[0006] Given the pivotal role of PCSK9 in the pathogenesis of hypercholesterolemia and cardiovascular disease, new and improved therapeutic approaches that target PCSK9 expression are needed.

[0007] The present disclosure provides systems and compositions for epigenetic modification (herein "epigenetic editors" or "epigenetic editing systems") and methods using same to generate epigenetic modifications in PCSK9, including host cells and organisms. [Means for solving the problem]

[0008] In some embodiments, the present disclosure provides a system for inhibiting transcription of the human PCSK9 gene in a human cell, optionally a human hepatocyte, comprising: a) a DNA methyltransferase (DNMT) domain and / or a domain that recruits DNMTs, optionally wherein the DNMT domain and / or recruiter domain comprises a DNMT3A domain and / or a DNMT3L domain, and optionally wherein the recruited DNMT is DNMT3A; and Transcriptional repressor domain collectively including, one or more fusion proteins, each of which is linked to a DNA-binding domain that binds to a target region in the human PCSK9 gene; or b) one or more nucleic acid molecules encoding one or more fusion proteins The present invention provides a system including:

[0009] In some embodiments, the DNA-binding domain binds to a target sequence in SEQ ID NO: 1488 or 1489. In certain embodiments, the DNA-binding domain targets the fusion protein to one or more sequences in the PCSK9 gene selected from SEQ ID NOs: 700-747 and 1036-1261.

[0010] In some embodiments, the DNA-binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain. For example, the DNA-binding domain may comprise a dCas9 domain, and the system may further comprise (i) one or more guide RNAs (e.g., comprising any one of SEQ ID NOs: 1262-1487), or (ii) a nucleic acid molecule encoding the one or more guide RNAs. In certain embodiments, the dCas domain comprises a sequence having at least 90% identity to a dCas9 sequence, e.g., SEQ ID NO: 12 or 13.

[0011] In some embodiments, the fusion protein comprises a dead CRISPR Cas (dCas) domain, and the system comprises one or more PCSK9-binding guide RNAs (gRNAs) provided herein. In some embodiments, the system comprises a single gRNA. In some embodiments, the system comprises two gRNAs. In some embodiments, the system comprises three gRNAs. In some embodiments, the system comprises four gRNAs. In some embodiments, the system comprises five or more gRNAs. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 2. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 7. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 8. In some embodiments, the system comprises an sgRNA selected from the gRNAs provided in Table 10. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 12.

[0012] In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA009 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA009. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA003 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA003. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA093 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA093. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA011 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA011. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA007 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA007. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA077 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA077. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA113 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA113. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA004 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA004. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA008 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA008. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA012 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA012. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA111 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA111. In some embodiments, the system includes a gRNA that includes a gRNA targeting sequence for gRNA005 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA005.In some embodiments, the system includes a gRNA that includes the gRNA targeting sequence of gRNA013 in Table 10, or a gRNA that binds to the same target domain sequence as gRNA013.

[0013] In some embodiments, the system includes fusion protein 9 variant 1 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 9 variant 2 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 10 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 11 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 12 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 13 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 14 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 15 (Example 12) and gRNA g041. In some embodiments, the system includes fusion protein 9 variant 1 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 9 variant 2 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 10 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 11 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 12 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 13 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 14 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 15 (Example 12) and gRNA g049. In some embodiments, the system includes fusion protein 9 variant 1 (Example 12) and gRNA g041 and g049. In some embodiments, the system includes fusion protein 9 variant 2 (Example 12) and gRNA g041 and g049.In some embodiments, the system includes fusion protein 10 (Example 12) and gRNAs g041 and g049. In some embodiments, the system includes fusion protein 11 (Example 12) and gRNAs g041 and g049. In some embodiments, the system includes fusion protein 12 (Example 12) and gRNAs g041 and g049. In some embodiments, the system includes fusion protein 13 (Example 12) and gRNAs g041 and g049. In some embodiments, the system includes fusion protein 14 (Example 12) and gRNAs g041 and g049. In some embodiments, the system includes fusion protein 15 (Example 12) and gRNAs g041 and g049.

[0014] In some embodiments, the DNA-binding domain comprises a ZFP domain that targets a nucleotide sequence selected from SEQ ID NOs: 700 to 747. In certain embodiments, the ZFP domain comprises the amino acid sequences F1 to F6, in order, of any one of ZF001 to ZF048 shown in Table 1.

[0015] In some embodiments, the DNMT3A domain comprises a sequence having at least 90% identity to SEQ ID NO: 574 or 575.

[0016] The DNMT3L domain can comprise, for example, a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 578-581. In some embodiments, the DNMT3L domain comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 582-603. In some embodiments, the DNMT3L domain comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 601-603.

[0017] In some embodiments, the transcriptional repressor domain comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 33-570. In certain embodiments, the transcriptional repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. The KRAB domain can comprise, for example, a sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 89, 116, 245, and 255. In some embodiments, the transcriptional repressor domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, optionally comprising the amino acid sequence of SEQ ID NO: 571 or 572. In certain embodiments, the transcriptional repressor domain is derived from KAP1, MECP2, HP1a / CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.

[0018] In some embodiments, the system comprises: a) comprising a DNMT3A domain, a DNMT3L domain, a transcriptional repressor domain, and a DNA binding domain; optionally, one or both of the DNMT3A domain and the DNMT3L domain are human; Optionally, the DNA binding domain comprises a dead CRISPR Cas domain or a ZFP domain. a fusion protein, or b) a nucleic acid molecule encoding the fusion protein Includes:

[0019] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA-binding domain, a third peptide linker, and a transcriptional repressor domain. For example, the fusion protein may comprise, from N-terminus to C-terminus, a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a first nuclear localization signal (NLS), a DNA-binding domain, a second NLS, a third peptide linker, and a transcriptional repressor domain. The fusion protein may comprise, from N-terminus to C-terminus, a first NLS, a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA-binding domain, a third peptide linker, a transcriptional repressor domain, and a second NLS. The fusion protein may comprise, from N-terminus to C-terminus, a first and second NLS, a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA-binding domain, a third peptide linker, a transcriptional repressor domain, and a third and fourth NLS. In certain embodiments, the transcriptional repressor domain is a KRAB domain, e.g., a human KOX1, ZFP28, ZN627, or ZIM3 KRAB domain. In certain embodiments, one or both of the second and third peptide linkers is an XTEN linker, which may be selected from XTEN80 (e.g., SEQ ID NO: 643) and XTEN16 (e.g., SEQ ID NO: 638), e.g., the second peptide linker is XTEN80 and the third peptide linker is XTEN16.

[0020] In some embodiments, the fusion protein can comprise, 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 certain embodiments, the fusion protein comprises SEQ ID NO: 658 or a sequence at least 90% identical thereto. In certain embodiments, the fusion protein comprises SEQ ID NO: 1495 or a sequence at least 90% identical thereto.

[0021] 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 ZFP domain, a second NLS, an XTEN16 linker, and a human KOX1 KRAB domain. In certain embodiments, the fusion protein comprises SEQ ID NO: 659, or a sequence at least 90% identical thereto, and optionally, the ZFP comprises, in order, the F1-F6 amino acid sequence of any one of ZF001-ZF048 set forth in Table 1. In certain embodiments, the fusion protein comprises SEQ ID NO: 1496, or a sequence at least 90% identical thereto, and optionally, the ZFP comprises, in order, the F1-F6 amino acid sequence of any one of ZF001-ZF048 set forth in Table 1.

[0022] In some embodiments, the fusion protein comprises, from N- to C-terminus, a first and second NLS, 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 a third and fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 660, or a sequence at least 90% identical thereto.

[0023] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and second NLS, 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 KOX1 KRAB domain, and a third and fourth NLS.

[0024] In some embodiments, the fusion protein comprises, from N- to C-terminus, a first and second NLS, 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 a third and fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 661, or a sequence at least 90% identical thereto.

[0025] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and second NLS, 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 ZFP28 KRAB domain, and a third and fourth NLS.

[0026] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and second NLS, 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 a third and fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 662, or a sequence at least 90% identical thereto.

[0027] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and second NLS, 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 ZN627 KRAB domain, and a third and fourth NLS.

[0028] In some embodiments, the fusion protein comprises, from N- to C-terminus, a first and second NLS, 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 a third and fourth NLS. In certain embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 663, or a sequence at least 90% identical thereto.

[0029] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and second NLS, 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 a third and fourth NLS.

[0030] In some embodiments, at least one of the NLSs in the fusion proteins described herein is an SV40 NLS (eg, SEQ ID NO: 644).

[0031] In some embodiments, the system comprises: a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting a DNMT3A domain; a second fusion protein comprising a second DNA-binding domain and comprising or recruiting a DNMT3L domain; a third fusion protein that includes a third DNA-binding domain and that includes or recruits a transcriptional repressor domain; or b) one or more nucleic acid molecules encoding the fusion protein Includes:

[0032] The present disclosure also provides a human cell, or a progeny of the cell, comprising the system described herein. In some embodiments, the cell is a hepatocyte.

[0033] The present disclosure also provides pharmaceutical compositions comprising the system described herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises a lipid nanoparticle (LNP) comprising the system, and / or the DNA-binding domain is a dCas domain, and the LNP further comprises one or more gRNAs.

[0034] The present disclosure also provides methods of treating a patient in need thereof, the method comprising administering (e.g., intravenously) to the patient a system or pharmaceutical composition described herein. In some embodiments, the patient has heart disease; elevated low-density lipoprotein cholesterol (LDL-C) or hypercholesterolemia; is at risk for developing myocardial infarction, stroke, or unstable angina; and / or has primary hyperlipidemia (e.g., heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH)).

[0035] The present disclosure also provides a system or pharmaceutical composition described herein for use in treating a patient in need thereof, e.g., in a method described herein.

[0036] The present disclosure also provides for the use of the systems described herein in the manufacture of a medicament for treating a patient in need thereof, e.g., for treatment in the methods described herein.

[0037] The present disclosure also provides articles and kits that include the systems described herein.

[0038] Other features, objects, and advantages of the disclosed methods and compositions will become apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and specific aspects of the disclosed methods and compositions, is provided for purposes of illustration only, and not limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows the predicted binding positions of ZF proteins and computationally designed gRNAs on the PCSK9 gene. [Figure 2] A scatter plot showing relative PCSK9 expression (y-axis) in cells treated with CRISPR-off(DNMT3A-3L-dCas9-KRAB) at day 7. The genomic distance from the gRNA target site to the PCSK9 TSS is shown on the x-axis. [Figure 3] FIG. 1 shows the overlap of the top 40 gRNAs with the PCSK9 gene. [Figure 4A] 1 is a bar graph showing the levels of secreted PCSK9 at days 7 and 28 after treatment with the indicated gRNAs. The dashed line indicates silencing achieved by wild-type (WT) Cas9. [Figure 4B] Scatter plot showing the correlation between PCSK9 mRNA expression and PCSK9 protein secretion in cells after treatment with gRNA. CRISPRi(dCas9-KRAB) represents the dCas9-KRAB fusion protein. [Figure 5] 1 is a line graph showing silencing of PCSK9 after treatment with CRISPRi (dCas9-KRAB), CRISPR-off (DNMT3A-3L-dCas9-KRAB), and the indicated gRNAs. [Figure 6] 1 is a bar graph showing PCSK9 secretion in cells treated with CRISPRoff and simvastatin compared to cells treated with the CRISPRoff system alone. [Figure 7]1 is a bar graph showing the reduction of PCSK9 secretion in Huh7 hepatoma cells treated with CRISPRoff and a given gRNA. [Figure 8] Scatter plot showing the activity and toxicity of 247 PCSK9-targeting ZF proteins. Relative PCSK9 expression is shown on the x-axis, and the corresponding cell count compared to pUC and off-target controls is shown on the y-axis. The diagonal line represents a 1:1 correlation between relative PCSK9 expression and cell count. [Figure 9] Scatter plot showing relative PCSK9 expression (y-axis) by cells treated with the ZF-off(DNMT3A-3L-ZF-KRAB) construct and the corresponding targeted genomic distance (x-axis) relative to the PCSK9 transcription start site (TSS). [Figure 10]

[0023] Figure 1 shows the entire human PCSK9 locus, flanked by 35.5 kb and 7 kb of the upstream and downstream genomic regions (67.5 kb), respectively, introduced into and expressed in transgenic mice. The transgenic mouse line expresses human PCSK9 under the control of its own (human) endogenous promoter. [Figure 11] Figure 11A shows a schematic of a fusion protein construct with a variant NLS configuration, and Figure 11B shows a schematic of an additional fusion protein construct with a variant KRAB domain. [Figure 12A] 12A-12B are graphs showing the percentage of PCSK9 protein levels measured after treatment with fusion protein constructs with various NLS configurations in HeLa cells using 6.25 ng of RNA (FIG. 12A) or 2.5 ng of RNA (FIG. 12B). Human and mouse DNMT3L sequences are indicated as h3L and m3L, respectively. [Figure 12B]12A-12B are graphs showing the percentage of PCSK9 protein levels measured after treatment with fusion protein constructs with various NLS configurations in HeLa cells using 6.25 ng of RNA (FIG. 12A) or 2.5 ng of RNA (FIG. 12B). Human and mouse DNMT3L sequences are indicated as h3L and m3L, respectively. [Figure 13] 10 is a graph showing that a construct with a 2X NLS is 3-fold more efficient than CRISPR-off in silencing mPcsk9 in Hepa1-6 cells. [Figure 14A] FIG. 14A is a graph showing that constructs with 2X NLS are more efficient than CRISPR-off in silencing mPcsk9 in Huh7 cells. [Figure 14B] Figures 14B-14C are graphs showing that constructs with 2X NLS are also more efficient than CRISPR-off in silencing mPcsk9 in Huh7 cells at different doses at both day 5 (Figure 14B) and day 15 (Figure 14C). [Figure 14C] Figures 14B-14C are graphs showing that constructs with 2X NLS are also more efficient than CRISPR-off in silencing mPcsk9 in Huh7 cells at different doses at both day 5 (Figure 14B) and day 15 (Figure 14C). [Figure 15]

[0023] Figure 14 is a graph showing that in a CRISPR-off-like format in which dCas9 is replaced with zinc fingers in Huh7 cells, 2X NLS provides improvement across multiple ZFs. [Figure 16] 1 is a set of graphs showing that methylation of the CLTA4 promoter with bacterial DNMT proteins can induce epigenetic silencing of the locus. [Figure 17]1 is a set of graphs showing the methylation profile at the VIM3 locus of cells treated with different constructs harboring bacterial DNA methyltransferase fused to dCas9 at day 30. Samples treated with M. SssI are 20% methylated. [Figure 18] 10 is a set of graphs showing methylation profiles by hybridization capture at the CLTA locus of cells comparing M. SssI with mouse DNMT3A / 3L in dCas9 fusions at day 29. [Figure 19A] 19A and 19B are a set of graphs showing alternative KRAB domains tested for episilencing activity against CRISPR-off using 0.5 ng of effector DNA with CLTA-GFP as a marker (FIG. 19A), 3 ng of effector DNA with GFP as a marker (FIG. 19B), and 0.5 ng of effector DNA with GFP as a marker (FIG. 19C). [Figure 19B] 19A and 19B are a set of graphs showing alternative KRAB domains tested for episilencing activity against CRISPR-off using 0.5 ng of effector DNA with CLTA-GFP as a marker (FIG. 19A), 3 ng of effector DNA with GFP as a marker (FIG. 19B), and 0.5 ng of effector DNA with GFP as a marker (FIG. 19C). [Figure 19C] 19A and 19B are a set of graphs showing alternative KRAB domains tested for episilencing activity against CRISPR-off using 0.5 ng of effector DNA with CLTA-GFP as a marker (FIG. 19A), 3 ng of effector DNA with GFP as a marker (FIG. 19B), and 0.5 ng of effector DNA with GFP as a marker (FIG. 19C). [Figure 19D] FIG. 19D shows the results after 30 days using various nanogram amounts of effector DNA. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure provides an epigenetic editor for regulating the expression of the PCSK9 gene. By altering the expression of PCSK9, the systems, compositions, and methods described herein can be used to treat conditions such as hypercholesterolemia (e.g., heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), familial hypercholesterolemia (HF), or established atherosclerotic cardiovascular disease (ASCVD)), or renal insufficiency (RI). Unless otherwise indicated, "PCSK9" herein refers to human PCSK9. The human PCSK9 gene sequence can be found in Ensembl Accession No. ENSG00000169174. This epigenetic editor has several advantages over other genome manipulation methods, including reversibility, reduced risk of translocations, and sustained, heritable silencing.

[0041] In some embodiments, the region of the human PCSK9 gene targeted for epigenetic modulation is approximately 2 kb in length, approximately ±1 kb from the PCSK9 TSS. In certain embodiments, this region has the nucleotide sequence of SEQ ID NO: 1488. In some embodiments, the targeted PCSK9 region is approximately 1069 bp in length, approximately ±500 bp from the PCSK9 TSS. In certain embodiments, the targeted region has the nucleotide sequence of SEQ ID NO: 1489. The PCSK9 TSS is located at #chr1:55039548 in genomic GRCh38.

[0042] In some embodiments, the epigenetic editors described herein comprise one or more fusion proteins, each comprising a DNA-binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments, when the DNA-binding domain is a polynucleotide-guided DNA-binding domain, the epigenetic editor may further comprise one or more guide polynucleotides. The DNA-binding domains, effector domains, and guide polynucleotides of the epigenetic editors described herein may be selected in any functional combination from, for example, those described below.

[0043] The epigenetic editors described herein may be transiently expressed in a host cell or integrated into the genome of the host cell, and such cells and their progeny are also contemplated by the present disclosure. Both transiently expressed epigenetic editors and integrated epigenetic editors or components thereof may result in stable epigenetic modifications. For example, after introducing an epigenetic editor described herein into a host cell, the target gene in the host cell may be stably or permanently suppressed or silenced. In some embodiments, the expression of the target gene is reduced or silenced compared to the expression level in the absence of the epigenetic editor 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, or for the entire lifespan of the cell or the subject carrying the cell. The epigenetic modification may be inherited by the progeny of the host cell into which the epigenetic editor was introduced.

[0044] The epigenetic editor can be introduced into a patient (e.g., a human patient) in need thereof, for example, into the patient's hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and hepatic sinusoidal endothelial cells.

