Compositions and methods for epigenetic modulation of pcsk9 expression

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 337,164, filed on May 1, 2022, entitled “Compositions and Methods for Epigenetic Regulation of PCSK9 Expression,” U.S. Provisional Application No. 63 / 337,167, filed on May 1, 2022, entitled “Compositions and Methods for Epigenetic Regulation of PCSK9 Expression,” and U.S. Provisional Application No. 63 / 355,083, filed on June 23, 2022, entitled “Compositions and Methods for Epigenetic Regulation of PCSK9 Expression,” the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0003] Reference Electronic Sequence Listing

[0004] 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 Art

[0005] For more than a decade, genome editing has been considered a promising therapeutic approach for the treatment of genetic diseases. However, manipulation at the DNA level using traditional genetic editors remains risky given the potential for undesired double-strand DNA breaks, heterogeneous repair (including large and small insertions and deletions at the intended sites), and toxicity. In contrast, targeted epigenetic modifications offer the potential to alter gene expression without causing double-strand break-induced genotoxicity.

[0006] A promising candidate for epigenetic silencing is the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene. PCSK9 is a key target for the treatment of heart disease, which is the leading cause of death worldwide (Berberich et al., Nature Rev Cardiol. (2019) 16(1):9-20). The human PCSK9 gene, located on chromosome 1, has approximately 94% and 80% homology with its cynomolgus monkey and mouse counterparts, respectively. The gene has a CpG island in the promoter region and is far away from other genes and cis-regulatory features. PCSK9 protein is mainly produced by the liver.

[0007] In humans, PCSK9 plays a key role in regulating the circulating levels of low-density lipoprotein (LDL) particles due to binding to the LDL receptor (LDLR). LDLR reduces the circulating concentration of LDL particles by mediating the endocytosis and degradation of LDL particles in cells. In the absence of PCSK9, or if the interaction of PCSK9 with LDLR is blocked, the rate of LDLR recycling 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 the endocytosed LDLR binds to PCSK9, the LDLR is degraded together with its passenger LDL particles. Clinical and genetic studies have determined that circulating LDL causes 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 can reduce 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 helps increase the recycling of LDLR, which will lead to lower blood LDL particle concentrations.

[0008] Given the critical role of PCSK9 in the pathogenesis of hypercholesterolemia and cardiovascular disease, new and improved therapies targeting PCSK9 expression are needed. SUMMARY OF THE INVENTION

[0010] The present disclosure provides systems and compositions for epigenetic modification (herein "epigenetic editors" or "epigenetic editing systems"), and methods of using the same to produce epigenetic modifications at PCSK9, including in host cells and organisms.

[0011] In some aspects, the present disclosure provides a system for inhibiting transcription of a human PCSK9 gene in a human cell (optionally a human hepatocyte), comprising:

[0012] a) one or more fusion proteins, which together comprise

[0013] a DNA methyltransferase (DNMT) domain and / or a domain that recruits DNMTs, optionally wherein the DNMT domain and / or the recruitment domain comprises a DNMT3A domain and / or a DNMT3L domain, and optionally wherein the recruited DNMT is DNMT3A, and

[0014] Transcription repressor domain,

[0015] Each domain is linked to a DNA binding domain that binds to a target region in the human PCSK9 gene; or

[0016] b) one or more nucleic acid molecules encoding said one or more fusion proteins.

[0017] 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 NO: 700-747 and 1036-1261.

[0018] 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 can comprise a dCas9 domain, and the system can 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 one or more guide RNAs. In certain embodiments, the dCas domain comprises a dCas9 sequence, such as a sequence having at least 90% identity to SEQ ID NO: 12 or 13.

[0019] In some embodiments, the fusion protein comprises a death CRISPR Cas (dCas) domain, and the system comprises one or more guide RNAs (gRNAs) that bind PCSK9 as provided herein. In some embodiments, the system comprises a single gRNA. In some embodiments, the system comprises 2 gRNAs. In some embodiments, the system comprises 3 gRNAs. In some embodiments, the system comprises 4 gRNAs. In some embodiments, the system comprises 5 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.

[0020] In some embodiments, the system comprises a gRNA containing the gRNA targeting sequence of gRNA009 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA009. In some embodiments, the system comprises a gRNA containing the gRNA targeting sequence of gRNA003 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA003. In some embodiments, the system comprises a gRNA containing the gRNA targeting sequence of gRNA093 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA093. In some embodiments, the system comprises a gRNA containing the gRNA targeting sequence of gRNA011 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA011. In some embodiments, the system comprises a gRNA containing the gRNA targeting sequence of gRNA007 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA007. In some embodiments, the system comprises a gRNA containing a gRNA targeting sequence of gRNA077 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA077. In some embodiments, the system comprises a gRNA containing a gRNA targeting sequence of gRNA113 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA113. In some embodiments, the system comprises a gRNA containing a gRNA targeting sequence of gRNA004 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA004. In some embodiments, the system comprises a gRNA containing a gRNA targeting sequence of gRNA008 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA008. In some embodiments, the system comprises a gRNA containing a gRNA targeting sequence of gRNA012 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA012. In some embodiments, the system comprises a gRNA comprising a gRNA targeting sequence of gRNA111 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA111. In some embodiments, the system comprises a gRNA comprising a gRNA targeting sequence of gRNA005 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA005. In some embodiments, the system comprises a gRNA comprising a gRNA targeting sequence of gRNA013 of Table 10, or a gRNA that binds to the same target domain sequence as gRNA013.

[0021] In some embodiments, the system comprises fusion protein 9 variant 1 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 9 variant 2 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 10 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 11 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 12 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 13 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 14 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 15 (Example 12) and gRNA g041. In some embodiments, the system comprises fusion protein 9 variant 1 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 9 variant 2 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 10 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 11 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 12 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 13 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 14 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 15 (Example 12) and gRNA g049. In some embodiments, the system comprises fusion protein 9 variant 1 (Example 12) and gRNA g041 and g049. In some embodiments, the system comprises fusion protein 9 variant 2 (Example 12) and gRNA g041 and g049. In some embodiments, the system comprises fusion protein 10 (Example 12) and gRNA g041 and g049. In some embodiments, the system comprises fusion protein 11 (Example 12) and gRNA g041 and g049. In some embodiments, the system comprises fusion protein 12 (Example 12) and gRNA g041 and g049. In some embodiments, the system comprises fusion protein 13 (Example 12) and gRNA g041 and g049. In some embodiments, the system comprises fusion protein 14 (Example 12) and gRNA g041 and g049. In some embodiments, the system comprises fusion protein 15 (Example 12) and gRNA g041 and g049.

[0022] In some embodiments, the DNA binding domain comprises a ZFP domain that targets a nucleotide sequence selected from SEQ ID NOs: 700-747. In certain embodiments, the ZFP domain comprises, in order, the F1-F6 amino acid sequence of any one of ZF001 to ZF048 as shown in Table 1.

[0023] In some embodiments, the DNMT3A domain comprises a sequence that is at least 90% identical to SEQ ID NO:574 or 575.

[0024] 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.

[0025] In some embodiments, the transcriptional repression domain comprises a sequence having at least 90% identity to a sequence selected from SEQ ID NO: 33-570. In certain embodiments, the transcriptional repression domain is a KRAB domain derived from KOX1, ZIM3, ZFP28 or ZN627. The KRAB domain may comprise, for example, a sequence having at least 90% identity to a sequence selected from SEQ ID NO: 89, 116, 245 and 255. In some embodiments, the transcriptional repression domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises an amino acid sequence of SEQ ID NO: 571 or 572. In certain embodiments, the transcriptional repression domain is derived from KAP1, MECP2, HP1a / CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1 or SCML2.

[0026] In some embodiments, the system comprises

[0027] a) a fusion protein comprising a DNMT3A domain, a DNMT3L domain, a transcription repression domain and a DNA binding domain,

[0028] Optionally, wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and

[0029] Optionally, wherein the DNA binding domain is a death CRISPR Cas domain or a ZFP domain; or

[0030] b) A nucleic acid molecule encoding a fusion protein.

[0031] In certain embodiments, the fusion protein comprises a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA binding domain, a third peptide linker, and a transcription repression domain from the N-terminus to the C-terminus. For example, the fusion protein may comprise 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 transcription repression domain from the N-terminus to the C-terminus. The fusion protein may comprise 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 transcription repression domain, and a second NLS from the N-terminus to the C-terminus. The fusion protein may comprise a first and a 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 transcription repression domain, and a third and a fourth NLS from the N-terminus to the C-terminus. In a specific embodiment, the transcriptional repression domain is a KRAB domain, such as human KOX1, ZFP28, ZN627 or ZIM3 KRAB domain. In a specific embodiment, one or both of the second and third peptide linkers are XTEN linkers, which can be selected from XTEN80 (e.g., SEQ ID NO: 643) and XTEN16 (e.g., SEQ ID NO: 638), for example, wherein the second peptide linker is XTEN80, and the third peptide linker is XTEN16.