[0045] I. DNA binding domain The epigenetic editors described herein may include one or more DNA-binding domains that direct the effector domain of the epigenetic editor to a target sequence within or near the PCSK9 locus. The DNA-binding domains described herein may be, for example, polynucleotide-guided DNA-binding domains, zinc finger protein (ZFP) domains, transcription activator-like effector (TALE) domains, meganuclease DNA-binding domains, etc. Examples of DNA-binding domains can be found in U.S. Patent No. 11,162,114, which is incorporated herein by reference in its entirety.

[0046] In some embodiments, the DNA-binding domains described herein are encoded by their native coding sequences, while in other embodiments, the DNA-binding domains are encoded by nucleotide sequences that have been codon-optimized for optimal expression in human cells.

[0047] A. Polynucleotide-guided DNA-binding domain In some embodiments, the DNA-binding domain herein can be a protein domain that is directed to a target site in the PCSK9 locus by a guide nucleic acid sequence (e.g., a guide RNA sequence). In certain embodiments, the protein domain can be derived from a CRISPR-associated nuclease, such as a class I or II CRISPR-associated nuclease. In some embodiments, the protein domain can be derived from a Cas nuclease, such as type II, type IIA, type IIB, type IIC, type V, or type VI Cas nuclease. In certain embodiments, the protein domain is selected from the group consisting of 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, C The Cas nuclease may be derived from a Class II Cas nuclease selected from sn2, 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 homologs and modified versions thereof. "Derived from" is used to mean that the protein domain comprises the full-length polypeptide sequence of the parent protein or a variant thereof (e.g., having deletions, insertions, and / or substitutions of amino acid residues). The variant retains the desired function of the parent protein (e.g., the ability to form a complex with a guide nucleic acid sequence and target DNA).

[0048] In some embodiments, the CRISPR-associated protein domain can be a Cas9 domain described herein. Cas9 can refer to, for example, a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wild-type Cas9 polypeptide described herein. In some embodiments, the wild-type polypeptide is Cas9 from Streptococcus pyogenes (NCBI Reference No. NC_002737.2 (SEQ ID NO: 1)) and / or UniProt Reference No. Q99ZW2 (SEQ ID NO: 2). In some embodiments, the wild-type polypeptide is Cas9 from Staphylococcus aureus (SEQ ID NO: 3). In some embodiments, the CRISPR-associated protein domain is a Cpf1 domain or protein, or a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wild-type Cpf1 polypeptide described herein (e.g., Cpf1 from Francisella novicida (UniProt Reference No. U2UMQ6 or SEQ ID NO: 4)). In certain embodiments, the CRISPR-associated protein domain can be a modified form, fusion, or chimera of the wild-type protein, or any combination thereof, that includes an alteration, e.g., deletion, insertion, or substitution, of one or more amino acid residues.

[0049] Cas9 sequences and structures of variant Cas9 orthologs have been described for a variety of organisms. Exemplary organisms from which the Cas9 domains herein may be derived include 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, and the like. multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Strepto- sporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitiridacens selenitireducens), Exiguobacterium sibiricumsibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp. sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldas caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilushalophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus Cas9 sequences from organisms and loci disclosed by Chylinski et al., RNA Biol. (2013) 10(5):726-37 are also included.

[0050] In some embodiments, the Cas9 domain is derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 domain is derived from Staphylococcus aureus (SaCas9).

[0051] Other Cas domains are also contemplated for use in the epigenetic editors herein, including, for example, those derived from CasX (Cas12E) (e.g., SEQ ID NO: 5), CasY (Cas12d) (e.g., SEQ ID NO: 6), Casφ (CasPhi) (e.g., SEQ ID NO: 7), Cas12f1 (Cas14a) (e.g., SEQ ID NO: 8), Cas12f2 (Cas14b) (e.g., SEQ ID NO: 9), Cas12f3 (Cas14c) (e.g., SEQ ID NO: 10), and C2c8 (e.g., SEQ ID NO: 11).

[0052] With respect to epigenetic editing, a protein domain derived from a nuclease (e.g., a Cas9 or Cpfl domain) may or may not have reduced nuclease activity through mutation, such that the protein domain does not cleave DNA or has reduced DNA cleavage activity, while retaining the ability to form a complex with a guide nucleic acid sequence (e.g., a guide RNA) and 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 wild-type domain. In some embodiments, the CRISPR-associated protein domains described herein are catalytically inactive ("dead"). Examples of such domains include dCas9 ("dead" Cas9), dCpf1, ddCpf1, dCasPhi, ddCas12a, dLbCpf1, and dFnCpf1. The dCas9 protein domain can contain one, two, or more mutations that abrogate its nuclease activity, for example, compared to wild-type Cas9. The DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while 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 the case of RuvC1) and H840A (in the case of HNH) completely inactivate the nuclease activity of SpCas9. SaCas9 can also be inactivated by the mutations D10A and N580A. In some embodiments, dCas9 contains at least one mutation in the HNH subdomain and / or RuvC1 subdomain that reduces or abolishes nuclease activity. In some embodiments, dCas9 contains only the RuvC1 subdomain or only the HNH subdomain.It should be understood that any mutation that inactivates the RuvC1 and / or HNH domain, for example, an insertion, deletion, or single or multiple amino acid substitution in the RuvC1 domain and / or HNH domain, can be included in the dCas9 herein.

[0053] In some embodiments, the dCas9 protein herein comprises a mutation at a position corresponding to position D10 (e.g., D10A), position H840 (e.g., H840A), or both, of the wild-type SpCas9 sequence, as numbered in the sequence provided in UniProt Accession No. Q99ZW2 (SEQ ID NO: 2). In certain embodiments, the dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).

[0054] In some embodiments, the dCas9 proteins described herein comprise mutations at positions corresponding to positions D10 (e.g., D10A), N580 (e.g., N580A), or both, of the wild-type SaCas9 sequence (e.g., SEQ ID NO: 3). In certain embodiments, the dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO: 13).

[0055] Additional suitable mutations that inactivate Cas9 will be apparent to those of skill in the art based on this disclosure and knowledge in the art 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 mutation that reduces or abolishes the nuclease activity of any Cas9 described herein (e.g., a mutation corresponding to any of the Cas9 mutations described herein).

[0056] The dCpf1 protein domain can contain one, two, or more mutations that reduce or abolish its nuclease activity compared to wild-type Cpf1. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have the HNH endonuclease domain, and the N-terminus of Cpf1 does not have the alpha-helical recognition lobe of Cas9. In some embodiments, dCpf1 contains one or more mutations corresponding to D917A, E1006A, or D1255A numbered in the sequence of the Francisella novicida Cpf1 protein (FnCpf1, SEQ ID NO: 4). In certain embodiments, the dCpfl protein comprises a mutation corresponding to positions D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A, or a corresponding mutation in any of the Cpfl amino acid sequences described herein. In some embodiments, dCpfl comprises a D917A mutation. In certain embodiments, dCpfl comprises the amino acid sequence of dFnCpfl (SEQ ID NO: 14).

[0057] Additional nuclease-inactive CRISPR-associated protein domains contemplated herein include, for example, those derived from dNmeCas9 (e.g., SEQ ID NO: 15), dCjCas9 (e.g., SEQ ID NO: 16), dSt1Cas9 (e.g., SEQ ID NO: 17), dSt3Cas9 (e.g., SEQ ID NO: 18), dLbCpf1 (e.g., SEQ ID NO: 19), dAsCpf1 (e.g., SEQ ID NO: 20), denAsCpf1 (e.g., SEQ ID NO: 21), dHFAsCpf1 (e.g., SEQ ID NO: 22), dRVRAsCpf1 (e.g., SEQ ID NO: 23), dRRAsCpf1 (e.g., SEQ ID NO: 24), dCasX (e.g., SEQ ID NO: 25), and dCasPhi (e.g., SEQ ID NO: 26).

[0058] In some embodiments, the Cas9 domains described herein can be high-fidelity Cas9 domains that contain one or more mutations that reduce electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA, e.g., to confer increased target binding specificity. In certain embodiments, the high-fidelity Cas9 domains can be nuclease-inactive, as described herein.

[0059] The CRISPR-associated protein domains described herein can recognize protospacer adjacent motif (PAM) sequences in target genes. A "PAM" sequence is typically a 2-6 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. CRISPR-associated protein domains can recognize either naturally occurring or canonical PAM sequences, or can 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 to non-canonical PAM sequences are described in Kleinstiver et al., Nature (2015) 523(7561):481-5 and Kleinstiver et al., Nat Biotechnol. (2015) 33:1293-8. Such Cas9 domains include those derived from, for example, "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: 27), nuclease-inactive EQR SpCas9 (e.g., SEQ ID NO: 28), nuclease-inactive VQR SpCas9 (e.g., SEQ ID NO: 29), nuclease-inactive SpG Cas9 (e.g., SEQ ID NO: 30), nuclease-inactive SpRY Cas9 (e.g., SEQ ID NO: 31), and nuclease-inactive KKH SaCas9 (e.g., SEQ ID NO: 32). Another example is Francisella novicida Cas9 engineered to recognize 5'-YG-3' (where "Y" is a pyrimidine).

[0060] 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.

[0061] Guide RNAs that can be used with the CRISPR-associated protein domains herein are further described in Section II below.

[0062] B. Zinc finger protein domain In some embodiments, the DNA-binding domain of the epigenetic editors described herein comprises a zinc finger protein (ZFP) domain (or "ZF domain" as used herein). ZFPs are proteins with at least one zinc finger that bind to DNA in a sequence-specific manner. A "zinc finger" (ZF) or "zinc finger motif" (ZF motif) refers to a polypeptide domain containing a beta-beta-alpha (ββα) protein fold stabilized by zinc ions. A ZF binds two to four nucleotide base pairs, typically three or four base pairs (contiguous or non-contiguous). Each ZF typically contains approximately 30 amino acids. A ZFP domain can contain multiple ZFs that make tandem contacts with their target nucleic acid sequence. Tandem arrays of ZFs can be engineered to generate artificial ZFPs that bind to desired nucleic acid targets. ZFPs can be rationally designed using databases containing triplet (or quadruplet) nucleotide sequences and individual ZF amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of a ZF that binds to a particular triplet or quadruplet sequence. See, e.g., U.S. Patent Nos. 6,453,242, 6,534,261, and 8,772,453.

[0063] ZFPs are widespread in eukaryotic cells and can belong to, for example, the C2H2 class, CCHC class, PHD class, or RING class. An exemplary motif that characterizes one class of these proteins (the C2H2 class) is -Cys-(X) 2-4 -Cys-(X) 12 -His-(X) 3-5-His- (SEQ ID NO: 657), where X is any independently selected amino acid. In some embodiments, the ZFP domains herein can comprise a ZF array comprising consecutive C2H2-ZFs, each of which contacts three or more consecutive nucleotides.

[0064] The ZFP domains of the epigenetic editors described herein can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more ZFs. The ZFP domains can comprise an array of two-finger or three-finger units, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more units, where each unit binds to 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 12-14 nucleotides. In some embodiments, a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18-21 nucleotides.

[0065] In some embodiments, the ZFs in the ZFP domains described herein are connected via a peptide linker. The peptide linker can be 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, the linker comprises 5 or more amino acids. In some embodiments, the linker comprises 7 to 17 amino acids. The linker can be flexible or rigid.

[0066] In some embodiments, the zinc finger array comprises the sequence: SRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH [linker] FQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH [linker] PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTHLRGS (SEQ ID NO: 650) or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, where "XXXXXXX" represents amino acids of the ZF recognition helix that confer DNA-binding specificity to the zinc finger, and each X may be independently selected. In the above sequences, the italicized "XX" may be TR, LR, or LK, and "[linker]" represents a linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 651), which can be used when the subsites targeted by the ZFs are adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO: 652), which can be used when there are bases between the subsites targeted by the zinc fingers. The two shown linkers may be the same or different.

[0067] A ZFP domain herein can comprise an array of two or more adjacent ZFs that are directly adjacent to each other (e.g., separated by a short (canonical) linker sequence) or separated by a long flexible or structured polypeptide sequence. In some embodiments, directly adjacent fingers bind to contiguous nucleic acid sequences, i.e., adjacent trinucleotides / triplets. In some embodiments, adjacent fingers cross-link with each other between their respective target triplets, which can serve to strengthen or enhance target sequence recognition and result in binding of overlapping sequences. In some embodiments, distal ZFs within a ZFP domain can recognize (or bind to) non-contiguous nucleotide sequences.

[0068] The amino acid sequences of the ZF DNA recognition helices of exemplary ZFP domains herein and their PCSK9 target sequences are shown in Table 1 below, where the numbers in parentheses are SEQ ID NOs.

[0069] [Table 1] TIFF2025516294000003.tif247167TIFF2025516294000004.tif150167

[0070] In some embodiments, the ZFP domain of the epigenetic editor binds to a target sequence selected from any one of SEQ ID NOs: 700-747. In further embodiments, the ZFP domain comprises the F1-F6 amino acid sequences, in order, of any one of ZF001-ZF048 shown in Table 1. The F1-F6 amino acid sequences may be placed within the ZF framework sequence of SEQ ID NO: 650 or any other ZF framework known in the art.

[0071] C.TALE In some embodiments, the DNA-binding domain of the epigenetic editors described herein comprises a transcription activator-like effector (TALE) domain. TALE DNA-binding domains contain a highly conserved sequence of approximately 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 to virtually 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.

[0072] 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, such as that derived from Natronobacterium gregoryi (NgAgo). NgAgo is a 5'-phosphorylated ssDNA-guided endonuclease that is guided to its target site by gDNA, creating a double-strand break there. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer adjacent motif (PAM). Therefore, by using nuclease-inactive NgAgo (dNgAgo), the range of bases that can be targeted can be greatly expanded. The characterization and use of NgAgo is described, for example, 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.

[0073] In some embodiments, the DNA-binding domain comprises an inactivated nuclease, e.g., an inactivated meganuclease. Additional non-limiting examples of DNA-binding domains include a tetracycline-controlled repressor (tetR) DNA-binding domain, a leucine zipper, a helix-loop-helix (HLH) domain, a helix-turn-helix domain, a β-sheet motif, a steroid receptor motif, a bZIP domain, a homeodomain, and an AT hook.

[0074] II. Guide Polynucleotide The epigenetic editors described herein that include a polynucleotide-guided DNA-binding domain can also include a guide polynucleotide that can form a complex with the DNA-binding domain. The guide polynucleotide can include RNA, DNA, or a mixture of both. For example, if the polynucleotide-guided DNA-binding domain is a CRISPR-associated protein domain, the guide polynucleotide can be a guide RNA (gRNA). "Guide RNA" or "gRNA" refers to a nucleic acid that can hybridize to a target sequence and guide the binding of a CRISPR-Cas complex to the target sequence. Methods for using guide polynucleotide sequences in conjunction 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.

[0075] A guide polynucleotide sequence (e.g., a gRNA sequence) may comprise two portions: 1) a nucleotide sequence comprising a "targeting sequence" that is complementary to a target nucleic acid sequence ("target sequence"), e.g., a nucleic acid sequence contained in a genomic target site, and 2) a nucleotide sequence that binds to a polynucleotide-guided DNA-binding domain (e.g., a CRISPR-Cas protein domain). The nucleotide sequence of 1) may comprise a targeting sequence that is 100% complementary to a genomic nucleic acid sequence, e.g., a nucleic acid sequence contained in a genomic target site, and thus may hybridize to the target nucleic acid sequence. The nucleotide sequence of 1) may be referred to, for example, as a crispr RNA or crRNA. The nucleotide sequence of 2) may be referred to as a scaffold sequence of the guide nucleic acid, e.g., a tracrRNA, or an activation region of the guide nucleic acid, and may comprise a stem-loop structure. The above portions 1) and 2) 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, the guide polynucleotide comprises portions 1) and 2) connected by a linker. In some embodiments, the guide polynucleotide comprises portions 1) and 2) connected by a non-nucleic acid linker, e.g., a peptide linker or a chemical linker.

[0076] Portion 2 (the scaffold sequence) of the guide polynucleotide described herein can be, for example, that described in Jinek et al., Science (2012) 337:816-21, U.S. Patent Application Publication No. 2016 / 0208288, or U.S. Patent Application Publication No. 2016 / 0200779. Variants of portion 2) are also contemplated by the present disclosure. For example, the tetraloop and stem-loop of the gRNA scaffold (tracrRNA) sequence may be modified to include an RNA aptamer that can be bound by a specific protein domain. In some embodiments, such modified gRNAs can be used to promote the recruitment of repression or activation domains fused to RNA aptamers that interact with proteins.

[0077] The gRNAs provided herein typically comprise a targeting domain and a binding domain. The targeting domain (also referred to as a "targeting sequence") may comprise a nucleic acid sequence that binds to a target site, e.g., a genomic nucleic acid molecule within a cell. The target site may be a double-stranded DNA sequence comprising a PAM sequence and a target sequence located on the same strand as the PAM sequence and directly adjacent to it. The targeting domain of a gRNA may comprise an RNA sequence corresponding to the target sequence, i.e., it is similar to the sequence of the target domain and may have one or more mismatches, but typically comprises an RNA sequence instead of a DNA sequence. The targeting domain of a gRNA may therefore base-pair (with full or partial complementarity) with the sequence of the double-stranded target site that is complementary to the target sequence and, therefore, with the strand complementary to the strand containing the PAM sequence. It will be understood that the targeting domain of a gRNA typically does not comprise a sequence similar to the PAM sequence. It will further be understood that the location of the PAM may be 5' or 3' of the target sequence, depending on the nuclease utilized. For example, the PAM is typically located 3' of the target sequence for Cas9 nucleases and 5' of the target sequence for Cas12a nucleases. For examples of PAM locations and mechanisms by which gRNAs bind to target sites, see, e.g., Figure 1 in Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6, which is incorporated herein by reference. For additional examples and explanations of mechanisms of gRNA targeting to RNA-guided nuclease target sites, see Fu et al., Nat Biotechnol (2014) 32(3):279-84 and Sternberg et al., Nature (2014) 507(7490):62-7, each of which is incorporated herein by reference.

[0078] In some embodiments, the targeting domain sequence comprises 17-30 nucleotides and corresponds perfectly to the target sequence (i.e., does not have any mismatched nucleotides). In some embodiments, however, the targeting domain sequence may contain one or more, typically four or fewer, mismatches, e.g., one, two, three, or four mismatches. Because the targeting domain is part of a gRNA, which is an RNA molecule, it will typically contain ribonucleotides, whereas a DNA-targeting domain will contain deoxyribonucleotides.

[0079] An exemplary illustration of a Cas9 target site comprising a 22-nucleotide targeting domain and an NGG PAM sequence, and a gRNA comprising a targeting domain that corresponds perfectly to the target sequence (and thus base-pairs with perfect complementarity to the DNA strand complementary to the strand containing the target sequence and PAM), is provided below. [ Target Domain (DNA) ] [PAM] 5'-NNNNNNNNNNNNNNNNNNNNN-NNGG-3' (DNA) 3'-NNNNNNNNNNNNNNNNNNNNN-NNCC-5' (DNA) | | | | | | | | | | | | | | | | | | | | | | 5'-NNNNNNNNNNNNNNNNNNNNN-N-[gRNA scaffold]-3' (RNA) [Targeting domain (RNA)] [Binding domain]

[0080] An exemplary illustration of a Casl2a target site containing a 22-nucleotide targeting domain and a TTN PAM sequence, as well as a gRNA containing a targeting domain that corresponds perfectly to the target sequence (and thus base-pairs with perfect complementarity to the DNA strand complementary to the strand containing the target sequence and PAM), is provided below. [PAM] [Target domain (DNA)] 5'-TTNNNNNNNNNNNNNNNNNN-NNNN-3' (DNA) 3'-AANNNNNNNNNNNNNNNNN-NNNN-5' (DNA) | | | | | | | | | | | | | | | | | | | | | | 5'-[gRNAスキャフォールド]-NNNNNNNNNNNNNNNNNNNN-N-3' (RNA) [ PubMed ] [ PubMed ]

[0081] Without wishing to be bound by theory, it is believed that, at least in some embodiments, the length of the targeting domain and its complementarity with the target sequence contribute to the specificity of the interaction between the gRNA / Cas9 molecular complex and the target nucleic acid. In some embodiments, the targeting domain of a gRNA provided herein is 5-50 nucleotides in length. In some embodiments, the targeting domain is 15-25 nucleotides in length. In some embodiments, the targeting domain is 18-22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In certain embodiments, the targeting domain completely corresponds to a target sequence provided herein or a portion thereof without any mismatches. In some embodiments, the targeting domain of a gRNA provided herein contains one mismatch to a target sequence provided herein. In some embodiments, the targeting domain contains two mismatches to the target sequence. In some embodiments, the targeting domain contains three mismatches to the target sequence.

[0082] Methods for designing, selecting, and validating gRNA are described herein and are known in the art.Software tools can be used to optimize the gRNA corresponding to target DNA sequence, for example, to minimize the total off-target activity throughout the genome.For example, DNA sequence search algorithms can be used to identify the target sequence in the crRNA of the gRNA for use with Cas9.Exemplary gRNA design tools include those described in Bae et al., Bioinformatics (2014) 30:1473-5.

[0083] The guide polynucleotides (e.g., gRNAs) described herein can 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 different optimal targeting sequence lengths. Thus, the spacer sequence can comprise, for example, 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, the guide polynucleotide (e.g., gRNA) comprises 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. A guide polynucleotide is a nucleotide sequence of at least 10 (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) consecutive nucleotides complementary to the target sequence. In some embodiments, the guide polynucleotides described herein can be truncated by, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more nucleotides.

[0084] In certain embodiments, the 3' end of the PCSK9 target sequence is immediately adjacent to a PAM sequence (e.g., a canonical PAM sequence, e.g., NGG for SpCas9). The degree of complementarity between the targeting sequence of the guide polynucleotide (e.g., the spacer sequence of the gRNA) and the target sequence can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the targeting sequence and the target sequence can be 100% complementary. In other embodiments, the targeting sequence and the target sequence can contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

[0085] A guide polynucleotide (e.g., gRNA) may be modified, for example, by chemical and synthetic modifications. Modified gRNAs can include, for example, modification or replacement of one or both of the non-linked phosphate oxygens and / or one or more of the linking phosphate oxygens in the phosphodiester backbone linkage, modification of the ribose sugar (e.g., the 2' hydroxyl of the ribose sugar), modification of a phosphate moiety, modification or replacement of a naturally occurring nucleobase, modification or replacement of the ribose-phosphate backbone, modification of the 3' 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.

[0086] 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-acetal ester, or bicyclic nucleotides, such as locked nucleic acids (LNA), 2'-(5-constrained ethyl (S-cEt)), constrained MOE, or 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC). 2'-O-methyl and / or 2'-fluoro modifications may increase the binding affinity and / or nuclease stability of the gRNA oligonucleotide.

[0087] In some embodiments, one or more phosphate groups of a gRNA may be chemically modified. Examples of chemical modifications to phosphate groups include, but are not limited to, phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modifications. In some embodiments, a guide polynucleotide described herein may contain one, two, three, or more PS linkages at or near the 5' and / or 3' ends, and the PS linkages may be contiguous or non-contiguous.

[0088] In some embodiments, the gRNAs herein comprise a mixture of ribonucleotides and deoxyribonucleotides and / or one or more PS linkages.

[0089] 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 pyrimidines, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups. Chemical modifications may be made to the spacer region, the tracr RNA region, the stem-loop, or any combination thereof.

[0090] Table 2 below lists exemplary gRNA target sequences for epigenetic modification of human PCSK9, as well as the coordinates of the start and end of the targeted site on human chromosome 1 (SEQ: SEQ ID NO:). The table also shows the distance from the start coordinate to the TSS coordinate of the PCSK9 gene.

[0091] [Table 2] TIFF2025516294000006.tif255166TIFF2025516294000007.tif255166TIFF2025516294000008.tif255166TIFF2025516294000009.tif118166

[0092] In some embodiments, the gRNA herein does not comprise the sequence CCCGCACCUUGGCGCAGCGG (SEQ ID NO: 1490).

[0093] Any tracr sequence known in the art is contemplated for the gRNAs described herein. In some embodiments, the gRNAs described herein have a tracr sequence set forth in Table 3 below, or a tracr sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequences set forth below (SEQ: SEQ ID NO:).

[0094] [Table 3]

[0095] In some embodiments, the gRNA herein is provided to a cell directly (e.g., through an RNP complex together with a CRISPR-associated protein domain). In some embodiments, the gRNA is provided to a cell through an expression vector (e.g., a plasmid vector or a viral vector) that is introduced into the cell, and the cell then expresses the gRNA from the expression vector. Methods for introducing gRNAs and expression vectors into cells are well known in the art.

[0096] III. Effector Domain The epigenetic editors described herein include one or more effector protein domains (also referred to herein as "epigenetic effector domains" or "effector domains") that effect epigenetic modifications of a target gene. Epigenetic editors with one or more effector domains can modulate the expression of a target gene without altering the nucleic acid sequence of the target gene. In some embodiments, the effector domains described herein can provide suppression or silencing of expression of a target gene, such as PCSK9, by, for example, repressing transcription or by modifying or remodeling chromatin. Such effector domains are also referred to herein as "repression domains," "repressor domains," or "epigenetic repressor domains." Non-limiting examples of chemical modifications that can be mediated by effector domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, sumoylation, and / or ubiquitination of DNA or histone residues.

[0097] In some embodiments, the effector domain of an epigenetic editor described herein can effect histone tail modification, for example, by adding or removing an activity mark to the histone tail.

[0098] In some embodiments, the effector domain of an epigenetic editor described herein may comprise or recruit a transcription-associated protein, e.g., a transcription repressor. The transcription-associated protein may be endogenous or exogenous.

[0099] In some embodiments, the effector domain of an epigenetic editor described herein can include, for example, a protein that directly or indirectly blocks access of a transcription factor to a gene of interest having a target sequence.

[0100] The effector domain can be a full-length protein or a fragment thereof ("functional domain") that retains epigenetic effector function. Functional domains capable of modulating (e.g., suppressing) gene expression can be derived from larger proteins. For example, functional domains capable of reducing target gene expression can be identified based on the sequence of a repressor protein. The amino acid sequence of a protein that modulates gene expression can be obtained from available genome browsers, such as the UCSD genome browser or the Ensembl genome browser. Protein annotation databases, such as UniProt or Pfam, can be used to identify functional domains within a full-length protein sequence. As a starting point, the largest sequence encompassing all regions identified by different databases can be tested for gene expression modulation activity. Various truncated forms can then be tested to identify a minimal functional unit.

[0101] Variants of the effector domains described herein are also contemplated by the present disclosure. A variant refers to, for example, a polypeptide having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wild-type effector domain described herein. In certain embodiments, a 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 wild-type effector domain.

[0102] In some embodiments, an effector domain described herein can comprise a fusion of two or more effector domains (e.g., KOX1 KRAB and ZIM3). An effector domain can comprise, for example, a fusion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector domains, e.g., an effector domain described herein. In certain embodiments, an effector domain comprises a fusion of a truncated form of one effector domain and a second effector domain. In certain embodiments, an effector domain comprises a fusion of truncated forms of two effector domains (e.g., a fusion of the N-terminal and C-terminal portions of two effector domains).

[0103] In some embodiments, the epigenetic editors described herein can include one effector domain, two effector domains, three effector domains, four effector domains, five effector domains, six effector domains, seven effector domains, eight effector domains, nine effector domains, ten effector domains, or more. In certain embodiments, the epigenetic editors include one or more fusion proteins (e.g., one, two, or three fusion proteins), each having 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 can induce a combination of epigenetic modifications, such as transcriptional 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 ubiquitination, or DNA methylation and histone sumoylation.

[0104] In certain embodiments, the effector domains described herein (e.g., DNMT3A and / or DNMT3L) are encoded by the nucleotide sequence found in the native genome (e.g., human or mouse) for that effector domain. In other embodiments, the effector domains described herein are encoded by nucleotide sequences that have been codon-optimized for optimal expression in human cells.

[0105] Effector domains described herein can include, for example, transcriptional repressors, DNA methyltransferases, and / or histone modifiers, as further detailed below.

[0106] A. Transcriptional repressors In some embodiments, the epigenetic effector domains described herein mediate the suppression of target gene expression (e.g., transcription). The effector domain may include, for example, a Kruppel-associated box (KRAB) repressor domain, a repressor element silencing transcription factor (REST) ​​repressor domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, an FKHR (forkhead gene in rhabdosarcoma) repressor domain, an EGR-1 (early growth response gene product-1) repressor domain, an ets2 repressor factor repressor domain (ERD), a MAD smSIN3-interacting domain (SID), a WRPW motif of a hairy-related basic helix-loop-helix (bHLH) repressor protein, an HP1 alpha chromoshadow repressor domain, an HP1 beta repressor domain, or any combination thereof. The effector domain may recruit, for example, through a scaffold protein, one or more protein domains that repress expression of a target gene. In some embodiments, the effector domain may recruit or interact with a scaffold protein domain that recruits a PRMT protein, an HDAC protein, a SETDB1 protein, or a NuRD protein domain.

[0107] 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 can be found, for example, in Ecco et al., Development (2017) 144(15):2719-29 and Lambert et al., Cell (2018) 172:650-65.

[0108] In certain embodiments, the effector domain comprises a repressor domain (e.g., KRAB) from KOX1 / ZNF10, KOX8 / ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain is selected from the group consisting of 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, ZNF556, ZNF558, ZNF559 ... The repressor domain may comprise a repressor domain (e.g., KRAB) derived from 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, or any combination thereof. For example, the repressor domain may be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In certain embodiments, the repressor domain is a ZIM3 KRAB domain. In further embodiments, the effector domain is derived from a human protein, such as human ZIM3, human KOX1, human ZFP28, or human ZN627.

[0109] Exemplary effector domain sequences, or protein sequences containing them, that can reduce or silence target gene expression are provided in Table 4 below (SEQ: SEQ ID NO:). Further examples of repressors and transcriptional repressor domains can be found, for example, in PCT Patent Application Publication No. 2021 / 226077 and Tycko et al., Cell (2020) 183(7):2020-35, each of which is incorporated herein by reference in its entirety.

[0110] [Table 4] TIFF2025516294000012.tif253162TIFF2025516294000013.tif255163TIFF20255162940 00014.tif255163TIFF2025516294000015.tif255163TIFF2025516294000016.tif217163

[0111] Functional analogs of any one of the above-listed proteins, i.e., molecules that retain the same or substantially the same biological function (e.g., 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the protein's transcription factor function), are encompassed by the present disclosure. For example, functional analogs can be isoforms or variants of the above-listed proteins, including, for example, portions of the above proteins with or without additional amino acid residues and / or including mutations to the above proteins. In some embodiments, functional analogs have at least 75, 80, 85, 90, 95, 98, or 99% sequence identity to one of the sequences listed in Table 4. Homologs, orthologs, and mutants of the above-listed proteins are also contemplated.

[0112] In certain embodiments, the epigenetic editors described herein include 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, whereby the parent protein is a human protein. In certain embodiments, the epigenetic editors described herein include a domain derived from KOX1, ZIM3, ZFP28, and / or ZN627, whereby the parent protein is a human protein. In certain embodiments, the epigenetic editor may include a KRAB domain derived from KOX1 (ZNF10), e.g., human KOX1. In certain embodiments, the epigenetic editor may include a KRAB domain derived from ZIM3 (ZNF657 or ZNF264), e.g., human ZIM3. In certain embodiments, an epigenetic editor can include a KRAB domain from ZFP28, e.g., human ZFP28. In certain embodiments, an epigenetic editor can include a KRAB domain from ZN627, e.g., human ZN627. In certain embodiments, an epigenetic editor described herein can include a CDYL2, e.g., human CDYL2, and / or a TOX domain (e.g., a human TOX domain), in combination with a KOX1 KRAB domain (e.g., a human KOX1 KRAB domain).

[0113] In certain embodiments, an epigenetic effector described herein comprises a repressor domain derived from KOX1 / ZNF10 (SEQ ID NO: 89). For example, the repressor domain can comprise the sequence of SEQ ID NO: 89, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 89.

[0114] In certain embodiments, the epigenetic effectors described herein comprise a repressor domain derived from KOX1 / ZNF10, as shown in Table 5 below.

[0115] [Table 5]

[0116] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:565, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:565.

[0117] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:566, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:566.

[0118] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:567, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:567.

[0119] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:568, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:568.

[0120] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:569, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:569.

[0121] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:570, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:570.

[0122] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:571, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:571.

[0123] In certain embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO:572, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:572.

[0124] B. DNA methyltransferase In some embodiments, the effector domain of the epigenetic editor described herein alters target gene expression through DNA modification, e.g., methylation. Highly methylated areas of DNA tend to be less transcriptionally active than less methylated areas. DNA methylation occurs primarily at CpG sites (truncated forms of "C-phosphate-G-" or "cytosine-phosphate-guanine" sites). Many mammalian genes have promoter regions near or containing CpG islands (nucleic acid regions with a high frequency of CpG dinucleotides).

[0125] The effector domain described herein may be, for example, a DNA methyltransferase (DNMT) or its catalytic domain, or may be capable of recruiting a DNA methyltransferase. DNMTs include enzymes that catalyze the transfer of methyl groups to DNA nucleotides, such as canonical cytosine-5 DNMTs (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C), which catalyze the addition of methyl groups to genomic DNA. The term also encompasses non-canonical family members that do not themselves catalyze methylation but recruit (including activate) catalytically active DNMTs; a non-limiting example of such a DNMT is DNMT3L. See, e.g., Lyko, Nat Review (2018) 19:81-92. Unless otherwise indicated, a DNMT domain may refer to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT1, DNMT3A, and DNMT3B) or a catalytically inactive DNMT (e.g., DNMT3L). DNMTs can suppress the expression of target genes through the recruitment of inhibitory 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.

[0126] In some embodiments, the DNMTs described herein can be animal DNMTs (eg, mammalian DNMTs), plant DNMTs, fungal DNMTs, or bacterial DNMTs. Bacterial DNMTs can be obtained from bacterial species (e.g., cocci, bacilli, spiral bacteria, or intracellular Gram-positive or Gram-negative bacteria). In certain embodiments, the bacterial species is Mycoplasmatales bacterium, Mycoplasma marinum, or Spiroplasma chinense. In certain embodiments, the bacterial species is M. penetrans, S. monbiae, H. parainfluenzae, A. luteus, H. aegyptius, H. haemolyticus, Moraxella, E. coli, T. aquaticus, C. crescentus, In certain embodiments, the epigenetic editor described herein is not C. crescentus or C. difficile. In certain embodiments, the epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO: 601, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 601. In certain embodiments, the epigenetic editor described herein comprises SEQ ID NO: 602, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 602. In certain embodiments, the epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO:603, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:603.

[0127] In certain embodiments, the DNMT in the epigenetic editors described herein can include, for example, DNMT1, DNMT3A, DNMT3B, and / or DNMT3C. In some embodiments, the DNMT is a mammalian (e.g., human or mouse) DNMT. In certain embodiments, the DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, the epigenetic editors described herein include a DNMT3A domain comprising SEQ ID NO:574, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:574. In certain embodiments, an epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 575, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 575. In some embodiments, the DNMT3A domain can have a mutation at, for example, position H739 (e.g., H739A or H739E), position R771 (e.g., R771L), and / or position R836 (e.g., R836A or R836Q), or any combination thereof (numbering according to SEQ ID NO: 574).

[0128] In some embodiments, the effector domain described herein can be a DNMT-like domain. As used herein, a "DNMT-like domain" refers to a regulator of DNMTs that can activate or recruit other DNMT domains but does not itself have methylation activity. In some embodiments, the DNMT-like domain is a mammalian (e.g., human or mouse) DNMT-like domain. In certain embodiments, the DNMT-like domain is DNMT3L, which can be, for example, human DNMT3L or mouse DNMT3L. In certain embodiments, the epigenetic editor described herein comprises a DNMT3L 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 certain embodiments, an epigenetic editor herein comprises a DNMT3L domain comprising SEQ ID NO: 579, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 579. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 580, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 580. 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 some embodiments, the DNMT3L domain can have a corresponding mutation, e.g., at position D226 (e.g., D226V), position Q268 (e.g., Q268K), or both (numbering according to SEQ ID NO: 578).