[0032] In some embodiments, the fusion protein may comprise 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 from N-terminus to C-terminus. 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.

[0033] 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, optionally wherein the ZFP comprises, in sequence, the F1-F6 amino acid sequence of any one of ZF001 to ZF048 as shown in Table 1. In certain embodiments, the fusion protein comprises SEQ ID NO: 1496, or a sequence at least 90% identical thereto, optionally wherein the ZFP comprises, in sequence, the F1-F6 amino acid sequence of any one of ZF001 to ZF048 as shown in Table 1.

[0034] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 KOX1KRAB domain, and a third and a fourth NLS. In a specific embodiment, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 660, or a sequence at least 90% identical thereto.

[0035] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

[0036] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 ZFP28KRAB domain, and a third and a fourth NLS. In a specific embodiment, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 661, or a sequence at least 90% identical thereto.

[0037] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

[0038] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 ZN627KRAB domain, and a third and a fourth NLS. In a specific embodiment, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 662, or a sequence at least 90% identical thereto.

[0039] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

[0040] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 ZIM3KRAB domain, and a third and a fourth NLS. In a specific embodiment, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 663, or a sequence at least 90% identical thereto.

[0041] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

[0042] In some embodiments, at least one of the NLSs in the fusion proteins described herein is an SV40 NLS (eg, SEQ ID NO: 644).

[0043] In some embodiments, the system comprises:

[0044] a) a first fusion protein comprising a first DNA binding domain and comprising or recruiting a DNMT3A domain,

[0045] a second fusion protein comprising a second DNA binding domain and comprising or recruiting a DNMT3L domain, and

[0046] a third fusion protein comprising a third DNA binding domain and comprising or recruiting a transcriptional repressor domain; or

[0047] b) one or more nucleic acid molecules encoding the fusion protein.

[0048] The present disclosure also provides a human cell or progeny of the cell comprising the system described herein. In some embodiments, the cell is a hepatocyte.

[0049] The present disclosure also provides a pharmaceutical composition 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.

[0050] The present disclosure also provides a method of treating a patient in need thereof, comprising administering to the patient a system or pharmaceutical composition described herein (e.g., intravenously). In some embodiments, the patient has heart disease; elevated low-density lipoprotein cholesterol (LDL-C) or hypercholesterolemia; is at risk of developing myocardial infarction, stroke, or unstable angina; and / or has primary hyperlipidemia (e.g., heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH)).

[0051] The present disclosure also provides a system or pharmaceutical composition described herein for use in treating a patient in need thereof, such as in the methods described herein.

[0052] The present disclosure also provides for use of the systems described herein in the preparation of a medicament for treating a patient in need thereof, such as in the methods described herein.

[0053] The present disclosure also provides articles of manufacture and kits comprising the systems described herein.

[0054] Other features, purposes and advantages of the disclosed methods and compositions are apparent in the following detailed description. However, it should be understood that the detailed description, although indicating examples and embodiments of the disclosed methods and compositions, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art based on the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is a diagram showing the predicted binding locations of ZF proteins and computationally designed gRNAs on the PCSK9 gene.

[0056] Figure 2 is 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.

[0057] Figure 3is a graph showing the overlap of the top 40 gRNAs with the PCSK9 gene.

[0058] Figure 4A is a bar graph showing secreted PCSK9 levels at day 7 and day 28 after treatment with the indicated gRNAs. The dashed line shows silencing achieved by wild-type (WT) Cas9.

[0059] Figure 4B is a scatter plot showing the correlation between PCSK9 mRNA expression and PCSK9 protein secretion in cells treated with gRNA. CRISPRi(dCas9-KRAB) stands for dCas9-KRAB fusion protein.

[0060] Figure 5 are line graphs showing PCSK9 silencing after treatment with CRISPRi (dCas9-KRAB), CRISPR-off (DNMT3A-3L-dCas9-KRAB), and the indicated gRNAs.

[0061] Figure 6 is a bar graph showing PCSK9 secretion in cells treated with CRISPRoff and simvastatin compared to cells treated with the CRISPRoff system alone.

[0062] Figure 7 is a bar graph showing the reduction of PCSK9 secretion in Huh7 hepatoma cells treated with CRISPRoff and the given gRNAs.

[0063] Figure 8 is a scatter plot showing the activity and toxicity of 247 ZF proteins targeting PCSK9. Relative PCSK9 expression is shown on the x-axis and the corresponding cell counts relative to pUC and off-target controls are shown on the y-axis. The diagonal line indicates a 1:1 correlation between relative PCSK9 expression and cell counts.

[0064] Fig. 9 is a scatter plot showing relative PCSK9 expression (y-axis) of cells treated with ZF-off (DNMT3A-3L-ZF-KRAB) constructs and the corresponding targeted genomic distance relative to the PCSK9 transcription start site (TSS) (x-axis).

[0065] Fig.10 is a diagram showing that the entire human PCSK9 locus flanked by 35.5 kb and 7 kb upstream and downstream genomic regions (67.5 kb) was introduced into and expressed in transgenic mice. The transgenic mice express human PCSK9 under the control of its own (human) endogenous promoter.

[0066] Fig.11ASchematic representation of fusion protein constructs with variant NLS configurations is shown. Fig. 11B Schematic representations of additional fusion protein constructs with variant KRAB domains are shown.

[0067] Figures 12A-12B It is shown that using 6.25ng RNA ( Fig. 12A ) or 2.5ng RNA ( Fig. 12B ) Graph showing the percentage of PCSK9 protein levels measured after treatment with fusion protein constructs with various NLS positions in HeLa cells. Human and mouse DNMT3L sequences are denoted as h3L and m3L, respectively.

[0068] Fig.13 is a graph showing that the construct with 2X NLS is 3-fold more efficient than CRISPR-off in silencing mPcsk9 in Hepa1-6 cells.

[0069] Fig.14A is a graph showing that constructs with 2X NLS are more efficient than CRISPR-off in silencing mPcsk9 in Huh7 cells. Figures 14B-14C is shown on the 5th day ( Fig. 14B ) and Day 15 ( Fig. 14C ) Constructs with 2XNLS were also more effective than CRISPR-off in silencing mPcsk9 in Huh7 cells at different doses.

[0070] Fig.15 is a graph showing that 2XNLS provides improvements in multiple ZFs in a CRISPR-off-like format in Huh7 cells where dCas9 is replaced with zinc fingers.

[0071] Fig.16 is a set of graphs showing that methylation of the CTLA4 promoter with bacterial DNMT proteins can induce epigenetic silencing of the locus.

[0072] Fig.17 is a set of graphs showing the methylation profile at the VIM3 locus of cells treated with different constructs carrying a bacterial DNA methyltransferase fused to dCas9 at day 30. Samples treated with M.SssI were methylated by 20%.

[0073] Fig.18 is a set of graphs showing methylation profiles captured by hybridization at the CLTA locus of cells at day 29 comparing M.SssI in dCas9 fusions to murine DNMT3A / 3L.

[0074] Figures 19A-19D The results show that when 0.5 ng of effector DNA ( Fig.19A ), 3ng effector DNA with GFP as marker ( Fig.19B ) and 0.5 ng of effector DNA with GFP as marker ( Fig.19C ) A set of diagrams showing alternative KRAB domains tested for apparent silencing activity against CRISPR-off. Fig.19D Results after 30 days using different nanogram amounts of effector DNA are shown. DETAILED DESCRIPTION OF THE INVENTION

[0076] The present disclosure provides an epigenetic editor for regulating PCSK9 gene expression. By changing 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" refers to human PCSK9 in this article. Human PCSK9 gene sequences can be found in Ensembl accession number ENSG00000169174. Compared with other genome engineering methods, the epigenetic editor has several advantages, including reversibility, reduced risk of translocation and persistent heritable silencing.

[0077] In some embodiments, the region of the human PCSK9 gene targeted for epigenetic regulation is about 2 kb in length and is about + / - 1 kb of the PCSK9 TSS. In certain embodiments, the region has the nucleotide sequence of SEQ ID NO: 1488. In some embodiments, the targeted PCSK9 region is about 1069 bp in length and is about + / - 500 bp of the PCSK9 TSS. In certain embodiments, the targeted region has the nucleotide sequence of SEQ ID NO: 1489. The TSS of PCSK9 is located at #chr1: 55039548 of genomic GRCh38.

[0078] In some embodiments, an epigenetic editor as described herein may include one or more fusion proteins, wherein each fusion protein includes a DNA binding domain, which is connected to one or more effector domains for epigenetic modification. In certain embodiments, wherein the DNA binding domain is a DNA binding domain guided by a polynucleotide, the epigenetic editor may further include one or more guidance polynucleotides. The DNA binding domain, effector domain and guidance polynucleotide of an epigenetic editor as described herein may be selected from, for example, those described below in any functional combination.

[0079] The epigenetic editor described herein can be transiently expressed in a host cell, or can be integrated into the genome of a host cell; the present disclosure also contemplates such cells and their offspring. Both transiently expressed and integrated epigenetic editors or their components can achieve stable epigenetic modifications. For example, after the epigenetic editor described herein is introduced into a host cell, the target gene in the host cell can be stably or permanently suppressed or silenced. In some embodiments, compared to the expression level in the absence of an epigenetic editor, the expression of the target gene is reduced or silenced for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or the entire life span of a cell or a subject carrying a cell. Epigenetic modifications can be inherited by the offspring of the host cell into which the epigenetic editor is introduced.

[0080] The epigenetic editors of the invention can be introduced into a patient (e.g., a human patient) in need thereof, for example, into the patient's hepatocytes, bile duct epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and sinusoidal endothelial cells.

[0081] I. DNA binding domain

[0082] 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. DNA binding domains as 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.

[0083] In some embodiments, the DNA binding domain described herein is encoded by its native coding sequence.In other embodiments, the DNA binding domain is encoded by a nucleotide sequence that has been codon optimized for optimal expression in human cells.

[0084] A. Polynucleotide-directed DNA binding domain

[0085] In some embodiments, the DNA binding domain herein can be a protein domain that is guided to a target site in a 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 class II CRISPR-associated nuclease. In some embodiments, the protein domain can be derived from a Cas nuclease, such as a type II, type IIA, type IIB, type IIC, type V, or type VI Cas nuclease. In certain embodiments, the protein domain can be derived from a class II Cas nuclease, and the class II Cas nuclease is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas14a, Cas14b, Cas14c, CasX, CasY, CasPhi, C2c4, C2c8, C2c9, C2c10, Csy1, Csy2, Cs Csf4, and homologues and modified versions thereof. "Derived from" is used to indicate that the protein domain comprises the complete polypeptide sequence of the parent protein, or comprises a variant thereof (e.g., having amino acid residue deletions, insertions and / or substitutions). The variant retains the desired function of the parent protein (e.g., the ability to form a complex with a guide nucleic acid sequence and a target DNA).

[0086] In some embodiments, the CRISPR-associated protein domain can be a Cas9 domain described herein. For example, Cas9 can refer to 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 with a wild-type Cas9 polypeptide described herein. In some embodiments, the wild-type polypeptide is Cas9 (NCBI Ref. No. NC_002737.2 (SEQ ID NO: 1)) and / or UniProt Ref. No. Q99ZW2 (SEQ ID NO: 2) from Streptococcus pyogenes. In some embodiments, the wild-type polypeptide is Cas9 (SEQ ID NO: 3) from Staphylococcus aureus. 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 Franscisella novicida (UniProt Ref. No. U2UMQ6 or SEQ ID NO: 4)). In certain embodiments, the CRISPR-associated protein domain may be a modified form of a wild-type protein comprising one or more amino acid residue changes, such as deletions, insertions, or substitutions; fusions or chimeras; or any combination thereof.

[0087] Cas9 sequences and structures of variant Cas9 orthologs of various organisms have been described. Exemplary organisms from which the Cas9 domains herein may be derived include, but are not limited to, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.), Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella neocidin, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viride viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderialesbacterium, Polaromonas naphthalenivorans, Polaromonas sibiricum sp.), Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacilluscaldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus varrooxidans watsoni), Pseudoalteromonas shaloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleuschthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina. Cas9 sequences also include sequences from organisms and loci disclosed in Chylinski et al., RNA Biol. (2013) 10(5):726-37. .

[0088] In some embodiments, the Cas9 domain is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 domain is from Staphylococcus aureus (SaCas9).

[0089] Other Cas domains are also contemplated for use in epigenetic editors herein. These include, for example, CasX (Cas12E) (e.g., SEQ ID NO: 5), CasY (Cas12d) (e.g., SEQ ID NO: 6), Cas domains of (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).

[0090] For epigenetic editing, a nuclease-derived protein domain (e.g., Cas9 or Cpf1 domain) may have reduced nuclease activity or no nuclease activity by mutation, so that the protein domain does not cut DNA or has reduced DNA cutting activity while retaining the ability to complex with a guide nucleic acid sequence (e.g., guide RNA) and a target DNA. For example, compared to a wild-type domain, 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%. In some embodiments, the CRISPR-associated protein domain described herein is catalytically inactive ("death"). Examples of such domains include, for example, dCas9 ("death" Cas9), dCpf1, ddCpf1, dCasPhi, ddCas12a, dLbCpf1, and dFnCpf1. For example, compared to wild-type Cas9, the dCas9 protein domain may include one, two or more mutations that eliminate its nuclease activity. It is known that the DNA cleavage domain of Cas9 includes two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the chain complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary chain. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A (in RuvC1) and H840A (in HNH) completely inactivate the nuclease activity of SpCas9. Similarly, SaCas9 can be inactivated by mutations D10A and N580A. In some embodiments, dCas9 includes at least one mutation that reduces or eliminates nuclease activity in the HNH subdomain and / or the RuvC1 subdomain. In some embodiments, dCas9 includes only the RuvC1 subdomain or only the HNH subdomain. It should be understood that any mutation that inactivates the RuvC1 and / or HNH domains may be included in the dCas9 herein, for example, insertions, deletions, or single or multiple amino acid substitutions in the RuvC1 domain and / or the HNH domain.

[0091] In some embodiments, the dCas9 protein herein comprises a mutation at position D10 (e.g., D10A), H840 (e.g., H840A), or both corresponding to the wild-type SpCas9 sequence, as numbered in the sequence provided by UniProt Accession No. Q99ZW2 (SEQ ID NO: 2). In a specific embodiment, the dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).

[0092] In some embodiments, a dCas9 protein as described herein comprises a mutation at position D10 (e.g., D10A), N580 (e.g., N580A), or both corresponding to a wild-type SaCas9 sequence (e.g., SEQ ID NO: 3). In a specific embodiment, the dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO: 13).

[0093] Based on this disclosure and knowledge in the art, additional suitable mutations that inactivate Cas9 will be apparent to those skilled 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. This disclosure contemplates any mutation that reduces or eliminates the nuclease activity of any Cas9 described herein (e.g., a mutation corresponding to any Cas9 mutation described herein).

[0094] Compared to wild-type Cpf1, the dCpf1 protein domain may comprise one, two or more mutations that reduce or eliminate its nuclease activity. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have a HNH endonuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. In some embodiments, dCpf1 comprises one or more mutations corresponding to positions D917A, E1006A, or D1255A as numbered in the sequence of the Francisella novocidalis Cpf1 protein (FnCpf1; SEQ ID NO: 4). In certain embodiments, the dCpf1 protein comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A, or a corresponding mutation in any Cpf1 amino acid sequence described herein. In some embodiments, dCpf1 comprises a D917A mutation. In a specific embodiment, dCpf1 comprises the amino acid sequence of dFnCpf1 (SEQ ID NO: 14).

[0095] Additional nuclease-inactivated CRISPR-associated protein domains contemplated herein include nuclease-inactivated CRISPR-associated protein domains from, e.g., 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).

[0096] In some embodiments, the Cas9 domain described herein can be a high-fidelity Cas9 domain, e.g., comprising one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA to confer increased target binding specificity. In certain embodiments, the high-fidelity Cas9 domain can be nuclease-inactivated as described herein.

[0097] The CRISPR-associated protein domains described herein can recognize a protospacer adjacent motif (PAM) sequence in a target gene. A "PAM" sequence is typically a 2 to 6 bp DNA sequence immediately following the sequence targeted by the CRISPR-associated protein domain. The PAM sequence is required for CRISPR protein binding and cleavage, but is not part of the target sequence. The CRISPR-associated protein domain can recognize a naturally occurring or typical PAM sequence, or can have an altered PAM specificity. CRISPR-associated protein domains that bind to non-classical PAM sequences have been described in the art. For example, Cas9 domains that bind to atypical PAM sequences have been described in Kleinstiver et al., Nature (2015) 523(7561):481-5 and Kleinstiver et al., Nat Biotechnol. (2015) 33:1293-8. Such Cas9 domains can include, for example, Cas9 domains from "VRER" SpCas9, "EQR" SpCas9, "VQR" SpCas9, "SpG Cas9", "SpRYCas9", and "KKH" SaCas9. Also contemplated are nuclease-inactive forms of these Cas9 domains, 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 a new Francisella Cas9 engineered to recognize 5'-YG-3' (where "Y" is a pyrimidine).

[0098] Additional suitable CRISPR-associated proteins, orthologs, and variants, including nuclease-inactive variants and sequences, will be apparent to those skilled in the art based on this disclosure.

[0099] Guide RNAs that can be used in conjunction with the CRISPR-associated protein domains herein are further described in Section II below.