[0129] In certain embodiments, the epigenetic editors described herein may include both DNMT and DNMT-like effector domains. For example, the epigenetic editor may include a DNMT3A-3L domain, where DNMT3A and DNMT3L may be covalently linked. In other embodiments, the epigenetic editors described herein may include an effector domain that includes only a DNMT3A domain (e.g., human DNMT3A) or only a DNMT-like domain (e.g., DNMT3L, which may be human or mouse DNMT3L).

[0130] Table 6 below provides exemplary DNMTs that may be part of the epigenetic effector domains described herein, or from which the effector domains of the epigenetic editors described herein may be derived.

[0131] [Table 6] TIFF2025516294000019.tif254163TIFF2025516294000020.tif66162

[0132] Functional analogs of any one of the above-listed proteins, i.e., molecules that retain the same or substantially the same biological function (e.g., 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the protein's DNA methylation or recruitment function), are encompassed by the present disclosure. For example, functional analogs can be isoforms or variants of the above-listed proteins, e.g., including portions of the above proteins with or without additional amino acid residues and / or including mutations to the above proteins. In some embodiments, functional analogs have at least 75, 80, 85, 90, 95, 98, or 99% sequence identity to one of the sequences listed in Table 6. In some embodiments, the effector domain herein comprises only a functional domain (or a functional analog thereof) of the above-listed proteins, e.g., the catalytic domain or the recruitment domain. In some embodiments, the effector domain herein comprises one or more epigenetic effector domains selected from Table 6, or functional homologs, orthologs, or variants thereof.

[0133] As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is identical to a parent protein (e.g., human or mouse DNMT3A or DNMT3L) or a functional analog thereof (e.g., having a functional fragment of the parent protein, e.g., a catalytic fragment or a recruitment fragment, and / or having a mutation that improves the activity of the DNMT protein).

[0134] The epigenetic editors herein can effect methylation at, for example, 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 within a target gene or chromosome. The CpG dinucleotide sequences may be located within or near the target gene in a CpG island, or may be located in a region that is not a CpG island. A CpG island generally refers to a nucleic acid sequence or chromosomal region that contains a high frequency of CpG dinucleotides. For example, a CpG island may contain at least 50% GC content. A 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, the observed-to-expected CpG ratio is determined by the number of CpGs x (length of sequence) / (number of Cs x number of Gs). In some embodiments, a CpG island has an observed-to-expected CpG ratio of at least 60%, 70%, 80%, 90%, or more. A CpG island may be, for example, a sequence or region of at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, only 1, or fewer than 2, fewer than 3, fewer than 4, fewer than 5, fewer than 6, fewer than 7, fewer than 8, fewer than 9, fewer than 10, fewer than 11, fewer than 12, fewer than 13, fewer than 14, fewer than 15, fewer than 16, fewer than 17, fewer than 18, fewer than 19, fewer than 20, fewer than 30, fewer than 40, or fewer than 50 CpG dinucleotides are methylated by the epigenetic editor.

[0135] In some embodiments, the epigenetic editors herein effect methylation in hypomethylated nucleic acid sequences, i.e., sequences that may lack a methyl group on a 5-methylcytosine nucleotide (e.g., in a CpG) compared to a standard control. Hypomethylation can occur, for example, in aged cells or cancer (e.g., early stages of neoplasia) compared to young cells or non-cancerous cells, respectively.

[0136] In some embodiments, the epigenetic editors described herein induce methylation in hypermethylated nucleic acid sequences.

[0137] In some embodiments, methylation can be introduced by an epigenetic editor at sites other than CpG dinucleotides. For example, a target gene sequence can be methylated at the C nucleotide of a CpA, CpT, or CpC sequence. In some embodiments, the epigenetic editor includes a DNMT3A domain, resulting in methylation at CpG, CpA, CpT, CpC sequences, or any combination thereof. In some embodiments, the epigenetic editor includes a DNMT3A domain that lacks the regulatory subdomain and retains only the catalytic domain. In some embodiments, an epigenetic editor including a DNMT3A catalytic domain results in methylation exclusively at CpG sequences. In some embodiments, an epigenetic editor including a DNMT3A domain containing a mutation, e.g., an R836A or R836Q mutation (numbering according to SEQ ID NO: 574), has higher methylation activity at CpA, CpC, and / or CpT sequences compared to an epigenetic editor including a wild-type DNMT3A domain.

[0138] C. Histone Modification Factors In some embodiments, the effector domain of the epigenetic editor herein mediates histone modification. Histone modifications play a structural and biochemical role in gene transcription, for example, by forming or disrupting nucleosome structures that bind to histones and prevent gene transcription. Histone modifications can include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, sumoylation, and the like, at their N-termini ("histone tails"). These modifications maintain or specifically transform chromatin structure, thereby controlling responses occurring on chromosomal DNA, such as gene expression, DNA replication, and DNA repair. Post-translational modifications of histones are an epigenetic regulatory mechanism and are believed to be essential for gene regulation in eukaryotic cells. Recent studies have revealed that chromatin remodeling factors, such as SWI / SNF, RSC, NURF, and NRD, which modify nucleosome structure to facilitate the access of transcription factors to DNA, histone acetyltransferases (HATs) that regulate the acetylation status of histones, and histone deacetylases (HDACs) act as key regulators.

[0139] In particular, the unstructured N-terminus of histones can be modified by acetylation, deacetylation, methylation, ubiquitination, phosphorylation, sumoylation, ribosylation, citrullination, O-GlcNAcylation, or crotonylation, or any combination thereof. For example, histone acetyltransferases (HATs) use acetyl-CoA as a cofactor to catalyze the transfer of an acetyl group to the epsilon-amino group of a lysine side chain. This neutralizes the positive charge of the lysine, weakening the interaction between the histone and DNA, thereby opening the chromosome for transcription factor binding and initiating transcription. Acetylation of the K14 and K9 lysines of histone H3 by histone acetyltransferase enzymes may be associated with 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 can be associated with heterochromatin, or transcriptionally silent chromatin.

[0140] In certain embodiments, the 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 certain embodiments, the effector domain comprises a histone-lysine-N-methyltransferase SETDB1 domain.

[0141] In some embodiments, the effector domain comprises a histone deacetylase protein domain. In certain embodiments, the effector domain comprises an HDAC family protein domain, such as an HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In certain embodiments, the effector domain comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.

[0142] D. 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. The KAP1 protein in the epigenetic editors herein forms a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in modulating gene expression in the cellular environment. For example, KAP1 can be recruited by the KRAB domain of a transcriptional repressor. The KAP1 protein domain can interact with or recruit one or more protein complexes that reduce or silence gene expression. In some embodiments, KAP1 interacts with or recruits histone deacetylase proteins, histone-lysine methyltransferase proteins, chromatin remodeling proteins, and / or heterochromatin proteins. For example, the KAP1 protein domain may interact with or recruit heterochromatin protein 1 (HP1) protein, SETDB1 protein, HDAC protein, and / or NuRD protein complex components. In some embodiments, the KAP1 protein domain interacts with or recruits ZFP90 protein (e.g., ZFP90 isoform 2) and / or FOXP3 protein. An exemplary KAP1 amino acid sequence is set forth in SEQ ID NO:629.

[0143] 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 within a target gene (which may or may not be in a CpG island of the target gene). The MECP2 protein domain in the epigenetic editors described herein may induce a condensed chromatin structure, thereby reducing or silencing expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein may interact with histone deacetylases (e.g., HDACs), thereby repressing or silencing expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein may block access of transcription factors or transcriptional activators to a target sequence, thereby repressing or silencing expression of the target gene. An exemplary MECP2 amino acid sequence is set forth in SEQ ID NO: 630.

[0144] Effector domains of the epigenetic editors described herein include, for example, a chromoshadow domain, a ubiquitin-2-like Rad60 SUMO-like (Rad60-SLD / SUMO) domain, a chromatin organization modifier domain (Chromo) domain, and Yaf2 / RYBP. C-terminal binding motif domain (YAF2_RYBP), CBX family C-terminal motif domain (CBX7_C), zinc finger C3HC4 type (RING finger) domain (ZF-C3HC4_2), cytochrome b5 domain (Cyt-b5), helix-loop-helix domain (HLH), helix-heparin-helix motif domain (e.g., HHH_3), high mobility group box domain (HMG-box), basic leucine zipper domain (e.g., bZIP_1 or bZIP_2), Myb_DNA binding domain, homeodomain, MYM-type zinc finger domain with FCS sequence domain (ZF-FCS), interferon regulatory factor 2-binding protein Protein zinc finger domain (IRF-2BP1_2), SSX repressor domain (SSXRD), B-box type zinc finger domain (ZF-B_box), CXXC zinc finger domain (ZF-CXXC), regulator of chromosome condensation 1 domain (RCC1), SRC homology 3 domain (SH3_9), sterile alpha motif domain (SAM_1), sterile alpha motif domain (SAM_2), sterile alpha motif / pointed domain (SAM_PNT), vestibulial / Tondu family domain (Vg_Tdu), LIM domain, RNA recognition motif domain (RRM_1), paired amphipathic helix domain (PAH), proteasome ATPase OB C-terminal domain (Prot_ATP_ID_OB), nerve homology 2 domain (NHR2), hinge domain of cleavage stimulatory factor subunit 2 (CSTF2_hinge), PPAR gamma N-terminal region domain (PPARgamma_N), CDC48 N-terminal domain (CDC48_2), WD40 repeat domain (WD40), Fip1 motif domain (Fip1), PDZ domain (PDZ_6), von Willebrand factor type C domain (VWC), NAB conserved region 1 domain (NCD1), S1RNA binding domain (S1), HNF3 C-terminal domain (HNF_C), Tudor domain (Tudor_2), histone-like transcription factor (CBF / NF-Y) and archaeal histone domain (CBFD_NFYB_HMF), zinc finger protein domain (DUF3669), EGF-like domain (cEGF), GATA zinc finger domain (GATA), TEA / ATTS domain (TEA), phorbol ester / diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), transactivation domain of the FOXO protein family (FOXO-TAD), homeobox KN domain (Homeobox_KN), BED zinc finger domain (ZF-BED), zinc finger of C3HC4-type RING domain (ZF-C3HC4_4), RAD51 interaction motif domain (RAD51_interact), p55 binding region of methyl-CpG-binding domain protein MBD (MBDa), Notch domain, Raf-like Ras binding domain (RBD), Spin / Ssty family domain (Spin-Ssty), PHD finger domain (PHD_3), low density lipoprotein receptor domain class A (Ldl_recept_a), CS domain, DM DNA binding domain, and QLQ domain are also contemplated.

[0145] In some embodiments, the effector domain is a protein domain comprising a YAF2_RYBP domain, or its 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 certain embodiments, the YAF2_RYBP domain may comprise a 32-amino acid Yaf2 / RYBP C-terminal binding motif domain (32 aa RYBP).

[0146] In some embodiments, the effector domain comprises a protein domain selected from the group consisting of a SUMO3 domain, a chromodomain derived from M-phase phosphoprotein 8 (MPP8), a chromoshadow domain derived from chromobox 1 (CBX1), and a SAM_1 / SPM domain derived from Scm polycomb group protein homolog 1 (SCMH1).

[0147] In some embodiments, the effector domain comprises the HNF3 C-terminal domain (HNF_C). The HNF_C domain can be derived from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif.

[0148] 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 the DNA repair factor HERC2 E3 ligase, a variant SH3 domain (SH3_9) from Bridging Integrator 1 (BIN1), an HMG box domain from the transcription factor TOX, or a ZF-C3HC4_2 RING finger domain from the polycomb component PCGF2, a chromodomain-helicase-DNA binding protein 3 (CHD3) domain, or a ZNF783 domain.

[0149] IV. Epigenetic Editor For example, provided herein are epigenetic editors (i.e., epigenetic editing systems) that use any combination of one or more DNA-binding domains described herein and one or more effector domains (e.g., epigenetic repressor domains) described herein to direct epigenetic modifications to a target sequence in a gene of interest. The DNA-binding domain (in cooperation with a guide polynucleotide, e.g., one described herein, where the DNA-binding domain is a DNA-binding domain guided by the polynucleotide) directs the effector domain to epigenetically modify the target sequence, resulting in persistent gene suppression or silencing that can be inherited across generations of cells. In some embodiments, the epigenetic editors described herein can reversibly or irreversibly suppress or silence genes in cells.

[0150] In certain embodiments, the epigenetic editors described herein comprise one or more fusion proteins, each comprising (1) a DNA-binding domain and (2) an effector domain. The effector domains may be present on one or more fusion proteins comprised by the epigenetic editor. For example, a single fusion protein may comprise all effector domains with a DNA-binding domain. Alternatively, the effector domains, or subsets thereof, may be present on separate fusion proteins (which may be the same or different), each with a DNA-binding domain. The fusion proteins 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, "fusion protein" refers to a chimeric protein in which two or more coding sequences (e.g., for DNA-binding domains and / or effector domains) are joined directly or indirectly by covalent or non-covalent bonds.

[0151] In some embodiments, the epigenetic editors described herein include two, three, four, five, six, seven, eight, nine, ten, or more effector (e.g., repressor) domains, which may be the same or different. In certain embodiments, two or more of the effector domains function synergistically. Combinations of effector domains may include a DNA methylation domain, a histone deacetylation domain, a histone methylation domain, and / or a scaffold domain that recruits any of the above. For example, the epigenetic editors described herein may include one or more transcriptional repressor domains (e.g., KRAB domains, e.g., KOX1, ZIM3, ZFP28, or ZN627 KRAB) in combination with one or more DNA methylation domains (e.g., DNMT domains) and / or recruiter domains (e.g., DNMT3L domains). Such epigenetic editors can include, for example, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, the epigenetic editor further includes 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, the epigenetic editors described herein can include a CDYL2 and / or TOX domain in combination with a KRAB domain (e.g., a KOX1 KRAB domain).

[0152] A. Linker The fusion proteins described herein can include one or more linkers connecting the components of the epigenetic editor. The linkers can be peptide or non-peptide linkers.

[0153] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are peptide linkers, i.e., linkers that include a peptide moiety. Peptide linkers can be of any length applicable to the epigenetic editor fusion proteins described herein. In some embodiments, a linker can include a peptide of 1 to 200 (e.g., 1 to 80) amino acids. In some embodiments, the linker is 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 1 00, 20-150, 20-200, 30-40, 30-50, 30-60, 30-80, 30-100, 30-150, 30-200, 40-50, 40-60, 40-80, 40-100, 40-150, 40-200, 50-60, 50-80, 50-100, 50-150, 50-200, 60-80, 60-100, 60-150, 60-200, 80-100, 80-150, 80-200, 100-150, 100-200, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. For example, the peptide linker can be 4, 5, 16, 20, 24, 27, 32, 40, 64, 92, or 104 amino acids in length. The peptide linker can be a flexible or rigid linker.In certain embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 631-637 and 664-665, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0154] In certain embodiments, the peptide linker is an XTEN linker. Such a linker can comprise a portion of the XTEN sequence, an unstructured hydrophilic polypeptide consisting only of the residues G, S, P, T, E, and A (Schellenberger et al., Nat Biotechnol (2009) 27(1):1186-90). As used herein, the term "XTEN" refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. XTEN linkers are typically unstructured and contain a limited set of naturally occurring amino acids. Fusion of XTEN to a protein alters its hydrodynamic properties and reduces the clearance and degradation rate of the fusion protein. These XTEN fusion proteins are produced using recombinant technology and are degraded by natural pathways without the need for chemical modification. XTEN linkers can be, for example, 5, 10, 16, 20, 26, or 80 amino acids in length. In some embodiments, the XTEN linker is 16 amino acids in length. In some embodiments, the XTEN linker is 80 amino acids in length. In certain embodiments, the XTEN linker can be XTEN10, XTEN16, XTEN20, or XTEN80. In certain embodiments, the XTEN linker can comprise the amino acid sequence of any one of SEQ ID NOs: 638-643, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 638. In certain embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 643.

[0155] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are non-peptide linkers. For example, the linker can be a carbon bond, a disulfide bond, or a 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.

[0156] In some embodiments, one or more linkers utilized in the epigenetic editors provided herein are polymers (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker may include, for example, a monomer, dimer, or polymer of an aminoalkanoic acid, a monomer, dimer, or polymer of an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.), an aminohexanoic acid (Ahx), 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 a functionalized moiety that facilitates 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.

[0157] A variety of linker lengths and flexibilities can be utilized between any two components of an epigenetic editor (e.g., between an effector domain (e.g., a repressor domain) and a DNA-binding domain (e.g., a Cas9 domain), between a first effector domain and a second effector domain, etc.). Linkers can range from very flexible linkers, e.g., glycine / serine-rich linkers, to more rigid linkers to achieve an optimal length for effector domain activity for a particular application. In some embodiments, a more flexible linker is a glycine / serine-rich linker (GS-rich linker), in which more than 45% (e.g., more than 48%, more than 50%, more than 55%, more than 60%, more than 70%, more than 80%, or more than 90%) of the residues are glycine or serine residues. Non-limiting examples of GS-rich linkers are (GGGGS)n (SEQ ID NO: 664), (G)n, and the W linker (SEQ ID NO: 637). In some embodiments, more rigid linkers are of the form (EAAAK)n (SEQ ID NO: 665), (SGGS)n (SEQ ID NO: 631 and (XP)n). In the above formulas for flexible and rigid linkers, n can be any integer from 1 to 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 motif, where n is 1, 3, or 7. In some embodiments, the linker comprises a (GGGGS)n motif, where n is 4 (SEQ ID NO: 636).

[0158] In some embodiments, the linker in an epigenetic editor described herein comprises a nuclear localization signal, e.g., having the amino acid sequence of any one of SEQ ID NOs: 644 to 649. In some embodiments, the linker in an epigenetic editor described herein comprises an expression tag, e.g., a detectable tag, e.g., green fluorescent protein.

[0159] B. Nuclear localization signal The fusion proteins described herein may contain one or more nuclear localization signals, and in certain embodiments, may contain two or more nuclear localization signals. For example, a fusion protein may contain one, two, three, four, five, six, seven, eight, nine, ten, or more nuclear localization signals. As used herein, a "nuclear localization signal" (NLS) is an amino acid sequence that directs a protein to the nucleus. In certain embodiments, the NLS may be an SV40 NLS (e.g., having the amino acid sequence of SEQ ID NO: 644). A fusion protein may contain an NLS at its N-terminus, C-terminus, or both, and / or the NLS may be embedded in the center of the fusion protein (e.g., at the N-terminus or C-terminus of the DNA-binding domain or effector domain).