[0100] B. Zinc finger protein domain

[0101] In some embodiments, the DNA binding domain of the epigenetic editor described herein comprises a zinc finger protein (ZFP) domain (or "ZF domain" as used herein). ZFP is a protein having at least one zinc finger, and binds to DNA in a sequence-specific manner. "Zinc finger" (ZF) or "zinc finger motif" (ZF motif) refers to a polypeptide domain comprising a β-β-α (ββα)-protein fold stabilized by zinc ions. ZF binds 2 to 4 base pairs of nucleotides, typically 3 or 4 base pairs (continuous or non-continuous). Each ZF typically comprises about 30 amino acids. The ZFP domain may contain a plurality of ZFs in series contact with its target nucleic acid sequence. Tandem arrays of ZFs may be engineered to produce artificial ZFPs that bind to desired nucleic acid targets. ZFPs may be rationally designed using a database comprising triplet (or quadruple) nucleotide sequences and single ZF amino acid sequences, wherein each triplet or quadruple nucleotide sequence is associated with one or more amino acid sequences of ZFs that bind to a specific triplet or quadruple sequence. See, e.g., U.S. Patents 6,453,242, 6,534,261, and 8,772,453.

[0102] ZFPs are widely present in eukaryotic cells and may 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), wherein X is any independently selected amino acid. In some embodiments, the ZFP domain herein may comprise a ZF array comprising consecutive C2H2-ZFs each contacting three or more consecutive nucleotides.

[0103] The ZFP domain of the epigenetic editor described herein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more ZFs. The ZFP domain may include an array of double-finger or three-finger units, such as 3, 4, 5, 6, 7, 8, 9 or 10 or more units, wherein each unit binds to a subsite in a target sequence. In some embodiments, a ZFP domain comprising at least three ZFs recognizes a target DNA sequence of 9 or 10 nucleotides. In some embodiments, a ZFP domain comprising at least four ZFs recognizes a target DNA sequence of 12 to 14 nucleotides. In some embodiments, a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18 to 21 nucleotides.

[0104] In some embodiments, the ZFs in the ZFP domains described herein are connected via peptide linkers. The length of the peptide linker can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. In some embodiments, the linker comprises 5 or more amino acids. In some embodiments, the linker comprises 7-17 amino acids. The linker can be flexible or rigid.

[0105] In some embodiments, the zinc finger array may have the following sequence:

[0106] 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, wherein "XXXXXXX" represents the amino acids of the ZF recognition helix that confer zinc finger DNA binding specificity; each X can be independently selected. In the above sequence, italic "XX" can be TR, LR or LK, and "[Linker]" represents the linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 651); this linker can be used when the subsites targeted by the ZF are adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO: 652); this linker can be used when there are bases between the subsites targeted by the zinc fingers. The two linkers shown can be the same or different.

[0107] The 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 (typical) linker sequence), or separated by a longer, flexible or structured polypeptide sequence. In some embodiments, directly adjacent fingers bind to a continuous nucleic acid sequence, i.e., to adjacent trinucleotides / triplets. In some embodiments, adjacent fingers cross-bind between each other's respective target triplets, which can help to strengthen or enhance the recognition of the target sequence and result in the binding of overlapping sequences. In some embodiments, a long-range ZF within a ZFP domain can recognize (or bind to) a non-contiguous nucleotide sequence.

[0108] The amino acid sequences of the ZF DNA recognition helices of the exemplary ZFP domains herein and their PCSK9 target sequences are shown in Table 1 below, wherein the numbers in brackets represent SEQ ID NOs.

[0109] Table 1. ZF sequences of exemplary ZFP domains targeting PCSK9

[0110]

[0111]

[0112]

[0113] 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, in sequence, the F1-F6 amino acid sequence of any one of ZF001-ZF048 as shown in Table 1. The F1-F6 amino acid sequence can be placed within the ZF framework sequence of SEQ ID NO: 650, or within any other ZF framework known in the art.

[0114] C.TALE

[0115] In some embodiments, the DNA binding domain of the epigenetic editor described herein comprises a transcription activator-like effector (TALE) domain. The DNA binding domain of TALE comprises a highly conserved sequence of about 33-34 amino acids, with repeated variable di-residues (RVDs) at positions 12 and 13 that are essential for recognizing specific nucleotides. TALE can be engineered to bind to almost any desired DNA sequence. Methods for programming TALE 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.

[0116] D. Other DNA binding domains

[0117] The epigenetic editors described herein contemplate other DNA binding domains. In some embodiments, the DNA binding domain comprises, for example, an argonaute protein domain from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease that is guided to its target site by 5' phosphorylated ssDNA (gDNA), where it produces a double-strand break. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer adjacent motif (PAM). Therefore, the use of nuclease-inactivated NgAgo (dNgAgo) can greatly expand the bases that can be targeted. Characterization and use of NgAgo have been described in, for example, 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.

[0118] In some embodiments, the DNA binding domain comprises an inactivated nuclease, such as an inactivated meganuclease. Additional non-limiting examples of DNA binding domains include tetracycline-controlled repressor (tetR) DNA binding domains, leucine zippers, helix-loop-helix (HLH) domains, helix-turn-helix domains, β-sheet motifs, steroid receptor motifs, bZIP domains, homeotic domains, and AT hooks.

[0119] II. Guide polynucleotide

[0120] The epigenetic editor comprising a DNA binding domain guided by a polynucleotide as described herein may also include a guiding polynucleotide capable of forming a complex with the DNA binding domain. The guiding polynucleotide may include RNA, DNA, or a mixture of the two. For example, in the case where the DNA binding domain guided by the polynucleotide is a CRISPR-associated protein domain, the guiding polynucleotide may be a guiding RNA (gRNA). "Guiding RNA" or "gRNA" refers to a nucleic acid that can hybridize with a target sequence and guide the CRISPR-Cas complex to bind to the target sequence. The method of using a guiding polynucleotide sequence with a programmable DNA binding protein (e.g., a CRISPR-associated protein domain) for site-specific DNA targeting (e.g., modifying a genome) is known in the art.

[0121] The guide polynucleotide sequence (e.g., gRNA sequence) can include two parts: 1) a nucleotide sequence comprising a "targeting sequence" complementary to a target nucleic acid sequence ("target sequence") (e.g., complementary to a nucleic acid sequence contained in a genomic target site); and 2) a nucleotide sequence of a DNA binding domain (e.g., a CRISPR-Cas protein domain) that binds to a polynucleotide guide. The nucleotide sequence in 1) can include 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 can therefore hybridize with the target nucleic acid sequence. The nucleotide sequence in 1) can be referred to as, for example, crispr RNA or crRNA. The nucleotide sequence in 2) can be referred to as a scaffold sequence of a guide nucleic acid (e.g., tracrRNA) or an activation region of a guide nucleic acid, and can include a stem-loop structure. Parts 1) and 2) as described above can be fused to form a single guide (e.g., a single guide RNA or sgRNA), or can be on two separate nucleic acid molecules. In some embodiments, the guide polynucleotide comprises parts 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 (eg, a peptide linker or a chemical linker).

[0122] Part 2 (scaffold sequence) of the guide polynucleotide as described herein can be, for example, Jinek et al., Science (2012) 337: 816-21; U.S. Patent Publication 2016 / 0208288; or U.S. Patent Publication 2016 / 0200779. The present disclosure also contemplates variants of part 2). For example, the tetraloop and stem loop of the gRNA scaffold (tracrRNA) sequence can be modified to include an RNA aptamer, which can be bound by a specific protein domain. In some embodiments, such modified gRNAs can be used to promote the recruitment of an inhibitory or activating domain fused to a protein-interacting RNA aptamer.

[0123] The gRNA provided herein generally comprises a targeting domain and a binding domain. The targeting domain (also referred to as "targeting sequence") may comprise a nucleic acid sequence that binds to a target site (e.g., a genomic nucleic acid molecule in a cell). The target site may be a double-stranded DNA sequence comprising a PAM sequence and a target sequence, which is located on the same chain as the PAM sequence and is directly adjacent. The targeting domain of a gRNA may comprise an RNA sequence corresponding to a target sequence, that is, it is similar to the sequence of the target domain, sometimes with one or more mispairings, but generally comprises an RNA sequence rather than a DNA sequence. Therefore, the targeting domain of a gRNA may be paired with a sequence base of a double-stranded target site complementary to the target sequence (completely or partially complementary), and therefore with a chain base pairing complementary to the chain comprising the PAM sequence. It should be understood that the targeting domain of a gRNA generally does not include a sequence similar to a PAM sequence. It should also be understood that, depending on the nuclease employed, the position of the PAM may be 5' or 3' of the target sequence. For example, PAM is generally located at 3' of the target sequence of a Cas9 nuclease and 5' of the target sequence of a Cas12a nuclease. For a description of the location of the PAM and the mechanism of gRNA binding to the target site, see, for example, Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6 Figure 1 , which is incorporated herein by reference. For additional explanation and description of the mechanism of gRNA targeting target sites of RNA-guided nucleases, 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.

[0124] In some embodiments, the targeting domain sequence comprises 17 to 30 nucleotides and corresponds completely to the target sequence (i.e., without any mismatched nucleotides). However, in some embodiments, the targeting domain sequence may comprise one or more but generally no more than 4 mismatches, such as 1, 2, 3, or 4 mismatches. Since the targeting domain is part of the gRNA (which is an RNA molecule), it will generally comprise ribonucleotides, while the DNA targeting domain will comprise deoxyribonucleotides.

[0125] An exemplary illustration of a Cas9 target site comprising a 22 nucleotide target domain and an NGG PAM sequence, and a gRNA comprising a targeting domain that completely corresponds to the target sequence (and thus comprises base pairs having complete complementarity to a DNA strand that is complementary to the strand comprising the target sequence and the PAM) is provided below:

[0126]

[0127] An exemplary description of a Cas12a target site comprising a 22 nucleotide target domain and a TTN PAM sequence, and a gRNA comprising a targeting domain that completely corresponds to the target sequence (and thus comprises base pairs having complete complementarity to a DNA strand complementary to the strand comprising the target sequence and the PAM) is provided below:

[0128]

[0129] Although it is not desired to be bound by theory, at least in some embodiments, it is believed that the length of the targeting domain and the 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 length of the targeting domain of the gRNA provided herein is 5 to 50 nucleotides. In some embodiments, the length of the targeting domain is 15 to 25 nucleotides. In some embodiments, the length of the targeting domain is 18 to 22 nucleotides. In some embodiments, the length of the targeting domain is 19-21 nucleotides. In some embodiments, the length of the targeting domain is 15 nucleotides. In some embodiments, the length of the targeting domain is 16 nucleotides. In some embodiments, the length of the targeting domain is 17 nucleotides. In some embodiments, the length of the targeting domain is 18 nucleotides. In some embodiments, the length of the targeting domain is 19 nucleotides. In some embodiments, the length of the targeting domain is 20 nucleotides. In some embodiments, the length of the targeting domain is 21 nucleotides. In some embodiments, the length of the targeting domain is 22 nucleotides. In some embodiments, the length of the targeting domain is 23 nucleotides. In some embodiments, the length of the targeting domain is 24 nucleotides. In some embodiments, the length of the targeting domain is 25 nucleotides. In certain embodiments, the targeting domain corresponds completely to the target sequence or a portion thereof provided herein, without mismatches. In some embodiments, the targeting domain of the gRNA provided herein comprises 1 mismatch relative to the target sequence provided herein. In some embodiments, the targeting domain comprises 2 mismatches relative to the target sequence. In some embodiments, the target domain comprises 3 mismatches relative to the target sequence.

[0130] Methods for designing, selecting and verifying gRNA are described herein and are known in the art. Software tools can be used to optimize the gRNA corresponding to the target DNA sequence, for example to minimize the total off-target activity of the entire genome. For example, a DNA sequence search algorithm can be used to identify the target sequence in the crRNA of the gRNA used with Cas9. Exemplary gRNA design tools include those described in Bae et al., Bioinformatics (2014) 30: 1473-5.

[0131] Guidance polynucleotides (e.g., gRNA) described herein can have various lengths. In some embodiments, the length of the spacer or targeting sequence depends on the CRISPR-associated protein components of the epigenetic editor system used. For example, Cas proteins from different bacterial species have different optimal targeting sequence lengths. Therefore, the length of the spacer sequence can include, 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 some embodiments, the length of the spacer includes 10-24, 11-20, 11-16, 18-24, 19-21 or 20 nucleotides. In some embodiments, the length of the guide polynucleotide (e.g., gRNA) is 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 and comprises a spacer 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 that are complementary to the target sequence. In some embodiments, the guide polynucleotides described herein can be truncated, for example, by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more nucleotides.

[0132] In certain embodiments, the 3' end of the PCSK9 target sequence is adjacent to a PAM sequence (e.g., a typical PAM sequence, such as NGG of 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 a specific embodiment, 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.

[0133] The guide polynucleotide (e.g., gRNA) can be modified with, for example, chemical changes and synthetic modifications. For example, the modified gRNA can include changes or replacements of one or two non-linked phosphate oxygens and / or one or more linked phosphate oxygens in the phosphodiester backbone bond, changes in ribose (e.g., the 2' hydroxyl on ribose), changes in the phosphate moiety, modifications or replacements of naturally occurring nucleobases, modifications or replacements of the ribose-phosphate backbone, modifications of the 3' end and / or 5' end of the oligonucleotide, replacement or part of the terminal phosphate group, conjugation of a cap or linker, or any combination thereof.

[0134] In some embodiments, one or more ribose groups of the gRNA can be modified. Examples of chemical modifications of ribose groups 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 esters or bicyclic nucleotides, such as locked nucleic acids (LNA), 2'-(5-restricted ethyl (S-cEt)), restricted MOE or 2'-0,4'-C-aminomethylene bridged nucleic acids (2',4'-BNANC). 2'-O-methyl modifications and / or 2'-fluoro modifications can increase the binding affinity and / or nuclease stability of gRNA oligonucleotides.

[0135] In some embodiments, one or more phosphate groups of gRNA can be chemically modified. Examples of chemical modifications of phosphate groups include, but are not limited to, phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modifications. In some embodiments, the guidance polynucleotides described herein may include one, two, three, or more PS bonds at or near the 5' end and / or 3' end; PS bonds may be continuous or non-continuous.

[0136] In some embodiments, the gRNA herein comprises a mixture of ribonucleotides and deoxyribonucleotides and / or one or more PS bonds.

[0137] In some embodiments, one or more nucleobases of the gRNA may be chemically modified. Examples of chemically modified nucleobases include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups. Chemical modification may be performed in the spacer, tracr RNA region, stem loop, or any combination thereof.

[0138] Table 2 below lists exemplary gRNA target sequences for epigenetic modification of human PCSK9, and the coordinates of the start and end positions of the targeted sites on human chromosome 1 (SEQ: SEQ ID NO). The table also shows the distance of the PCSK9 gene from the start coordinates to the TSS coordinates.

[0139] Table 2. Exemplary target sequences of gRNAs targeting PCSK9

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] In some embodiments, the gRNA herein does not comprise the sequence CCCGCACCUUGGCGCAGCGG (SEQ ID NO: 1490).

[0146] Any tracr sequence known in the art is contemplated for use in the gRNAs described herein. In some embodiments, the gRNAs described herein have a tracr sequence as shown in Table 3 below, or a tracr sequence (SEQ: SEQ ID NO) that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the tracr sequence shown below.

[0147] Table 3. Exemplary TRACR sequences

[0148]

[0149] In some embodiments, the gRNA herein is provided directly to the cell (e.g., by an RNP complex with a CRISPR-associated protein domain). In some embodiments, the gRNA is provided to the cell by an expression vector (e.g., a plasmid vector or a viral vector) introduced into the cell, and then, the cell expresses the gRNA from the expression vector. Methods for introducing gRNA and expression vectors into cells are well known in the art.

[0150] III. Effector domain

[0151] The epigenetic editors described herein include one or more effector protein domains (also referred to as "epigenetic effector domains" or "effect domains", as used herein) that affect epigenetic modifications of target genes. An epigenetic editor with one or more effector domains can regulate the expression of a target gene without changing its nucleobase sequence. In some embodiments, the effector domains described herein can provide inhibition or silencing of expression of a target gene, such as PCSK9, for example, by inhibiting transcription or by modifying or remodeling chromatin. Such effector domains are also referred to herein as "inhibition domains", "repression domains" or "epigenetic repression 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.

[0152] In some embodiments, the effector domain of an epigenetic editor described herein can perform histone tail modifications, such as by adding or removing an active tag on a histone tail.

[0153] In some embodiments, the effector domain of the epigenetic editor described herein can comprise or recruit a transcription-related protein, such as a transcription repressor. The transcription-related protein can be endogenous or exogenous.

[0154] In some embodiments, the effector domain of an epigenetic editor described herein may, for example, comprise a protein that directly or indirectly blocks access of a transcription factor to a gene of interest containing a target sequence.

[0155] The effector domain can be a full-length protein or a fragment thereof ("functional domain") that retains epigenetic effector function. Functional domains that can regulate (e.g., inhibit) gene expression can be derived from larger proteins. For example, functional domains that can reduce target gene expression can be identified based on the sequence of repressor proteins. The amino acid sequence of proteins that regulate 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 full protein sequences. As a starting point, the gene expression regulating activity of the largest sequence containing all regions identified by different databases can be tested. Various truncations can then be tested to identify the minimum functional unit.

[0156] The present disclosure also contemplates variants of the effector domains described herein. Variants can, for example, refer to polypeptides having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence similarity to the wild-type effector domains described herein. In specific embodiments, the variants retain 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.

[0157] In some embodiments, the effector domain described herein may comprise a fusion of two or more effector domains (e.g., KOX1 KRAB and ZIM3). The effector domain may, for example, comprise a fusion of 2, 3, 4, 5, 6, 7, 8, 9 or 10 effector domains, such as the effector domains described herein. In certain embodiments, the effector domain comprises a fusion of a truncated effector domain and a second effector domain. In certain embodiments, the 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).