[0160] In some embodiments, the fusion protein may contain two NLSs. The fusion protein may contain two NLSs at its N-terminus or C-terminus. The fusion protein may contain one NLS located at its N-terminus and one NLS embedded in the center of the fusion protein, or one NLS located at its C-terminus and one NLS embedded in the center of the fusion protein. The fusion protein may contain two NLSs embedded in the center of the fusion protein.

[0161] In some embodiments, the fusion protein may comprise four NLSs. The fusion protein may comprise at least two (e.g., two, three, or four) NLSs at its N-terminus or C-terminus. The fusion protein may comprise at least one (e.g., one, two, three, or four) NLS embedded in the center of the fusion protein. In certain embodiments, the fusion protein may comprise two NLSs at its N-terminus and two NLSs at its C-terminus.

[0162] The NLS described herein can be an endogenous NLS sequence. In certain embodiments, the NLS described herein comprises the amino acid sequence of any one of SEQ ID NOs: 644-649, or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the selected sequence. In certain embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 644. Additional NLSs are known in the art.

[0163] In some embodiments, an epigenetic editor comprising a fusion protein comprising at least one NLS at the N-terminus and at least one NLS at the C-terminus may increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more, compared to an epigenetic editor having a corresponding fusion protein that does not have at least one NLS at the N-terminus and at least one NLS at the C-terminus.

[0164] In some embodiments, an epigenetic editor comprising a fusion protein comprising two NLSs at the N-terminus and two NLSs at the C-terminus may increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more, compared to an epigenetic editor having a corresponding fusion protein that does not have two NLSs at the N-terminus and two NLSs at the C-terminus.

[0165] C. Tag The epigenetic editors provided herein can include one or more additional sequences (tags) for tracking, detecting, and localizing the editor. In some embodiments, the epigenetic editor includes one, two, three, four, five, six, seven, eight, nine, ten, or more detectable tags. Each of the detectable tags can be the same or different.

[0166] For example, epigenetic editor fusion proteins can include a cytoplasmic localization sequence, an external export sequence, e.g., a nuclear export sequence, or other localization sequence, as well as a sequence tag useful for solubilizing, purifying, or detecting the fusion protein. Suitable protein tags provided herein include, but are not limited to, a biotin carboxylase carrier protein (BCCP) tag, a myc tag, a calmodulin tag, a FLAG tag, a hemagglutinin (HA) tag, a polyhistidine tag (also referred to as a histidine tag or His tag), a maltose-binding protein (MBP) tag, a nus tag, a glutathione-S-transferase (GST) tag, a green fluorescent protein (GFP) tag, a thioredoxin tag, an S tag, a Softag (e.g., Softag 1 or Softag 3), a strep tag, a biotin ligase tag, a FlAsH tag, a V5 tag, and an SBP tag. Additional suitable sequences will be apparent to those skilled in the art.

[0167] D. Fusion Protein Construction The components of the epigenetic editor fusion proteins described herein may be structured in different configurations. For example, the DNA binding domain may be at the C-terminus, the N-terminus, or 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 on both sides by epigenetic effector domains or additional domains. In some embodiments, where "DBD" represents the DNA binding domain and "ED" represents the effector domain, the epigenetic editor may be -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' Includes the configuration of.

[0168] In some embodiments, the 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 can be any of those described herein in any combination. The DBD, DNMT, and repressor domains can be in any configuration with any of said domains at the N-terminus, C-terminus, or center of the fusion protein. In some embodiments, the epigenetic editor comprises: N']-[DNMT domain]-[DBD]-[repressor domain]-[C' N']-[repressor domain]-[DBD]-[DNMT domain]-[C' N']-[DNMT domain]-[repressor domain]-[DBD]-[C' or N']-[repressor domain]-[DNMT domain]-[DBD]-[C' The present invention also includes fusion proteins having the following structure:

[0169] In some embodiments, the 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 structure, multiple connecting structures "]-[" may be the same, or each may be a different linker, tag, NLS, or peptide bond. In some embodiments, the DNMT domain may comprise any one of the domains in Table 6, or any combination or homolog thereof. In certain embodiments, the DNMT domain comprises DNMT3A or a truncated version thereof, DNMT3L or a truncated version thereof, or both. In certain embodiments, the DBD is a catalytically inactive polynucleotide-guided DNA binding domain (e.g., dCas9) or a ZFP domain. In certain embodiments, the repressor domain comprises any one of the domains shown in Table 4 or 5, or any combination or homolog thereof. For example, the repressor domain may be a KRAB domain. In certain embodiments, the repressor domain is a ZFP28, ZN627, KAP1, MeCP2, HP1b, CBX8, CDYL2, TOX, Tox3, Tox4, EED, RBBP4, RCOR1, or SCML2 domain, or a fusion of two of said domains (e.g., a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB). In certain embodiments, the repressor domain is a KRAB domain from ZFP28, ZN627, ZIM3, or KOX1.

[0170] In some embodiments, the epigenetic editor comprises: N']-[DNMT3A-DNMT3L]-[DBD]-[repressor]-[C' N']-[repressor]-[DBD]-[DNMT3A-DNMT3L]-[C' N']-[repressor]-[DBD]-[DNMT3A]-[C' N']-[DNMT3A]-[DBD]-[repressor]-[C' N']-[repressor]-[DBD]-[DNMT3A]-[DNMT3L]-[C' N']-[DNMT3A]-[DNMT3L]-[DBD]-[repressor]-[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 the connecting structure]-[ is any one of the linkers described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, repressor, DNMT3A, and DNMT3L domains can be any of those described herein in any combination. For example, the DNMT3A and DNMT3L domains can be selected from those in Table 6. In certain embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain, the repressor domain is a KRAB domain 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.

[0171] In some embodiments, the epigenetic editor comprises: 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' and wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and the connecting structure]-[ is any one of the linkers 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 can be any of those described herein in any combination. In certain 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.

[0172] Specific constructs contemplated herein include: DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1 KRAB (configuration 1), DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1 KRAB (construct 2); NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS (configuration 3), NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS (construct 4), NLS-NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS-NLS (construct 5), and NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS-NLS (configuration 6) Examples include: DNMT3L and DNMT3A can be derived from human parent proteins, mouse parent proteins, or any combination thereof. In certain embodiments, DNMT3L and DNMT3A are derived from mouse and human parent proteins (mDNMT3L and hDNMT3A), respectively. In certain embodiments, DNMT3L and DNMT3A are both derived from human parent proteins (hDNMT3L and hDNMT3A). In some embodiments, dCas9 is dSpCas9. In some embodiments, KOX1 is human KOX1. Any of configurations 1-6 in which the KOX1 KRAB domain is replaced by a ZFP28, ZN627, or ZIM3 KRAB domain are also contemplated. In some embodiments, ZFP28, ZN627, and ZIM3 are human ZFP28, ZN627, and ZIM3, respectively. In certain embodiments, the fusion construct comprises the configuration: NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS-NLS (configuration 7), NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1 KRAB-NLS-NLS (configuration 8), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZFP28 KRAB-NLS-NLS (configuration 9), NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZFP28 KRAB-NLS-NLS (construct 10), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZN627 KRAB-NLS-NLS (configuration 11), NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZN627 KRAB-NLS-NLS (construct 12), NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZIM3 KRAB-NLS-NLS (construct 13), or NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZIM3 KRAB-NLS-NLS (configuration 14) may have:

[0173] In certain embodiments, the fusion constructs described herein may have configuration 1 and may include SEQ ID NO:658, 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:658 below, the XTEN linker is underlined, the W linker is bold, underlined, and italicized, the NLS sequence is bold, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the dCas9 domain is bold and italicized, and the KOX1 KRAB domain is underlined and bold. TIFF2025516294000021.tif137150TIFF2025516294000022.tif47150(sequence number 658)

[0174] In certain embodiments, the fusion constructs described herein may comprise the sequence provided below (SEQ ID NO: 1495), 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: 1495 below, the XTEN linker is underlined, the W linker is bolded, underlined, and italicized, the NLS sequence is 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. TIFF2025516294000023.tif141149TIFF2025516294000024.tif42148(sequence number 1495)

[0175] In certain embodiments, the fusion constructs described herein may have configuration 2 and may include SEQ ID NO:659, 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:659 below, the XTEN linker is underlined, the W linker is bold, underlined, and italicized, the NLS sequence is bold and underlined, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the ZFP domain is bold, and the KOX1 KRAB domain is underlined and bold. The variable amino acids represented by X are amino acids in the DNA recognition helix of the zinc finger, and the italicized XX can be either TR, LR, or LK. TIFF2025516294000025.tif79148 (sequence number 659) In certain embodiments, the six "XXXXXX" regions in SEQ ID NO: 659 comprise the F1-F6 amino acid sequences, in order, set forth in Table 1 for any one of ZF001-ZF048. [Linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 651). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 652). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 651, and the other linker sequence may have the amino acid sequence of SEQ ID NO: 652.

[0176] In certain embodiments, the fusion constructs described herein may comprise the sequence provided below (SEQ ID NO: 1496), 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: 1496 below, the XTEN linker is underlined, the W linker is bold, underlined, and italicized, the NLS sequence is bold and underlined, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the ZFP domain is bold, and the KOX1 KRAB domain is underlined and bold. The variable amino acids represented by X are amino acids of the DNA recognition helix of the zinc finger, and the italicized XX can be either TR, LR, or LK. TIFF2025516294000026.tif79148 (sequence number 1496)

[0177] In certain embodiments, the six "XXXXXXX" regions in SEQ ID NO: 1496 comprise the F1-F6 amino acid sequences, in order, set forth in Table 1 for any one of ZF001-ZF048. [Linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 651). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 652). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 651, and the other linker sequence may have the amino acid sequence of SEQ ID NO: 652.

[0178] In some embodiments, the fusion protein may further comprise a Dnmt3A ADD domain downstream of the Dnmt3A domain sequence, for example, as disclosed in SEQ ID NO: 658, 659, 1495, or 1496 disclosed above. In some embodiments, the ADD sequence is located between the Dnmt3A and Dnmt3L sequences of the fusion protein. In some embodiments, the ADD sequence is at the C-terminus of the Dnmt3A domain. In some embodiments, the Dnmt3A sequence and the ADD sequence are separated by a linker, for example, a linker disclosed herein. In some embodiments, the ADD sequence and the Dnmt3L sequence are separated by a linker, for example, a linker disclosed herein. In some embodiments, the ADD domain has the sequence: MAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVL SLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL (SEQ ID NO: 1497) Includes:

[0179] In certain embodiments, the fusion constructs described herein may have configuration 7 and may include SEQ ID NO: 660, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0180] In certain embodiments, the fusion constructs described herein may have configuration 9 and may include SEQ ID NO: 661, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0181] In certain embodiments, the fusion constructs described herein may have configuration 11 and may include SEQ ID NO: 662, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0182] In certain embodiments, the fusion constructs described herein may have configuration 13 and may include SEQ ID NO: 663, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0183] In some embodiments, a fusion construct described herein (e.g., a fusion construct of any one of constructs 1-14) is within an expression construct that includes a WPRE sequence, a polyadenylation site, or both. In certain embodiments, the WPRE sequence is in the 3' non-coding region. In certain embodiments, the WPRE sequence is upstream from the polyadenylation site. In certain embodiments, an expression construct includes a fusion construct (e.g., any one of constructs 1-14) and a WPRE sequence in the 3' non-coding region upstream from the polyadenylation site.

[0184] In some embodiments, the fusion constructs described herein can have the sequence of any one of fusion proteins 1-12 shown in Example 12.

[0185] Multiple fusion proteins can be used to activate or suppress a target gene or multiple target genes. For example, an epigenetic editor fusion protein containing a DNA-binding domain (e.g., a dCas9 domain) and an effector domain can be co-delivered with two or more guide polynucleotides (e.g., gRNAs), each targeting a different target DNA sequence. The target sites of the two DNA-binding domains can be the same or adjacent to each other, or can be separated by, for example, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, about 600 base pairs, or more. In addition, when targeting double-stranded DNA, e.g., an endogenous gene locus, the guide polynucleotides can target the same strand or different strands (one or more plus strands and / or one or more minus strands).

[0186] V. Target Sequence The epigenetic editors herein can be directed to a target sequence in PCSK9 to result in epigenetic modification of the PCSK9 gene. As used herein, a "target sequence," "target site," or "target region" is a nucleic acid sequence present in a gene of interest; in some cases, the target sequence may be outside but near the gene of interest, where methylation of the target sequence or binding of the target sequence by a repressor represses expression of the gene. In some embodiments, the target sequence can be a hypomethylated or hypermethylated nucleic acid sequence.

[0187] The target sequence can be in any part of the target gene. In some embodiments, the target sequence is part of or near a non-coding 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 enhancer. In some embodiments, the target sequence is adjacent to, overlaps with, or encompasses a CpG island. In certain embodiments, the target sequence is located 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) adjacent to the PCSK9 TSS. In certain embodiments, the target sequence is located within 500 bp adjacent to the PCSK9 TSS. In certain embodiments, the target sequence is located within 1000 bp adjacent to the PCSK9 TSS.

[0188] 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, e.g., dCas9) and an effector domain. The guide polynucleotide sequence may be designed to have complementarity to the target sequence or identity to the opposite 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 the protospacer sequence in the target sequence. In certain embodiments, the guide polynucleotide comprises a spacer sequence that is 100% identical to the protospacer sequence in the target sequence.

[0189] In some embodiments, when the DNA binding domain of an epigenetic editor described herein is a zinc finger array, the target sequence can be recognized by said zinc finger array.

[0190] In some embodiments, when the DNA binding domain of the epigenetic editor described herein is a TALE, the target sequence can be recognized by said TALE.

[0191] The target sequence described herein can be specific to one copy of the target gene or one allele of the target gene. Thus, the epigenetic modification and modulation of its expression can be specific to one copy or one allele of the target gene. For example, the epigenetic editor can suppress the expression of a specific copy (e.g., a copy associated with a disease or condition, or one with a mutation associated with a disease or condition) that has a target sequence recognized by the DNA binding domain.

[0192] In some embodiments, the target PCSK9 genomic region can be contained within the sequence shown below (chr1:55038548-55040548), with or without the terminal A: (SEQ ID NO: 1488)

[0193] In some embodiments, the target sequence can be GRCh38 Chr1:55039228-55040296, as shown below: (SEQ ID NO: 1489)

[0194] VI. Epigenetic Modifications The epigenetic editors described herein can make sequence-specific epigenetic modifications (e.g., chemical modifications) of a target gene having a target sequence. Such epigenetic modulation can be safer and more easily reversible than modulation resulting from gene editing, for example, using the generation of DNA double-strand breaks. In some embodiments, the epigenetic modulation can reduce or silence the target gene. In some embodiments, the modification is at a specific site in the target sequence. In some embodiments, the modification is at a specific allele of the target gene. Thus, the epigenetic modification can result in modulation (e.g., reduction) of expression of one copy of the target gene having a specific allele, but not other copies of the target gene. In some embodiments, the specific allele is associated with a disease, condition, or disorder.

[0195] In some embodiments, the epigenetic modification reduces or stops transcription of a target gene having a target sequence. In some embodiments, the epigenetic modification reduces or stops transcription of a copy of the target gene having a specific allele recognized by the epigenetic editor. In some embodiments, the epigenetic editor reduces the expression level of or eliminates the expression of a protein encoded by the target gene. In some embodiments, the epigenetic editor reduces the expression level of or eliminates the expression of a protein encoded by a copy of the target gene having a specific allele recognized by the epigenetic editor. The target PCSK9 gene can be epigenetically modified in vitro, ex vivo, or in vivo.

[0196] The effector domain of the epigenetic editors described herein can alter (e.g., install or remove) chemical modifications in nucleotides of a target gene or in histones associated with the target gene. The chemical modifications can be at a single nucleotide or a single histone, or at 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, 1000, 1500, 2000, 2500, 3000, or more nucleotides.

[0197] In some embodiments, the effector domain of an epigenetic editor described herein can alter CpG dinucleotides within a target gene, in some embodiments, all CpG dinucleotides within 2000 bp, 1500 bp, 1000 bp, 500 bp, or 200 bp flanking the target sequence (e.g., within a modification site described herein) are altered according to a modification type described herein, 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 compared to the original state of the gene or the gene in a comparable cell not contacted with an 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 compared to the original state of the gene or the gene in a comparable cell not contacted with an epigenetic editor, in some embodiments, one single CpG dinucleotide is altered compared to the original state of the gene or the gene in a comparable cell not contacted with an epigenetic editor.

[0198] The effector domain of the epigenetic editors described herein can alter the histone modification state of histones associated with or bound to a target gene. For example, the effector domain can place a modification at one or more lysine residues in the histone tails of the histones associated with the target gene. In some embodiments, the effector domain can cause deacetylation of one or more histone tails of the 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 can cause H3K9, H3K27, or H4K20 methylation (e.g., one or more of H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3 methylation) in one or more histone tails associated with the target gene, thereby reducing or silencing expression of the target gene.

[0199] In some embodiments, all histone tails of histones bound to DNA nucleotides within 2000 bp, 1500 bp, 1000 bp, 500 bp, or 200 bp flanking the target sequence are altered according to a modification type described herein, compared to the original state of the chromosome or a chromosome in a comparable cell not contacted with an 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 compared to the original state of the chromosome or compared to a chromosome in a comparable cell not contacted with an 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 histone tails of the bound histones are altered compared to the original state of the chromosome or a chromosome in a comparable cell not contacted with an epigenetic editor. For example, one single histone tail of the bound histones can be altered compared to the original state of the chromosome or a chromosome in a comparable cell not contacted with an epigenetic editor. As another example, one single bound histone octamer can be altered compared to the original state of the chromosome or a chromosome in a comparable cell not contacted with an epigenetic editor.

[0200] The chemical modification placed at a target gene DNA nucleotide or histone residue can be at or near a target sequence in the target gene. In some embodiments, the effector domain of the epigenetic editor described herein changes the 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 a target sequence in the target gene. In some embodiments, the effector domain alters the chemical modification state of a nucleotide or a histone tail attached to a nucleotide that is 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 adjacent to the target sequence. As used herein, "adjacent to" refers to nucleotide positions 5' to the 5' end and 3' to the 3' end of a particular sequence, eg, a target sequence.