[0158] In some embodiments, the epigenetic editor described herein may include 1 effector domain, 2 effector domains, 3 effector domains, 4 effector domains, 5 effector domains, 6 effector domains, 7 effector domains, 8 effector domains, 9 effector domains, 10 effector domains or more. In certain embodiments, the epigenetic editor comprises one or more fusion proteins (e.g., one, two or three fusion proteins), each fusion protein having one or more effector domains (e.g., one, two or three effector domains) connected to a DNA binding domain. In some embodiments, the effector domain 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, DNA methylation and histone SUMOylation.

[0159] In certain embodiments, the effector domains described herein (e.g., DNMT3A and / or DNMT3L) are encoded by nucleotide sequences found in the native genome (e.g., human or mouse) of the 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.

[0160] The effector domains described herein may include, for example, transcriptional repressors, DNA methyltransferases, and / or histone modifiers, as described in further detail below.

[0161] A. Transcriptional repressors

[0162] In some embodiments, the epigenetic effector domain described herein mediates the repression of target gene expression (e.g., transcription). The effector domain may include, for example, a Krüppel-associated box (KRAB) repression domain, a repressor element silencing transcription factor (REST) ​​repression domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, a FKHR (forkhead in rhabdomyosarcoma gene) repression domain, an EGR-1 (early growth response gene product-1) repression domain, an ets2 repressor factor repression domain (ERD), a MAD smSIN3 interaction domain (SID), a WRPW motif of a hair-related basic helix-loop-helix (bHLH) repressor protein, an HP1α chromo-shadow repression domain, an HP1β repression domain, or any combination thereof. The effector domain may recruit one or more protein domains that inhibit target gene expression, for example, through a scaffold protein. In some embodiments, the effector domain can recruit a scaffold protein domain or interact with a scaffold protein domain that recruits a PRMT protein, an HDAC protein, a SETDB1 protein, or a NuRD protein domain.

[0163] 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 in, for example, Ecco et al., Development (2017) 144 (15): 2719-29 and Lambert et al., Cell (2018) 172: 650-65.

[0164] In certain embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from KOX1 / ZNF10, KOX8 / ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from ZIM3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF680, ZNF41, ZNF189, ZNF528, ZNF543, ZNF554, ZNF140, ZNF610, ZNF264, ZNF350, ZNF8, ZNF582, ZNF30, ZNF324, ZNF98, ZNF669, ZNF677, ZNF596, ZNF214, ZNF37, Z NF34, 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, a repression domain (e.g., KRAB). For example, the repression domain can be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In a specific embodiment, the repression domain is a ZIM3 KRAB domain. In additional embodiments, the effector domain is derived from a human protein, such as human ZIM3, human KOX1, human ZFP28, or human ZN627.

[0165] Sequences of exemplary effector domains that can reduce or silence target gene expression or protein sequences containing them are provided in Table 4 below (SEQ: SEQ ID NO). Further examples of repressors and transcriptional repression domains can be found in, for example, PCT Patent Publication WO 2021 / 226077 and Tycko et al., Cell (2020) 183 (7): 2020-35, each of which is incorporated herein by reference in its entirety.

[0166] Table 4. Exemplary effector domains that can reduce or silence gene expression

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175] The present disclosure encompasses functional analogs of any of the above proteins, i.e., molecules having the same or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the transcription factor function of the protein). For example, a functional analog can be an isoform or variant of the protein listed above, e.g., containing a portion of the above protein with or without additional amino acid residues and / or containing mutations relative to the above protein. In some embodiments, a functional analog has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with one of the sequences listed in Table 4. Homologues, orthologues, and mutants of the above proteins are also contemplated.

[0176] In certain embodiments, the epigenetic editor described herein includes a KRAB domain derived from KOX1, ZIM3, ZFP28 or ZN627, and / or an effector domain derived from KAP1, MECP2, HP1a, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1 or SCML2, optionally wherein the parent protein is a human protein. In a specific embodiment, the epigenetic editor described herein includes a domain derived from KOX1, ZIM3, ZFP28 and / or ZN627, optionally wherein the parent protein is a human protein. In certain embodiments, the epigenetic editor may include a KRAB domain derived from KOX1 (ZNF10) (eg, human KOX1). In certain embodiments, the epigenetic editor may include a KRAB domain derived from ZIM3 (ZNF657 or ZNF264) (eg, human ZIM3). In certain embodiments, the epigenetic editor may include a KRAB domain derived from ZFP28 (eg, human ZFP28). In certain embodiments, the epigenetic editor may include a KRAB domain derived from ZN627 (eg, human ZN627). In certain embodiments, the epigenetic editor described herein may include a CDYL2 (eg, human CDYL2) and / or a TOX domain (eg, human TOX domain) in combination with a KOX1 KRAB domain (eg, human KOX1 KRAB domain).

[0177] In certain embodiments, the epigenetic effectors described herein comprise a repression domain derived from KOX1 / ZNF10 (SEQ ID NO: 89). For example, the repression domain can comprise the sequence of SEQ ID NO: 89, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 89.

[0178] In certain embodiments, the epigenetic effectors described herein comprise a repression domain derived from KOX1 / ZNF10, as shown in Table 5 below:

[0179] Table 5. Exemplary effector domains derived from KOX1 / ZNF10

[0180]

[0181]

[0182] In specific embodiments, the repression domain can 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.

[0183] In specific embodiments, the repression domain can 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.

[0184] In specific embodiments, the repression domain can 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.

[0185] In specific embodiments, the repression domain can 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.

[0186] In specific embodiments, the repression domain can 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.

[0187] In specific embodiments, the repression domain can 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.

[0188] In specific embodiments, the repression domain can 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.

[0189] In specific embodiments, the repression domain can 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.

[0190] B. DNA methyltransferase

[0191] In some embodiments, the effector domain of the epigenetic editor described herein changes target gene expression by DNA modification such as methylation. Highly methylated regions of DNA tend to have lower transcriptional activity than lowly methylated regions. DNA methylation mainly occurs at CpG sites (abbreviation for "C-phospho-G-" or "cytosine-phospho-guanine" sites). Many mammalian genes have promoter regions near or including CpG islands (nucleic acid regions with high frequency CpG dinucleotides).

[0192] The effector domain described herein can be, for example, a DNA methyltransferase (DNMT) or its catalytic domain, or can be capable of recruiting a DNA methyltransferase. DNMT covers enzymes that catalyze methyl transfer to DNA nucleotides, such as typical cytosine-5 DNMTs (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C) that catalyze methyl addition to genomic DNA. The term also covers atypical family members that do not catalyze methylation per se but recruit (including activation) catalytically active DNMTs; a non-limiting example of such DNMTs is DNMT3L. See, for example, Lyko, Nat Review (2018) 19: 81-92. Unless otherwise indicated, a DNMT domain can refer to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT1, DNMT3A, and DNMT3B) or derived from a non-catalytically active DNMT (e.g., DNMT3L). DNMT can inhibit the expression of a target gene by recruiting inhibitory regulatory proteins. In some embodiments, 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 of A, T, or C.

[0193] In some embodiments, the DNMTs described herein may be animal DNMTs (e.g., mammalian DNMTs), plant DNMTs, fungal DNMTs, or bacterial DNMTs. Bacterial DNMTs may be obtained from bacterial species (e.g., cocci, bacillus, spiral bacteria, or intracellular gram-positive or gram-negative bacteria). In certain embodiments, the bacterial species is a Mycoplasmaales bacterium, Mycoplasma marinum, or Spiroplasma chinense. In certain embodiments, the bacterial species is not M. penetrans, S. monbiae, H. parainfluenzae, A. luteus, H. aegyptius, H. haemolyticus, Moraxella, E. coli, T. aquaticus, 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 that is 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 editors described herein comprise a DNMT domain comprising 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 editors described herein comprise a DNMT domain comprising SEQ ID NO: 603, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 603.

[0194] In certain embodiments, the DNMT in the epigenetic editor described herein may include, for example, DNMT1, DNMT3A, DNMT3B and / or DNMT3C. In some embodiments, DNMT is a mammalian (e.g., human or mouse) DNMT. In a specific embodiment, DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, the epigenetic editor described herein comprises a DNMT3A domain, which comprises SEQ ID NO: 574 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: 574. In certain embodiments, the epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 575 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: 575. In some embodiments, the DNMT3A domain may have, for example, a mutation at position H739 (such as H739A or H739E), R771 (such as R771L), and / or R836 (such as R836A or R836Q), or any combination thereof (numbered according to SEQ ID NO: 574).

[0195] In some embodiments, the effector domain described herein can be a DNMT-like domain. As used herein, a "DNMT-like domain" is a regulatory factor that can activate or recruit other DNMT domains, but does not have methylation activity itself. 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 includes a DNMT3L domain, and the DNMT3L domain includes SEQ ID NO: 578 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: 578. In certain embodiments, the 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, the 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, the epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 581, 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: 581. In some embodiments, the DNMT3L domain may have, for example, mutations corresponding to positions D226 (e.g., D226V), Q268 (e.g., Q268K), or both (numbered according to SEQ ID NO: 578).

[0196] In certain embodiments, the epigenetic editor herein may include both DNMT and DNMT-like effector domains. For example, the epigenetic editor may include a DNMT3A-3L domain, wherein DNMT3A and DNMT3L may be covalently linked. In other embodiments, the epigenetic editor 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).

[0197] Table 6 below provides exemplary DNMTs that can be part of an epigenetic effector domain described herein, or from which the effector domain of an epigenetic editor described herein can be derived.

[0198] Table 6. Exemplary DNMT sequences

[0199]

[0200]

[0201]

[0202] The present disclosure encompasses functional analogs of any of the above-mentioned proteins, i.e., molecules having the same or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the DNA methylation function or recruitment function of the protein). For example, a functional analog may be an isoform or variant of the above-mentioned protein, such as a portion of the above-mentioned protein containing or not having additional amino acid residues and / or containing mutations relative to the above-mentioned protein. In some embodiments, a functional analog has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with one of the sequences listed in Table 6. In some embodiments, the effector domain herein comprises only the functional domain of the above-mentioned protein (or its functional analog), such as a catalytic domain or a 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.

[0203] As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is the same as 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, such as a catalytic fragment or a recruitment fragment; and / or having a mutation that improves the activity of the DNMT protein).

[0204] The epigenetic editor herein can achieve 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 in a target gene or chromosome. The CpG dinucleotide sequence can be located within or near the target gene in the CpG island, or can be located in a region that is not a CpG island. CpG islands generally refer to nucleic acid sequences or chromosome regions containing high-frequency 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, such as an observed to expected CpG ratio of at least 60%. As used herein, the observed and expected CpG ratio is determined by the number of CpGs * (sequence length) / (number of Cs * number of Gs). In some embodiments, the CpG island has an observed and expected CpG ratio of at least 60%, 70%, 80%, 90% or more. The CpG island can 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 less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50 CpG dinucleotides are methylated by an epigenetic editor.

[0205] In some embodiments, the epigenetic editors herein achieve methylation at hypomethylated nucleic acid sequences (i.e., sequences that may lack methyl groups on 5-methylcytosine nucleotides (e.g., in CpG) compared to a standard control). For example, hypomethylation may occur in senescent cells or cancer (e.g., early stages of neoplasia) relative to young cells or non-cancerous cells, respectively.

[0206] In some embodiments, the epigenetic editors described herein induce methylation at a hypermethylated nucleic acid sequence.

[0207] In some embodiments, methylation can be introduced by an epigenetic editor at sites other than CpG dinucleotides.For example, the target gene sequence can be methylated at the C nucleotides of CpA, CpT or CpC sequences.In some embodiments, the epigenetic editor includes a DNMT3A domain and realizes methylation at CpG, CpA, CpT, CpC sequences or any combination thereof.In some embodiments, the epigenetic editor includes a DNMT3A domain lacking a regulatory subdomain and only maintaining a catalytic domain.In some embodiments, the epigenetic editor comprising a DNMT3A catalytic domain realizes methylation only at a CpG sequence.In some embodiments, compared with an epigenetic editor comprising a wild-type DNMT3A domain, an epigenetic editor comprising a DNMT3A domain containing a mutation (such as R836A or R836Q mutation (according to SEQ ID NO: 574 numbering)) has a higher methylation activity at CpA, CpC and / or CpT sequences.

[0208] C. Histone modifiers

[0209] In some embodiments, the effector domain of the epigenetic editor herein mediates histone modification. Histone modification plays a structural and biochemical role in gene transcription, for example, by forming or destroying a nucleosome structure bound to histones and preventing gene transcription. Histone modification can include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, SUMOylation, etc., for example, at its N-terminus ("histone tail"). These modifications maintain or specifically transform chromatin structure, thereby controlling reactions such as gene expression, DNA replication, DNA repair, etc. that occur on chromosome DNA. Post-translational modification of histones is an epigenetic regulatory mechanism, which is considered to be necessary for eukaryotic cell genetic regulation. Recent studies have shown that chromatin remodeling factors such as SWI / SNF, RSC, NURF, NRD, etc., promote transcription factors to approach DNA by modifying nucleosome structure; histone acetyltransferase (HAT), which regulates histone acetylation state; and histone deacetylase (HDAC), as an important regulatory factor.

[0210] In particular, the unstructured N-terminal of histone can be modified by acetylation, deacetylation, methylation, ubiquitination, phosphorylation, SUMOylation, ribosylation, citrullination, O-GlcNAcylation, crotonylation or any combination thereof. For example, histone acetyltransferase (HAT) utilizes acetyl-CoA as cofactor and catalyzes the transfer of acetyl group to the epsilon amino group of lysine side chain. This neutralizes the positive charge of lysine, weakens the interaction between histone and DNA, thereby opens chromosome so that transcription factor is combined and starts transcription. The acetylation of K14 and K9 lysine of histone H3 by histone acetyltransferase may be relevant to people's transcriptional ability. Lysine acetylation can directly or indirectly produce the binding site of the chromatin modifying enzyme regulating transcriptional activation. On the other hand, the histone methylation of histone H3 lysine 9 may be relevant to heterochromatin or transcriptional silent chromatin.

[0211] In certain embodiments, the effector domain of the 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 a specific embodiment, the effector domain comprises a histone-lysine-N-methyltransferase SETDB1 domain.

[0212] 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 HDAC1, HDAC3, HDAC5, HDAC7 or HDAC9 protein domain. In a specific embodiment, the effector domain comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.

[0213] D. Other effector domains

[0214] In some embodiments, the effector domain comprises a protein containing a three-part motif (TRIM28, TIF1-β or KAP1). In certain embodiments, the effector domain comprises one or more KAP1 proteins. The KAP1 protein in the epigenetic editor herein can form a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in regulating gene expression in the cell 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 can 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 a ZFP90 protein (eg, isoform 2 of ZFP90) and / or a FOXP3 protein. An exemplary KAP1 amino acid sequence is shown in SEQ ID NO:629.

[0215] In some embodiments, the effector domain comprises a protein domain that interacts with one or more DNA epigenetic markers or is recruited by one or more DNA epigenetic markers.For example, the effector domain may include a methyl CpG binding protein 2 (MECP2) protein that interacts with a methylated DNA nucleotide in a target gene (which may or may not be at the CpG island of the target gene). The MECP2 protein domain in the epigenetic editor described herein can induce a condensed chromatin structure, thereby reducing or silencing the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editor described herein can interact with histone deacetylase (eg, HDAC), thereby inhibiting or silencing the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editor described herein can block a transcription factor or a transcription activator from approaching the target sequence, thereby inhibiting or silencing the expression of the target gene. Exemplary MECP2 amino acid sequences are shown in SEQ ID NO:630.

[0216] Also contemplated as effector domains of the epigenetic editors described herein are, for example, chromoshadow domains, ubiquitin-2-like Rad60 SUMO-like (Rad60-SLD / SUMO) domains, chromatin organization modification domain (Chromo) domains, 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-hairpin-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, homeotic domain, MYM-type zinc finger domain with FCS sequence (ZF-FCS), interferon regulatory factor 2 binding protein zinc Finger domain (IRF-2BP1_2), SSX repression domain (SSXRD), B-box zinc finger domain (ZF-B_box), CXXC zinc finger domain (ZF-CXXC), 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 / spike domain (SAM_PNT), Vestigial / 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), neurohomology 2 domain (NHR2), hinge domain of cleaved 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 C-type domain (VWC), NAB conserved region 1 domain (NCD1), S1 RNA binding domain (S1), HNF3C-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), transcriptional activation domain of FOXO protein family (FOXO-TAD), homeobox KN domain (Ho meobox_KN), BED zinc finger domain (ZF-BED), RING domain of C3HC4 type zinc finger (ZF-C3HC4_4), RAD51 interaction motif domain (RAD51_interact), p55 binding region of N-ethyl-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.

[0217] In some embodiments, the effector domain is a protein domain comprising a YAF2_RYBP domain or a homeodomain or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is a PRD domain, a NKL domain, a HOXL domain or a LIM domain. In a specific embodiment, the YAF2_RYBP domain may comprise a 32 amino acid Yaf2 / RYBP C-terminal binding motif domain (32aa RYBP).

[0218] In some embodiments, the effector domain comprises a protein domain selected from the group consisting of a SUMO3 domain, a chromodomain from M phase phosphoprotein 8 (MPP8), a chromoshadow domain from Chromobox 1 (CBX1), and a SAM_1 / SPM domain from Scm polycomb group protein homolog 1 (SCMH1).

[0219] In some embodiments, the effector domain comprises a HNF3 C-terminal domain (HNF_C). The HNF_C domain may be from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif.

[0220] In some embodiments, the effector domain can include 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 the 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 chromatin domain-helicase-DNA binding protein 3 (CHD3) domain, or a ZNF783 domain.