[0201] In some embodiments, the effector domain mediates or induces a change in the chemical modification of a nucleotide or histone tail attached to a nucleotide distal to the target sequence. Such modification can be initiated near the target sequence and then extend to one or more nucleotides distal to the target sequence in the target gene. For example, the effector domain can initiate a change in the chemical modification state of one or more nucleotides or one or more histone residues attached to one or more nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 nucleotides adjacent to the target sequence, and the change in the chemical modification state can be extended to the target sequence in the target gene. for one or more nucleotides either upstream or downstream of the target sequence, for 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. In certain embodiments, chemical modifications may begin at less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides in the target gene and may extend to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides in the target gene. In some embodiments, chemical modifications extend to the entire nucleotides of the target gene. Additional proteins or transcription factors, such as transcription repressors, methyltransferases, or transcriptional regulatory scaffold proteins, may be involved in the extension of the chemical modifications. Alternatively, an epigenetic editor alone may be involved.

[0202] In some embodiments, the epigenetic editors described herein reduce 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 relative to a control cell, control tissue, or control subject (e.g., in the absence of the epigenetic editor), as measured by transcription of the target gene copy in a cell, tissue, or subject. In some embodiments, the epigenetic editors described herein reduce expression of a target gene copy 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 relative to a control cell, control tissue, or control subject, as measured by transcription of the target gene copy in a cell, tissue, or subject. In certain embodiments, the copy of the target gene has a specific sequence or allele that is recognized by the epigenetic editor. In certain embodiments, the epigenetically modified copy encodes a functional protein, and thus, the epigenetic editors disclosed herein can reduce or abolish the expression and / or function of the protein.For example, the epigenetic editors described herein may reduce the expression and / or function of a protein encoded by a target gene in a cell, tissue, or subject 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 comparison to a control cell, control tissue, or control subject.

[0203] Modulation of target gene expression can be assayed by determining any parameter that is indirectly or directly affected by expression of the target gene, such as 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 luciferase, CAT, beta-galactosidase, or GFP, changes in signal transduction, changes in phosphorylation and dephosphorylation, changes in receptor-ligand interactions, changes in second messengers such as cGMP, cAMP, IP3, and Ca2 +These include changes in the concentration of ATP, changes in cell growth, changes in angiogenesis, 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, such as measuring RNA or protein levels, measuring RNA stability, and / or determining downstream or reporter gene expression. Readouts can be, for example, by 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, etc.

[0204] Methods for determining the expression level of a gene, e.g., a target of an epigenetic editor, can include determining the transcript level of the gene by, for example, 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. The level of protein expressed from the gene can be determined by, for example, Western blotting, enzyme-linked immunosorbent assay, mass spectrometry, immunohistochemistry, or flow cytometry analysis. The level of gene expression product may be normalized to an internal standard, e.g., total messenger ribonucleic acid (mRNA), or the expression level of a particular gene, e.g., a housekeeping gene.

[0205] In some embodiments, the effect of an epigenetic editor in modulating target gene expression can be tested using a reporter system. For example, an epigenetic editor may be designed to target a reporter gene encoding a reporter protein, e.g., a fluorescent protein. Expression of the reporter gene in such a model system can be monitored, for example, by flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a cell population can be transfected with a vector carrying a reporter gene. The vector can be constructed such that the reporter gene is expressed when the vector is transfected into cells. Suitable reporter genes include genes encoding fluorescent proteins, e.g., green, yellow, cherry, cyan, or orange fluorescent proteins. A cell population carrying a reporter system can be transfected with DNA, mRNA, or a vector encoding an epigenetic editor targeted to the reporter gene.

[0206] VII. Pharmaceutical Compositions In one aspect, the present disclosure provides pharmaceutical compositions comprising as an active ingredient (or sole active ingredient) one or more epigenetic editors or components thereof (e.g., fusion proteins and / or guide polynucleotides) described herein, or nucleic acid molecules encoding said epigenetic editors or components thereof. For example, a pharmaceutical composition can comprise a nucleic acid molecule encoding a fusion protein (and, if applicable, guide polynucleotide) of an epigenetic editor described herein. In some embodiments, separate pharmaceutical compositions comprise the fusion protein and the guide polynucleotide. A pharmaceutical composition can also comprise cells that have undergone epigenetic modifications mediated or induced by an epigenetic editor provided herein.

[0207] Generally, the epigenetic editors or components thereof described herein of the present disclosure, or nucleic acid molecules encoding said epigenetic editors or components thereof, are suitable for administration as a formulation with one or more pharmaceutically acceptable excipients, for example, as described below.

[0208] The term "excipient" is used herein to describe any ingredient other than the compound of the present disclosure. The choice of excipient will largely depend on factors such as the specific mode of administration, the excipient's effect on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like. 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 isotonicity agents, such as 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.

[0209] Formulations of pharmaceutical compositions suitable for parenteral administration typically include 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 continuous administration.

[0210] VIII. Delivery method In some embodiments, the epigenetic editor or a component thereof is introduced into a target cell in the form of a nucleic acid molecule encoding the epigenetic editor or a component thereof; therefore, the pharmaceutical compositions herein comprise the nucleic acid molecule. Such nucleic acid molecules can be, for example, DNA, RNA, or mRNA, and / or modified nucleic acid sequences (e.g., having chemical modifications, a 5' cap, or one or more 3' modifications). In some embodiments, the nucleic acid molecule can be delivered as naked DNA or RNA, for example, using transfection or electroporation, or can be conjugated to a molecule (e.g., N-acetylgalactosamine) that facilitates uptake by the target cell. In some embodiments, the nucleic acid molecule can be included in a nucleic acid expression vector, which can 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. The vector may also include a sequence encoding a signal peptide (e.g., for nuclear, nucleolar, or mitochondrial localization) associated with (e.g., inserted or fused to) the protein-encoding sequence.

[0211] 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., vectors derived from retroviruses such as murine leukemia virus or spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. In certain embodiments, the vector is a plasmid or viral vector. Viral particles or virus-like particles (VLPs) can also be used to deliver nucleic acid molecules encoding the epigenetic editors or components thereof described herein. For example, "empty" viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0212] In certain embodiments, the epigenetic editors described herein or components thereof are encoded by nucleic acid sequences present in one or more viral vectors or suitable capsid proteins of any viral vector, including 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., Moloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., vectors based on HIV and FIV), and herpesviral vectors (e.g., HSV-2).

[0213] In some embodiments, delivery involves an adeno-associated virus (AAV) vector. AAV vector delivery can be particularly useful when the DNA binding domain of the epigenetic editor fusion protein is a zinc finger array. Without wishing to be bound by any theory, the small size of a zinc finger array compared to a large DNA binding domain, such as a Cas protein domain, may allow such fusion proteins to be conveniently packaged into a viral vector, such as an AAV vector.

[0214] Any AAV serotype, e.g., a human AAV serotype, can be used in the AAV vectors 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), and variants thereof. In some embodiments, the AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to wild-type AAV. In certain embodiments, AAV variants may be engineered so that their capsid proteins have reduced immunogenicity or enhanced transduction capacity in humans. In some cases, one or more regions of at least two different AAV serotypes are shuffled and reassembled to generate chimeric variants. For example, a chimeric AAV may contain inverted terminal repeats (ITRs) of a serotype that is heterologous compared to the capsid serotype. The resulting chimeric AAV may have different antigenic reactivity or recognition compared to its parent serotype. In some embodiments, a chimeric AAV variant contains amino acid sequences derived from two, three, four, five, or more different AAV serotypes.

[0215] 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 gene gun, lipid-mediated transfection, heat shock transfection, small DNA-mediated transfection, lipofection, cationic drug-mediated transfection, and transfection with liposomes, immunoliposomes, exosomes, or cationic facial amphiphiles (CFAs). In certain embodiments, one or more mRNAs encoding the epigenetic editor fusion proteins described herein may be co-electroporated with one or more guide polynucleotides (e.g., gRNAs) described herein. One important category of non-viral nucleic acid vectors is nanoparticles, which may be organic (e.g., lipid) or inorganic (e.g., gold). For example, organic (e.g., lipid and / or polymer) nanoparticles may be suitable for use as delivery vehicles in certain embodiments of the present disclosure.

[0216] In some embodiments, delivery is achieved using lipid nanoparticles (LNPs). LNP compositions are typically micrometers or smaller in size and may contain a lipid bilayer. In some embodiments, LNPs refer to any particle having a diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. In some embodiments, nanoparticles may range in size from 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 25 to 100 nm, 35 to 75 nm, or 25 to 60 nm. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.

[0217] The LNPs described herein can be made from cationic, anionic, or neutral lipids. In some embodiments, the LNPs can include a neutral lipid, such as the fusogenic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as a helper lipid to enhance transfection activity and nanoparticle stability. In some embodiments, the LNPs can include hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or lipid combination known in the art can be used to produce the LNPs. Lipids can be combined in any molar ratio to produce the LNPs. In some embodiments, the LNPs are liver-targeted (e.g., preferentially or specifically target the liver) LNPs.

[0218] Any type of cell can be targeted for delivery of the epigenetic editors described herein or components thereof. For example, the cell can be a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a mammalian (e.g., human) cell. Human cells can include, for example, hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and hepatic sinusoidal endothelial cells.

[0219] In some embodiments, the epigenetic editors described herein or components thereof are delivered to host cells for transient expression, e.g., via a transient expression vector. Transient expression of the epigenetic editor or components thereof can result in long-term or permanent epigenetic modification of the target gene. For example, the epigenetic modification can be stable for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more, or for 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months or more after introducing the epigenetic editor into the host cell. The epigenetic modification can be maintained after one or more mitotic and / or meiotic events of the host cell. In certain embodiments, the epigenetic modification is maintained across generations in progeny generated from or derived from the host cell.

[0220] IX. Therapeutic Uses of Epigenetic Editors The present disclosure also provides methods of treating or preventing a condition in a subject, the methods comprising administering to the subject an epigenetic editor or pharmaceutical composition described herein. The epigenetic editor can effect epigenetic modification of a target polynucleotide sequence within 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 expression of the target gene to an extent sufficient to achieve a desired effect, e.g., a therapeutically relevant effect, e.g., prevention or treatment of the disease, condition, or disorder.

[0221] In some embodiments, the subject is administered a system for modulating (e.g., inhibiting) expression of PCSK9, wherein the system comprises (1) a fusion protein of an epigenetic editor described herein and, if associated, a guide polynucleotide, or (2) a nucleic acid molecule encoding the fusion protein and, if associated, a guide polynucleotide.

[0222] "Treat," "treating," and "treatment" refer to a method of alleviating or abrogating at least one of a biological disorder and / or its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of symptoms of the disease, disorder, or condition. Furthermore, references to "treatment" herein include references to curative, palliative, and preventative treatment. In some embodiments, alleviating symptoms compared to comparable untreated controls can include a reduction in symptoms 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.

[0223] In some embodiments, the subject may be a mammal, e.g., a human, hi some embodiments, the subject is selected from non-human primates, e.g., chimpanzees, cynomolgus monkeys, macaques, and other ape and monkey species.

[0224] In some embodiments, the human patient has a condition selected from hypercholesterolemia (e.g., familial hypercholesterolemia, e.g., heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), or established atherosclerotic cardiovascular disease (ASCVD)), or renal insufficiency (RI).

[0225] In some embodiments, a patient to be treated with an epigenetic editor of the disclosure has undergone prior treatment for the condition to be treated (e.g., hypercholesterolemia (e.g., HeFH, HoFH, HF, or established ASCVD) or RI). In other embodiments, the patient has not undergone such prior treatment. In some embodiments, the patient has failed prior treatment for the condition (e.g., prior hypercholesterolemia treatment).

[0226] The epigenetic editors of the present disclosure can be administered to patients with the conditions described herein in a therapeutically effective amount. As used herein, "therapeutically effective amount" refers to the amount of therapeutic agent administered that will alleviate to some extent one or more of the symptoms of the disorder being treated and / or result in the clinical endpoint desired by a medical professional. The effective amount of a therapeutic agent can be measured by its ability to stabilize disease progression and / or alleviate symptoms, preferably reverse disease progression, in a patient. The ability of the epigenetic editors of the present disclosure to reduce or silence PCSK9 expression can be evaluated, for example, by in vitro assays described herein and in suitable animal models predictive of efficacy in humans. An appropriate dosage regimen will be selected to provide the optimal therapeutic response in each specific situation, administered, for example, as a single bolus or continuous infusion, with the possibility of adjusting the dosage as indicated by the exigencies of each case.

[0227] The epigenetic editors of the present disclosure can be administered without additional therapeutic treatment, i.e., as a standalone therapy (monotherapy). Alternatively, treatment with the epigenetic editors of the present disclosure can include at least one additional therapeutic treatment (combination therapy). In some embodiments, the additional therapeutic agent is any known in the art for treating hypercholesterolemia or RI. Therapeutic agents include, but are not limited to, statins, fibrates, HMG-CoA reductase inhibitors, niacin, bile acid modulators or sequestrants, cholesterol absorption inhibitors or modulators, CETP inhibitors, MTTP inhibitors, and PPAR agonists.

[0228] An epigenetic editor or a component thereof (or a nucleic acid molecule encoding an epigenetic editor or a component thereof) of the present disclosure can be administered by any method accepted in the art, for example, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, intralymphatically, or intraperitoneally. In certain embodiments, a pharmaceutical composition of the present disclosure is administered to a subject intravenously.

[0229] X.Definition As used herein, the term "nucleic acid" refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form, including DNA and RNA. A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, linked through the phosphate group. "Base" includes naturally occurring compounds, e.g., adenine, thymine, guanine, cytosine, uracil, inosine, and purines and pyrimidines, including natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modified versions incorporating new reactive groups, e.g., amines, alcohols, thiols, carboxylic acids, alkyl halides, and the like. Nucleic acids can contain known nucleotide analogs and / or modified backbone residues or linkages, which may be synthetic, naturally occurring, or non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are well known in the art and can result in nucleic acid molecules with enhanced cellular uptake, reduced immunogenicity, and / or increased stability in the presence of nucleases.

[0230] As used herein, an "isolated" or "purified" nucleic acid molecule is a nucleic acid molecule that exists away from its native environment. For example, an "isolated" or "purified" nucleic acid molecule (1) is separated from the nucleic acid of its source, genomic DNA or cellular RNA, and / or (2) is not found in nature. In some embodiments, an "isolated" or "purified" nucleic acid molecule is a recombinant nucleic acid molecule.

[0231] It will be understood that in addition to the specific proteins and nucleic acid molecules referred to herein, the present disclosure also contemplates the use of variants, derivatives, homologs, and fragments thereof. A variant of any given sequence can have a specific sequence of residues (whether amino acid or nucleic acid residues) that have been altered in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its intrinsic functions. A variant sequence can be obtained by adding, deleting, substituting, modifying, replacing, and / or varying at least one residue (in some embodiments, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, or 20 or less residues) present in the naturally occurring sequence. For certain proteins described herein (e.g., the KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), the present disclosure also contemplates either naturally occurring forms of the proteins or variants or homologs that retain at least one of their endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98%, or 99% of that function compared to the particular protein described).

[0232] Several exemplary fusion proteins encompassed by the present disclosure are provided herein. Those skilled in the art will understand that these exemplary proteins are non-limiting examples and that additional proteins are encompassed within the scope of the present disclosure. For example, when an exemplary fusion protein is provided that includes a particular domain, e.g., a mammalian DNMT3A, DNMT3L, and / or KRAB domain, e.g., a human or mouse DNMT3A, DNMT3L, and / or KRAB domain, those skilled in the art will recognize that, in some embodiments, a fusion protein having the same architecture but in which one or more of the mammalian domains are replaced with homologous domains from another mammal, e.g., one or more mouse domains are replaced with one or more human domains, is also encompassed by the present disclosure. For example, when an exemplary fusion protein is provided that includes a mouse DNMT3L domain, a fusion protein having the same architecture but in which the mouse DNMT3L domain is replaced with a human DNMT3L domain is also encompassed.

[0233] As used herein, a homolog of any polypeptide or nucleic acid sequence contemplated herein includes a sequence having a certain homology with the wild-type amino acid and nucleic acid sequence. Homologous sequences can include sequences, e.g., amino acid sequences, that may be at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 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 percentage of residues that are the same in two sequences when aligned for maximum correspondence. In some embodiments, the length of the 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 can be measured using sequence analysis software (e.g., the Genetics Computer Group's Sequence Analysis Software Package, 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, the BLAST program can be used, with a probability score of e-3 to e-100 indicating closely related sequences.

[0234] The percent identity of two nucleotide or polypeptide sequences is determined, for example, using BLAST® with default parameters (available at the website of the U.S. National Library of Medicine's National Center for Biotechnology Information). 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.

[0235] It will be understood that the numbering of specific positions or residues in a polypeptide sequence will depend on the particular protein and numbering scheme used. Numbering may differ, for example, between precursors of the mature protein and the mature protein itself, and sequence differences between species may affect numbering. One of skill in the art will be able to identify any homologous proteins and their respective residues in their respective encoding nucleic acids by methods well known in the art, such as sequence alignment and determination of homologous residues.

[0236] The terms "modulate" or "alter" refer to a change in the amount, degree, or extent of a function. For example, the epigenetic editors described herein can modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or repressing transcription of a gene operably linked to the promoter sequence. As another example, the epigenetic editors described herein can block RNA polymerase from transcribing a gene or inhibit translation of an mRNA transcript. The terms "inhibit," "suppress," "repress," "silencing," and the like, when used in reference to the epigenetic editors described herein or components thereof, refer to reducing or preventing the activity (e.g., transcription) of a nucleic acid sequence (e.g., a target gene) or protein compared to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or components thereof. The terms can include partially or completely blocking activity, or preventing or delaying activity. Inhibited activity can be, for example, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 75% less, 80% less, 85% less, 90% less, 91% less, 92% less, 93% less, 94% less, 95% less, 96% less, 97% less, 98% less, or 99% less than that of the control, or can be, for example, at least 1.5-, 2-, 3-, 4-, 5-, or 10-fold less than that of the control.

[0237] The term "about" or "approximately" means within an acceptable range of error for a 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 standard deviations, as is customary for a given value. When a particular value is described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable range of error for that particular value. It is understood that the ranges provided herein include all truncated forms of values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange 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 foregoing integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested sub-ranges" extending from either endpoint of the range are specifically contemplated. For example, nested sub-ranges of the exemplary range of 1 to 50 could include 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.

[0238] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings 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 the case of conflict, the present specification, including definitions, will control. Furthermore, unless otherwise required by context, singular terms shall include plural terms 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 the stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless otherwise indicated, the recitation of a list of elements herein includes any of the elements, alone or in any combination. The description of an embodiment herein includes that embodiment as a single embodiment or in combination with any other embodiment herein. All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety. To the extent that a reference incorporated by reference conflicts with the present disclosure contained herein, it is intended that the present specification take precedence and / or supersede any such conflicting material. Although several documents are cited herein, this citation does not constitute an acknowledgment that any of these documents form part of the common general knowledge in the art.

[0239] According to this disclosure, a backward reference in a dependent claim is intended as a shorthand for a direct and clear disclosure of the respective individual claim combination indicated by the backward reference. Furthermore, the headings herein are made for ease of organization and are not intended in any way to limit the scope of the compositions and methods recited in the claims.

[0240] Some protein sequences, e.g., some fusion protein sequences, provided herein contain peptide tags, e.g., His6 tags or DYKDDDDK (SEQ ID NO: 1528) tags, which are useful for detecting and / or purifying the tagged protein but do not affect the function of the protein. These and additional suitable peptide tags are well known to those of skill in the art. It will be apparent to those of skill in the art that the disclosed tags can be substituted with other suitable peptide tags, and that fusion proteins of the same or highly similar sequence but not containing such peptide tags, e.g., those in which the peptide tag has been truncated or produced without the peptide tag, are equally suitable for carrying out embodiments of the present disclosure.

[0241] In order that this disclosure may be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the disclosure in any way.

[0242] [Example] [Example 1] Fusion protein design and synthesis A fusion protein ("CRISPR-off") containing dCas9, DNMT3A, DNMT3L, and KOX1 KRAB was designed and constructed. From N- to C-terminus, this protein has the domains DNMT3A-linker-DNMT3L-XTEN80-NLS-dSpCas9-NLS-XTEN16-KOX1 KRAB (SEQ ID NOs: 658 and 1495). The CRISPR-off plasmid construct was described by Nunez (Nunez et al., Cell (2021) 184(9):2503-19) and ordered from Twist Biosciences.

[0243] A ZF fusion protein ("ZF-off") containing DNMT3A, 3L, and KOX1Krab was also constructed. This construct has the general structure DNMT3A-linker-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1Krab (SEQ ID NOs: 659 and 1496).

[0244] [Example 2] Selection of target PCSK9 sequences for gRNA epigenetic silencing gRNAs targeting ±1 kb from the PCSK9 TSS were computationally designed for human (GRCh38), mouse (mm10), and cynomolgus monkey (Macaca fascicularis) (5.0) PCSK9 using the Benchling gRNA platform (Benchling (2021), available from benchling.com). gRNAs containing poly-TTTT sequences were initially discarded. We performed gRNA off-target analysis using CasOFFinder (Bae et al., Bioinformatics (2014) 30(10):1473-5). gRNAs were discarded if they matched multiple locations across the genomes constructed for each independent species.

[0245] Cross-reactivity sequence analysis was performed on the human PCSK9 gRNA to annotate sequence mismatches with the cynomolgus monkey or mouse gRNA sequences. Specifically, gRNA sequence alignment was performed to identify the degree of DNA similarity at each nucleotide, including annotation of guides with up to 0, 1, or 2 nucleotide mismatches. A final set of 226 gRNA sequences was selected for PCSK9 primary screening in HeLa cells.

[0246] [Example 3] Selection of ZF target sites and design of ZF proteins for epigenetic silencing A library of two-finger ZFPs (2F units), each recognizing a 6-bp DNA site, was used to design a larger six-finger ZFP array targeting an 18-bp DNA binding site. The source of the 2F units was a set of three-finger zinc finger proteins that had been selected to bind to specific target sites using the bacterial two-hybrid (B2H) selection system (Hurt et al., PNAS (2003) 100:12271-6; Maeder et al., Mol Cell (2008) 31(2):294-301). A list of targetable DNA sites was created by generating all possible triplet combinations of the 6-bp binding sites represented in the library and allowing either 0 or 1 bp between the 6-bp target sites. To identify zinc finger target sites within PCSK9, sequences ±1 kb from the TSS (human (GRCh38)) were matched against this list. For each identified ZF target site, multiple ZF proteins could be designed. The six recognition helices used to generate the complete protein were designed by selecting two-finger units and considering several factors, including the known binding preference of zinc finger proteins, the frequency with which amino acids at positions 1, 2, 3, and 6 are selected to bind to the desired target base in the B2H selection system, avoiding amino acids at positions 1, 2, 3, and 6 that are selected to bind multiple different bases in B2H, and maintaining context-dependent binding by matching adjacent bases when possible. The complete ZF sequence was derived from the naturally occurring Zif268 protein, and the selected recognition helices were maintained in the sequence context selected in B2H (fingers 1-2 or fingers 2-3 of Zif268). The two-finger units were connected by the linker TGSQKP (SEQ ID NO: 651) when the 6-bp binding sites were contiguous, or by the linker TGGGGSQKP (SEQ ID NO: 652) when the 6-bp binding sites were spaced 1 bp apart. A final set of 209 ZFPs targeted to 49 different binding sites was selected for PCSK9 primary screening in HeLa cells.

[0247] FIG. 1 shows the overlap of gRNAs and zinc finger proteins mapped to the PCSK9 target region.

[0248] [Example 4] Guide RNA screening in HeLa cells Primary screening of gRNAs targeting PCSK9 was performed in HeLa cells. gRNA sequences were ordered from Twist Biosciences as DNA fragments with a u6 promoter sequence preceding the gRNA coding sequence.

[0249] HeLa cells were transfected with gRNA and CRISPR-off in DNA format. Six 96-well plates (Sigma-Aldrich catalog number M2936) were seeded with 12,000 HeLa cells (ATCC catalog number CCL-2) per well in standard culture medium containing DMEM (Thermo Fisher catalog number 11-965-092) supplemented with 10% fetal bovine serum (Thermo Fisher catalog number A4766) volume / volume, 1x GlutaMAX™ (Thermo Fisher catalog number 35050061), and 1x penicillin-streptomycin (Thermo Fisher catalog number 15140122). After plating, the cells were grown in a 37°C, 5% CO2 incubator for 24 hours. 25 ng of each gRNA fragment and 50 ng of CRISPR-off plasmid (SEQ ID NO: 658) were resuspended in DPBS buffer (Thermo Fisher catalog no. 14190144) to a concentration of 7.5 ng / μL. Additionally, 10 ng of EF1a:puromycin resistance plasmid was also added to the transfection mix to achieve a total payload of 85 ng of DNA. The transfection mixture was created by adding the resuspended DNA components to Mirus® TransIT®-LT1 Transfection Reagent (Mirus catalog no. MIR2300) according to the manufacturer's instructions. 10 μL of each transfection mixture was added in duplicate across a total of six screening plates. The positive control used was CRISPRi(dCas9-KRAB) with two gRNA targeting sites adjacent to the TSS. The two CRISPRi positive control gRNAs used were gRNA004 and gRNA005, annotated in the gRNA sequence table. These control conditions are referred to as "CRISPRi-1" and "CRISPRi-2," respectively, in the tables of primary screening data.Negative controls were CRISPR-off without gRNA, CRISPR-off with gRNA targeting a non-PCSK9 locus (CD151), and empty vector (pUC19, NEB catalog number N3041S).

[0250] Twenty-four hours after transfection, puromycin resistance selection was performed: the cell culture medium was completely aspirated, all wells were washed three times with DPBS buffer (Thermo Fisher catalog number 14190144), and 200 μL of 1 μg / μL puromycin was added to all wells of the screening plate.

[0251] Forty-eight hours after transfection, cells were passaged. The cell culture medium was completely aspirated, and all wells were washed three times with DPBS buffer (Thermo Fisher catalog number 14190144). Cells were enzymatically lifted by adding 25 μL of trypsin-EDTA (0.25%) (Thermo Fisher catalog number 25200056) for 5 minutes in a 37°C incubator. Trypsinized cells were resuspended 1:8 in fresh standard culture medium and replated at a 1:4 ratio 72 hours after the medium change.

[0252] To measure secreted PCSK9 protein levels, medium was collected 24 hours after medium change, and cell plates were assayed for relative cell counts using the Promega Cell Titer Glo™ protocol (catalog number G7570) according to the manufacturer's recommendations. PCSK9 protein levels were assessed using the LEGEND MAX™ Human PCSK9 ELISA Kit from BioLegend (catalog number 443107). The collected medium was plated, and all subsequent steps were performed strictly according to the manufacturer's recommendations. Final plate readings were taken at 450 nm on a Perkin Elmer® VICTOR® Nivo™ F instrument. GraphPad Prism software was used to fit the standard curve and interpolate unknowns. To correct for any variability in cell numbers per well, PCSK9 ELISA results were normalized by the Cell Titer Glo™ assay (Promega catalog number G7571) results.

[0253] Over 200 gRNAs were tested, of which 40 were identified as top sequences (Figure 2; top sequences are represented by dark circles). The sequences and efficacy of the tested gRNAs are shown in Table 7 (SEQ ID NO: 1). Relative PCSK9 secretion ("PCSK9% relative to control") represents the average PCSK9 protein level of the treated samples, expressed as a percentage of the average across all non-targeting gRNA (CD151) negative control conditions. Strong silencing of PCSK9 (30-40% relative to negative control levels) was observed in cells treated with several gRNA candidate treatments. The top 40 gRNAs with the best PCSK9 protein knockdown were selected to be ordered as sgRNAs for further follow-up studies.

[0254] [Table 7] TIFF2025516294000028.tif253165TIFF2025516294000029.tif253165TIFF2025516294 000030.tif252165TIFF2025516294000031.tif252165TIFF2025516294000032.tif66165

[0255] The best-performing gRNAs were found to align closely to the PCSK9 gene transcription start site (Figure 3).

[0256] Following this primary screen, a secondary screen was performed using the top 40 gRNAs in RNA form. The top 40 guides were chemically synthesized and co-transfected with in vitro transcribed mRNA encoding CRISPR-off, CRISPRi, or wild-type Cas9 constructs. Secreted PCSK9 levels were measured 7 and 28 days after transfection.

[0257] To generate in vitro transcribed CRISPR-off, CRISPRi, and wild-type Cas9 effector mRNAs, plasmid constructs encoding these proteins were linearized using the MfeI restriction enzyme from NEB® (catalog number R3589S). One microgram of linearized template was used to set up an in vitro transcription reaction using the T7 mScript™ Standard mRNA Production System from CellScript (catalog number C-MSC100625) according to the manufacturer's instructions. The resulting RNA had a Cap 1 structure at the 5' end and was 3' polyadenylated. The transcribed RNA was purified using the RNeasy® Mini Kit from Qiagen (catalog number 74104).

[0258] Purified terminally modified sgRNAs were obtained from Integrated DNA Technologies using standard desalting. Three nucleotides at the 5' end and three nucleotides at the 3' end of each guide were 2'-O-methyl modified. Three internucleoside linkages at the 3' end and three internucleoside linkages at the 5' end were phosphorothioate internucleoside linkages (Table 8, SEQ: SEQ ID NO:). In this table, mX (i.e., mA, mC, mG, or mU) represents a 2'-O-methyl modified ribonucleoside, rX (i.e., rA, rC, rG, or rU) indicates a natural ribonucleoside, and * indicates a phosphorothioate linkage. All internucleoside linkages that are not phosphorothioate linkages are phosphate linkages.

[0259] [Table 8] TIFF2025516294000034.tif231165TIFF2025516294000035.tif231164TIFF2025516294000036.tif231164 TIFF2025516294000037.tif231164TIFF2025516294000038.tif231164TIFF2025516294000039.tif160165

[0260] HeLa cells were reverse transfected with 25 ng of effector and 12.5 ng of sgRNA in a 96-well plate format using TransIT®-X2 Transfection Reagent (Mirus) (Cat. No. MIR6003). Conditioned medium was collected weekly for up to 4 weeks and used to measure secreted PCSK9 levels using the LEGEND MAX™ Human PCSK9 ELISA Kit (BioLegend) (Cat. No. 443107). ELISA data were normalized to cell number using the CellTiter-Glo® Kit (Promega) (Cat. No. G7571). PCSK9 silencing was transient with CRISPRi(dCas9-KRAB) and returned to baseline by day 28, whereas several sgRNAs cotransfected with CRISPR-off(DNMT3A-3L-dCas9-KRAB) constructs showed potent and sustained silencing (Figure 4A). Sixteen of the 40 guides tested with CRISPR-off showed silencing efficiency superior to wild-type Cas9 (Table 9).

[0261] [Table 9] TIFF2025516294000041.tif14164

[0262] At the 2-week time point, RNA was extracted using the Quick-RNA 96 Kit (Cat. No. R1053) from Zymo Research. qPCR was performed using the qScript XLT One-Step RT-qPCR ToughMix (Cat. No. 95134-500) from Quantabio and TaqMan assays (PCSK9: Hs00545399_m1, PPIA: Hs99999904_m1). PCSK9 levels were normalized to PPIA. Relative quantification was performed using the delta-delta Ct method.

[0263] The abrogation of PCSK9 secretion was found to correlate with mRNA silencing at day 14 (Fig. 4B).

[0264] ModRNA004 and modRNA111 were tested in HeLa cells to suppress PCSK9 secretion for 60 days (Figure 5). Wild-type Cas9 was co-transfected with modRNA180 as a positive control. Cells were treated with 25 ng of effector and 12.5 ng of gRNA. ModRNA004 and modRNA111 were shown to mediate sustained silencing of PCSK9, comparable to that achieved by wild-type Cas9 via gene editing in HeLa cells.

[0265] Furthermore, the results suggest that epigenetic silencing is maintained in HeLa cells treated with simvastatin. Statin treatment is known to increase PCSK9 secretion via a transcriptional mechanism. In epigenetically silenced HeLa cells, statin treatment did not increase PCSK9 secretion (Figure 6).

[0266] [Example 5] Guide RNA assay in Huh7 hepatoma cell line The Huh7 hepatoma cell line is amenable to high-throughput screening and transfection. The top 13 guides from the HeLa screen, which had either perfect homology or a single mismatch with the cynomolgus monkey PCSK9 gene, were tested in the Huh7 hepatoma cell line. Epigenetic silencing with the CRISPR-off construct was shown to be stable over a 7-day period (Figure 7).

[0267] [Example 6] Guide RNA assay in primary human and cynomolgus monkey hepatocytes Primary human and cynomolgus monkey HepatoPac® cultures from BioIVT were used to test the efficacy of gRNAs in primary hepatocytes. HepatoPac® cultures were maintained according to the manufacturer's recommendations. Briefly, HepatoPac® maintenance medium was thawed and completed within 30 minutes of cell arrival. Upon medium change, cells were allowed to acclimate for two days in a 37°C, 10% CO2 incubator. On the second day after receipt, LNPs containing CRISPR-off+sgRNA, GFP-mRNA, and wild-type CRISPR Cas9 were formulated at various concentrations using SPARK™ (Precision Nanosystems) and the hepato9 mRNA LNP Formulation Kit (catalog number NWS0016) according to the manufacturer's instructions. LNPs were then characterized for encapsulation efficiency and total mRNA payload delivery using the Quant-it® RiboGreen RNA Assay Kit. LNPs are then added to the culture medium with a specified amount of total mRNA to achieve clinically relevant levels of PCSK9 silencing. The medium is changed every other day for up to four weeks to assess the persistence and / or potential inheritability of silencing. PCSK9 silencing is assessed every seven days by measuring the level of PCSK9 secreted into the culture medium by ELISA. PCSK9 concentration is controlled for total hepatocytes using a human albumin ELISA (Thermo Fisher®). Data are then presented as total PCSK9 secretion as a percentage of the GFP-mRNA negative control. Specificity can be assessed by isolating primary human and cynomolgus monkey hepatocytes from mouse fibroblast feeder layers using a magnetic bead-based antibody approach (Miltenyi Biotec). After primary hepatocytes are separated from the feeder layer, the cells are processed for RNAseq evaluation and whole-genome bisulfite sequencing.

[0268] The top 13 gRNAs in RNA format were selected for testing in primary human hepatocytes (PHH). The top gRNAs were selected based on (i) PCSK9 silencing efficiency and persistence in HeLa cells, and (ii) whether they perfectly aligned with the human PCSK9 gene and had at most one mismatch with the non-human primate PCSK9 gene. Combinations of gRNAs were also tested to determine their efficacy and persistence. Negative controls were CRISPR-off alone, gRNA fragment alone (modRNA003), and CRISPRi alone. The positive control was CRISPRi co-transfected with modRNA004 (Table 10, SEQ: SEQ ID NO: NHP: non-human primate). All gRNAs tested were predicted to bind to both human and non-human primate PCSK9.

[0269] [Table 10]

[0270] Potent PCSK9 silencing is observed: for some gRNAs, a greater than 70% reduction in secreted PCSK9 is observed at day 7 (depending on transfection efficiency).

[0271] [Example 7] ZF assay in HeLa cells A total of 209 zinc finger proteins (architecture shown in SEQ ID NO: 659) were designed from a ZF library against 49 PCSK9 target sites (selected from GRCh38 chromosome 1 1, 55038548-55040548), which had no other exact matches in the human genome (GRCh38).

[0272] HeLa cells were transfected with the ZF-off construct in DNA format. Six 96-well plates (Sigma-Aldrich catalog number M2936) were seeded with 12,000 HeLa cells (ATCC catalog number CCL-2) per well in standard culture medium containing DMEM (Thermo Fisher catalog number 11-965-092) supplemented with 10% fetal bovine serum (Thermo Fisher catalog number A4766) volume / volume, 1x GlutaMAX™ (Thermo Fisher catalog number 35050061), and 1x penicillin-streptomycin (Thermo Fisher catalog number 15140122). After plating, the cells were grown for 24 hours in a 37°C incubator with 5% CO2. 10 ng of ZF-off plasmid was resuspended in DPBS buffer (Thermo Fisher catalog number 14190144) to a concentration of 7.5 ng / μL. In addition, 10 ng of EF1a:puromycin resistance plasmid and 65 ng of empty vector (pUC19) were also added to the transfection mix to achieve a total payload of 85 ng of DNA. The transfection mixture was created by adding the resuspended DNA to serum-free OPTI-MEM medium (Thermo Fisher® catalog number 31985062) and adding Mirus® TransIT®-LT1 Transfection Reagent (MIR2300) according to the manufacturer's instructions. 10 μL of the transfection mixture was added in duplicate to a total of six screening plates. The positive control was CRISPR-off (SEQ ID NO: 658) with high-performing gRNA (gRNA009). Negative controls were ZF-off and empty vector (pUC19, NEB catalog number N3041S) with a non-PCSK9 gene coordinate (CLTA).

[0273] ZF screening yielded hits with activity comparable to CRISPR (Figure 8). Candidates with high silencing efficiency were carried forward to follow-up experiments. Figure 9 shows the ZF screening results according to the distance to TSS. A total of 209 ZFs were screened, and their PCSK9 knockdown activity compared to the negative control is shown in Table 11 below.

[0274] [Table 11] TIFF2025516294000044.tif147158

[0275] Several ZF-off constructs were shown to be more effective at silencing PCSK9 than CRISPR-off in combination with wild-type Cas9 and gRNA003. The target sites of the ZFP domains and ZF sequences (F1–F6) in these ZF-off constructs are shown in Table 1.

[0276] [Example 8] Complete specificity screening of constructs in primary human hepatocytes The specificity of CRISPR-off and ZF-off constructs for silencing PCSK9 was tested in primary human hepatocytes. The specificity was assessed by RNA sequencing, methylation arrays, and whole-genome bisulfite sequencing assays. Genome-wide changes in expression and methylation after epigenetic editing were profiled compared to negative controls.

[0277] [Example 9] CpG methylation patterns CpG methylation patterns are investigated in human hepatocytes (e.g., primary cells or cell lines) treated with CRISPR-off or ZF-off. Hybrid capture assays are performed on bisulfite-treated DNA to investigate the methylation patterns at CpG sites induced by CRISPR-off or ZF-off in the 1 kb region surrounding the PCSK9 TSS.

[0278] [Example 10] Stable PCSK9 silencing by epigenetic editing in mice with wild-type PCSK9 We test the ability of CRISPR-off and ZF-off constructs to mediate epigenetic silencing of endogenous PCSK9 in vivo. Constructs are delivered using a single intravenous administration of mRNA (and gRNA for CRISPR-off silencing) formulated in LNPs. Silencing is tested in wild-type mice over a 2- to 6-month period. Readouts are serum PCSK9 and serum cholesterol levels. A subset of each cohort is selected for liver hematoxylin and eosin (H&E) staining and RNAseq analysis. For several constructs, robust, stable, and heritable PCSK9 silencing is observed.

[0279] [Example 11] Stable PCSK9 silencing by epigenetic editing in mice expressing transgenic human PCSK9 Three different mouse strains expressing transgenic human PCSK9 are used: hPCSK9-Tg (mPCSK9+ / -) heterozygous mice, hPCSK9-Tg (mPCSK9+ / +) homozygous mice, and hPCSK9-Tg (mPCSK9- / -) mice. The hPCSK9-Tg (mPCSK9- / -) mouse strain used is C57BL / 6J-Pcsk9- / -Tg (RP11-55M23-AbsI), which expresses human PCSK9 under the control of its own promoter (Figure 10). See, for example, Weider et al., J Biol Chem (2016) 291(32):16659-71.

[0280] CRISPR-off and ZF-off constructs will be tested. Constructs will be delivered via a single intravenous administration of mRNA / gRNA formulated in LNPs. Readouts include liver H&E staining, measurement of PCSK9 mRNA levels by RNAseq, and AST / ASL measurements. Efficacy will also be tested, including the durability of PCSK9 silencing over a 3-4 month period as measured by serum PCSK9 protein levels. Sustained and significant reductions in serum PCSK9 levels will be observed for several constructs.

[0281] Durability will be studied over 6-12 months. Readouts will be serum PCSK9 and serum cholesterol levels. A subset of the cohort will be selected for liver H&E and RNAseq analysis.

[0282] [Example 12] Fusion proteins with variant NLS configurations Several improved fusion protein constructs were developed using variant nuclear localization sequence (NLS) constructs to have significantly higher episilencing activity.

[0283] Several constructs with variant NLS domain configurations (Figures 11A and 11B) were constructed and tested at the PCSK9 locus in HeLa cells (Figures 12A-12B). Constructs were further tested at the PCSK9 locus in Hepa1-6 (Figure 13) and HuH7 (Figures 14A-14C and Figure 15). The amino acid and DNA sequences of exemplary fusion protein constructs are shown below.

[0284] [Table 12] TIFF2025516294000046.tif247170TIFF2025516294000047.tif245170TIFF2025516294000048.tif246170TIFF2025516294000049.tif240170TIFF2025516294000050.tif245170TIFF2025516294000051.tif245170TIFF2025516294000052.tif246170TIFF2025516294000053.tif245170TIFF2025516294000054.tif244170TIFF2025516294000055.tif244170TIFF2025516294000056.tif245170TIFF2025516294000057.tif246170TIFF2025516294000058.tif246170TIFF2025516294000059.tif246170TIFF2025516294000060.tif245170TIFF2025516294000061.tif246170TIFF2025516294000062.tif242170TIFF2025516294000063.tif246170TIFF2025516294000064.tif246170TIFF2025516294000065.tif245170TIFF2025516294000066.tif245170TIFF2025516294000067.tif248170TIFF2025516294000068.tif245170TIFF2025516294000069.tif246170TIFF2025516294000070.tif246170TIFF2025516294000071.tif245170TIFF2025516294000072.tif247170TIFF2025516294000073.tif247170TIFF2025516294000074.tif246170TIFF2025516294000075.tif243170TIFF2025516294000076.tif247170TIFF2025516294000077.tif248170TIFF2025516294000078.tif245170TIFF2025516294000079.tif160170.

[0285] The sequence from Figure 14A can be found below:

[0286] [Table 13]

[0287] The sequences from Figure 15 can be found below:

[0288] [Table 14] TIFF2025516294000082.tif246170TIFF2025516294000083.tif248170TIFF2025516294000084.tif22170

[0289] Cell culture and transfection HeLa (ATCC-CRM-CCL-2), Hepa1-6 (PCSK9-IRES-TdTomato), Huh7 (Sekisui XenoTech, LLC), and HEK293T Griptite (CLTA-GFP) cells were cultured in DMEM with 10% FBS. All experiments in HeLa and Huh7 cells were performed using chemically synthesized guide RNAs and in vitro transcribed effector constructs. HeLa cells were reverse transfected using TransIT-X2 transfection reagent (Mirus) (catalog number MIR6003). Huh7 cells were reverse transfected using MessengerMAX reagent (Invitrogen) (catalog number LMRNA003). Secreted PCSK9 levels were measured at the indicated time points using the LEGEND MAX™ Human PCSK9 ELISA Kit (Biolegend) (catalog number 443107). All ELISA data were normalized to cell number using the CellTiter-Glo kit from Promega (catalog no. G7571).

[0290] HEK293T Griptite cells, in which GFP was knocked in as an in-frame CLTA fusion into the CLTA locus, were co-transfected with the effector construct and a plasmid encoding a human CLTA guide RNA using TransIT-X2 transfection reagent (Mirus, catalog no. MIR6003). GFP expression, a surrogate for CLTA expression, was measured by FACS.

[0291] Hepa1-6 cells were co-transfected with the effector construct and a plasmid encoding mouse PCSK9 guide RNA using an SF Cell Line 96-well Nucleofector Kit (Cat. No. V4SC-2096, Program Code: CM-138) in an Amaxa 4D Nucleofector device from Lonza. At the indicated time points, cells were analyzed by FACS for TdTomato expression as a surrogate for PCSK9 levels.

[0292] In vitro transcription of effector constructs and synthetic gRNAs An in vitro transcription reaction was set up using 1 μg of linearized effector template using the T7 mScript™ Standard mRNA Production System from CellScript (Catalog No. C-MSC100625) according to the manufacturer's instructions to generate RNA with a Cap 1 structure at the 5' end and 3' polyadenylated. Terminally modified sgRNAs with three 2'O-methyl modified nucleotides with phosphorothioate linkages at both the 5' and 3' ends were obtained from Integrated DNA Technologies.

[0293] Methylation profiling Genomic DNA was extracted from each well of a 96-well culture plate using the DNAdvance DNA Extraction from Tissue Kit (Beckman Coulter). After quantification of genomic DNA using a High-Sensitivity DNA 1X Kit (Quant-IT), each genomic DNA sample was bisulfite converted using the EZ-96 DNA Methylation-Gold MagPrep Kit (Zymo Research) according to the manufacturer's instructions. For hybridization capture experiments, DNA libraries were prepared using the xGen™ Methyl-Seq DNA Library Prep Kit (IDT), and hybrid capture was performed using the xGen™ DNA Library Hybridization Capture Kit (IDT). For amplicon sequencing experiments, DNA libraries were prepared using the xGen™ Methyl-Seq DNA Library Prep Kit (IDT), and hybrid capture was performed using the xGen™ DNA Library Hybridization Capture Kit (IDT). Bisulfite-converted DNA from each sample was used to seed PCRs corresponding to each of the two VIM amplicons using the Platinum Taq kit (Invitrogen). Pooled products were cleaned using the AMPure XP kit (Beckman Coulter) and fragment size was assessed using D1000 screen tape on a Tapestation 4200 (Agilent) before being sequenced by a commercial service (Azenta).

[0294] [Example 13] Bacterial DNA methyltransferase In this experiment, a panel of bacterial proteins was screened for DNA methyltransferase activity in mammalian cells. These bacterial DNA methyltransferases (Table 12) were tested for their epigenetic silencing activity by fusing them to the dCas9 domain at their N-terminus using the experimental procedure in Example 1. These constructs were then transfected into a reporter cell line expressing GFP under the control of the mammalian promoter of CLTA4.

[0295] [Table 15]

[0296] M.SssI DNA methyltransferase can efficiently methylate DNA in mammalian cells (Figure 16), and silencing was stable for up to 30 days. The sequence from Figure 16 can be found below:

[0297] [Table 16]

[0298] We also analyzed the methylation profile of these cells on day 29 and confirmed 20% methylation of the target genes (Figures 17-18).

[0299] Three additional orthologous DNA methyltransferases predicted to be closely related to M. SssI were identified and tested for epigenetic silencing activity using the experimental procedures in Example 1 (Table 13).

[0300] [Table 17]

[0301] The DNA methyltransferases in Table 13 are predicted to have similar or improved function to M. SssI. Sequences are tested in the CRISPR-off context in place of mouse DNMT3A / DNMT3L and their function compared to the function of M. SssI DNA methyltransferase in silencing the PCSK9 locus in the HeLa TdTomato system to identify novel features and improved function.

[0302] [Example 14] Alternative KRAB domains In this example, fusion proteins were constructed with alternative KRAB domains (Table 14), which showed improved activity compared to CRISPR-off when tested using the experimental procedures in Example 1 (Figures 19A-19D).

[0303] [Table 18] TIFF2025516294000089.tif60168

[0304] [Example 15] ZIM3 fusion construct Novel fusions of ZIM3 and KOX1KRAB were generated. Both ZIM3 and KOX1KRAB are KRAB family proteins with extensive homology. Sequences representing the midpoint between ZIM3 and KOX1KRAB were designed. These KOX1KRAB and ZIM3 constructs encode small regions of KOX1KRAB and ZIM3 centered around the zinc finger domains of the proteins. The utilized regions of KOX1KRAB and ZIM3 are highly similar within the first approximately 75 bp of their sequences, but ZIM3 also contains a small alpha-helical region at the C-terminus that is absent in KOX1KRAB. The KOX1KRAB-FL sequence contains the KOX1KRAB sequence corresponding to this extra fragment, while the ZIM3 truncated form has this extra fragment removed from the ZIM3 sequence. The ZIM3 / KOX1KRAB chimera is a fusion of the N- and C-terminal fragments of the two proteins. Each ZIM3-like KOX1KRAB variant was assembled by first assembling the 100 closest homologs ("families") of each gene by BLASTing ZIM3 or KOX1KRAB proteins from non-human species, second identifying the three KOX1KRAB family members most similar to ZIM3 and the three ZIM3 family members most similar to KOX1KRAB, and third logically modifying the KOX1KRAB-FL sequence to resemble each set of three (Table 15).

[0305] [Table 19]

[0306] array The SEQ ID NOs (SEQ) for the nucleotide (nt) and amino acid (aa) sequences described in this disclosure are listed below.

[0307] [Table 20] TIFF2025516294000092.tif254165TIFF2025516294000093.tif254166TIFF2025516294000094.tif252166TIFF2025516294000095.tif254167TIFF2025516294000096.tif253165TIFF2025516294000097.tif255166TIFF2025516294000098.tif254165TIFF2025516294000099.tif253164TIFF2025516294000100.tif254165TIFF2025516294000101.tif249165TIFF2025516294000102.tif253166TIFF2025516294000103.tif254165TIFF2025516294000104.tif249166TIFF2025516294000105.tif254165TIFF2025516294000106.tif254167TIFF2025516294000107.tif246166TIFF2025516294000108.tif252168TIFF2025516294000109.tif248167TIFF2025516294000110.tif255165TIFF2025516294000111.tif254166TIFF2025516294000112.tif253165TIFF2025516294000113.tif254166TIFF2025516294000114.tif254167TIFF2025516294000115.tif253166TIFF2025516294000116.tif254166TIFF2025516294000117.tif251165TIFF2025516294000118.tif253166TIFF2025516294000119.tif250166TIFF2025516294000120.tif249166TIFF2025516294000121.tif250166TIFF2025516294000122.tif250168TIFF2025516294000123.tif248165TIFF2025516294000124.tif250165TIFF2025516294000125.tif249166TIFF2025516294000126.tif250166TIFF2025516294000127.tif249165TIFF2025516294000128.tif250166TIFF2025516294000129.tif249166TIFF2025516294000130.tif249166TIFF2025516294000131.tif249166TIFF2025516294000132.tif248166TIFF2025516294000133.tif248166TIFF2025516294000134.tif248166TIFF2025516294000135.tif250166TIFF2025516294000136.tif251166TIFF2025516294000137.tif255167TIFF2025516294000138.tif249164TIFF2025516294000139.tif254165TIFF2025516294000140.tif254166TIFF2025516294000141.tif255166TIFF2025516294000142.tif255166TIFF2025516294000143.tif254166TIFF2025516294000144.tif249165TIFF2025516294000145.tif255165TIFF2025516294000146.tif254165TIFF2025516294000147.tif253166TIFF2025516294000148.tif253166TIFF2025516294000149.tif253167TIFF2025516294000150.tif254166TIFF2025516294000151.tif254166TIFF2025516294000152.tif253166TIFF2025516294000153.tif84166.

Claims

1. A system for suppressing the transcription of the human PCSK9 gene in human cells, and in some cases in human liver cells, a)(i) A domain that has a DNA methyltransferase (DNMT) domain and / or a domain that recruits DNMT, wherein the DNMT domain and / or recruiter domain may include a DNMT3A domain and / or a DNMT3L domain, and (ii) transcriptional repressor domain, and (iii) DNA binding domain that binds to the target region in the human PCSK9 gene One or more fusion proteins, or b) One or more nucleic acid molecules encoding one or more fusion proteins Includes, A system in which a DNA-binding domain targets the fusion protein to one or more sequences in the human PCSK9 gene selected from sequence numbers 700-747 and 1036-1261.

2. The system according to claim 1, wherein the DNA-binding domain includes a dead CRISPR-Cas (dCas) domain.

3. The system according to claim 2, wherein the dCas domain is a dCas9 domain, and the system further comprises (i) one or more guide RNAs containing any one of sequence numbers 1262 to 1487, or (ii) nucleic acid molecules encoding one or more guide RNAs.

4. The system according to claim 2, wherein the dCas domain is a dCas9 domain, and the system further comprises (i) one or more guide RNAs comprising either SEQ ID NO: 1302 or 1310, or (ii) nucleic acid molecules encoding one or more guide RNAs.

5. The system according to claim 2, wherein the dCas domain comprises a sequence having at least 90% identity with the dCas9 sequence, and optionally with sequence number 12 or 13.

6. The system according to claim 1, wherein the DNMT3A domain comprises a sequence having at least 90% identity with sequence number 574 or 575.

7. The system according to claim 1, wherein the transcriptional repressor domain comprises a sequence having at least 90% identity with a sequence selected from sequence numbers 33 to 570.

8. The system according to claim 1, wherein the transcriptional repressor domain comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627.

9. The system according to claim 8, wherein the KRAB domain comprises a sequence having at least 90% identity with a sequence selected from sequence numbers 89, 116, 245, and 255.

10. The system a) comprising a DNMT3A domain, a DNMT3L domain, a transcriptional repressor domain, and a DNA-binding domain, In some cases, one or both of the DNMT3A and DNMT3L domains are human. In some cases, the DNA-binding domain may be a fusion protein containing a dead CRISPR-Cas domain, or b) Nucleic acid molecules encoding a fusion protein The system according to claim 1, including the following:

11. A human cell or cell offspring comprising the system described in any one of claims 1 to 10, wherein the cell is optionally a hepatocyte.

12. A pharmaceutical composition comprising the system described in any one of claims 1 to 10 and a pharmaceutically acceptable excipient, optionally, (a) The composition comprises lipid nanoparticles (LNPs) including a system, and / or (b) The DNA-binding domain is a dCas domain, and the LNP further contains one or more gRNAs. Pharmaceutical composition.

13. A composition comprising the system according to any one of claims 1 to 10 for use in treating patients requiring treatment.

14. The patient, Having heart disease, Having elevated low-density lipoprotein cholesterol (LDL-C) or hypercholesterolemia, At risk of developing myocardial infarction, stroke, or unstable angina, and / or Primary hyperlipidemia, and in some cases heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), The composition according to claim 13.

15. Use of the system according to any one of claims 1 to 10 in the manufacture of a pharmaceutical product for treating a patient in need of treatment.

Images