[0221] IV. Epigenetic editors

[0222] Provided herein is an epigenetic editor (i.e., an epigenetic editing system), which, for example, uses one or more DNA binding domains as described herein and one or more effector domains as described herein (e.g., epigenetic repression domains) to direct epigenetic modifications to a target sequence in a gene of interest in any combination. A DNA binding domain (coordinated with a guidance polynucleotide as described herein, wherein the DNA binding domain is a DNA binding domain guided by a polynucleotide) guides the effector domain epigenetically to modify the target sequence, resulting in gene suppression or silencing, which can be persistent and can be inherited across cell generations. In some aspects, the epigenetic editor described herein can reversibly or irreversibly suppress or silence genes in cells.

[0223] In a specific embodiment, the epigenetic editor described herein comprises one or more fusion proteins, each comprising (1) a DNA binding domain and (2) an effector domain. The effector domain may be on one or more fusion proteins contained in the epigenetic editor. For example, a single fusion protein may comprise all effector domains with a DNA binding domain. Alternatively, the effector domain or a subset thereof may be on a separate fusion protein, each fusion protein having a DNA binding domain (which may be the same or different). The fusion protein described herein may further comprise one or more linkers (e.g., a peptide linker), a detectable label, a nuclear localization signal (NLS) or any combination thereof. As used herein, "fusion protein" refers to a chimeric protein to which two or more coding sequences (e.g., a DNA binding domain and / or an effector domain) are directly or indirectly covalently or non-covalently linked.

[0224] In some embodiments, the epigenetic editor described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more effector (e.g., inhibition / repression) domains, which may be the same or different. In certain embodiments, two or more of the effector domains work together. The combination of effector domains may include a DNA methylation domain, a histone deacetylation domain, a histone methylation domain, and / or a scaffold domain recruiting any of the above. For example, the epigenetic editor described herein may include one or more transcriptional repression domains (e.g., KRAB domains such as KOX1, ZIM3, ZFP28, or ZN627 KRAB) and one or more DNA methylation domains (e.g., DNMT domains) and / or recruitment domains (e.g., DNMT3L domains). This epigenetic editor may include, for example, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, the epigenetic editor further comprises an additional effector domain (e.g., 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 editor described herein can include a combination of a CDYL2 and / or TOX domain and a KRAB domain (e.g., a KOX1KRAB domain).

[0225] A. Connector

[0226] A fusion protein as described herein may comprise one or more linkers connecting the components of the epigenetic editor. The linker may be a peptide or non-peptide linker.

[0227] In some embodiments, one or more linkers used in the epigenetic editors provided herein are peptide linkers, i.e., linkers comprising a peptide portion. The peptide linker can be any length suitable for the epigenetic editor fusion protein described herein. In some embodiments, the linker can comprise a peptide of 1 to 200 (e.g., 1 to 80) amino acids. In some embodiments, the linker comprises a length of 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 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids. Longer or shorter linkers are also contemplated. In some embodiments, the length of 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. For example, the length of the peptide linker can be 4, 5, 16, 20, 24, 27, 32, 40, 64, 92 or 104 amino acids. The peptide linker can be a flexible or rigid linker. In a specific embodiment, the peptide linker comprises an amino acid sequence of any one of SEQ ID NO: 631-637 and 664-665 or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0228] In certain embodiments, the peptide linker is an XTEN linker. Such a linker can include a portion of an XTEN sequence (Schellenberger et al., Nat Biotechnol (2009) 27 (1): 1186-90), which is an unstructured hydrophilic polypeptide consisting only of residues G, S, P, T, E and A. As used herein, the term "XTEN" refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. An XTEN linker is typically unstructured and includes a limited set of natural amino acids. The fusion of XTEN with protein changes its hydrodynamic properties and reduces the removal and degradation rate of the fusion protein. These XTEN fusion proteins are produced using recombinant technology, do not require chemical modification, and are degraded by natural pathways. The length of an XTEN linker can be, for example, 5, 10, 16, 20, 26 or 80 amino acids. In some embodiments, the length of an XTEN linker is 16 amino acids. In some embodiments, the length of an XTEN linker is 80 amino acids. In certain embodiments, an 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 specific embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 638. In specific embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 643.

[0229] In some embodiments, one or more linkers used 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 linker. In certain embodiments, the linker is a cyclic or non-cyclic, substituted or unsubstituted, or branched or non-branched aliphatic or heteroaliphatic linker.

[0230] In some embodiments, one or more linkers used in the epigenetic editors provided herein are polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker may include, for example, a monomer, dimer, or polymer of aminoalkanoic acid; aminoalkanoic acid (e.g., glycine, acetic acid, alanine, β-alanine, 3-aminopropionic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.); a monomer, dimer, or polymer of aminocaproic acid (Ahx); or a polyethylene glycol moiety (PEG); or an aryl or heteroaryl moiety. In certain embodiments, the linker may be based on a carbocyclic moiety (e.g., cyclopentane or cyclohexane) or a benzene ring. The linker may include a functionalized moiety to facilitate attachment of nucleophiles (e.g., thiol, amino) to the linker from the peptide. Any electrophilic agent may be used as part of a linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0231] Various linker lengths and flexibilities can be used 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 the 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, 50, 55, 60, 70, 80, or 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 W linkers (SEQ ID NO: 637). In some embodiments, the more rigid linker is in the form of (EAAAK)n (SEQ ID NO: 665), (SGGS)n (SEQ ID NO: 631, and (XP)n). In the formulas of the above flexible linkers and rigid linkers, n can be any integer between 1 and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (GGGGS)n motif, wherein n is 4 (SEQ ID NO: 636).

[0232] In some embodiments, the linker in the epigenetic editor described herein comprises a nuclear localization signal, e.g., an amino acid sequence having any one of SEQ ID NOs: 644-649. In some embodiments, the linker in the epigenetic editor described herein comprises an expression tag, e.g., a detectable tag, such as green fluorescent protein.

[0233] B. Nuclear localization signal

[0234] Fusion protein as described herein can include one or more nuclear localization signals, and in certain embodiments, can include two or more nuclear localization signals.For example, fusion protein can include 1,2,3,4,5,6,7,8,9,10 or more nuclear localization signals.As used herein, "nuclear localization signal" (NLS) is the amino acid sequence that directs protein to the nucleus.In certain embodiments, NLS can be SV40 NLS (for example, with SEQ ID NO:644 amino acid sequence).Fusion protein can include NLS at its N-terminal, C-terminal or both, and / or NLS can be embedded in the middle of fusion protein (for example, at N or C-terminal of DNA binding domain or effector domain).

[0235] In some embodiments, the fusion protein may include two NLSs. The fusion protein may include two NLSs at its N-terminus or C-terminus. The fusion protein may include one NLS at its N-terminus and one NLS embedded in the middle of the fusion protein, or one NLS at its C-terminus and one NLS embedded in the middle of the fusion protein. The fusion protein may include two NLSs embedded in the middle of the fusion protein.

[0236] In some embodiments, the fusion protein can include four NLSs. The fusion protein can include at least two (e.g., two, three, or four) NLSs at its N-terminus or C-terminus. The fusion protein can include at least one (e.g., one, two, three, or four) NLSs embedded in the middle of the fusion protein. In a specific embodiment, the fusion protein can include two NLSs at its N-terminus and two NLSs at its C-terminus.

[0237] 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 a specific embodiment, the NLS comprises the amino acid sequence of SEQ ID NO: 644. Additional NLSs are known in the art.

[0238] 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 can 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 with a corresponding fusion protein that does not have at least one NLS at the N-terminus and does not have at least one NLS at the C-terminus.

[0239] In some embodiments, an epigenetic editor comprising a fusion protein comprising two NLSs at the N-terminus and two NLSs at the C-terminus can 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 does not have two NLSs at the C-terminus.

[0240] C. Labels

[0241] The epigenetic editors provided herein may include one or more additional sequences ("tags") for tracking, detecting, and locating the editor. In some embodiments, the epigenetic editor includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more detectable tags. Each detectable tag may be the same or different.

[0242] For example, epigenetic editor fusion protein can include cytoplasm localization sequence, export sequence (such as nuclear export sequence) or other localization sequence, and can be used for dissolving, purifying or detecting sequence tag of fusion protein.Suitable protein tag provided herein includes but is not limited to biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, polyhistidine tag (also referred to as histidine tag or His- tag), maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S tag, Softag (for example, Softag 1 or Softag 3), strep tag, biotin ligase tag, FlAsH tag, V5 tag and SBP tag.Extra suitable sequence will be apparent to those skilled in the art.

[0243] D. Fusion protein configuration

[0244] The fusion proteins of the epigenetic editors described herein may have their components constructed 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 located at the C-terminus of the epigenetic editor. In some embodiments, the DNA binding domain is located at the N-terminus of the epigenetic editor. In some embodiments, the DNA binding domain is connected to one or more nuclear localization signals. In some embodiments, the DNA binding domain is flanked by epigenetic effector domains and / or additional domains on both sides. In some embodiments, wherein "DBD" indicates a DNA binding domain and "ED" indicates an effector domain, the epigenetic editor comprises the following configurations:

[0245] -N']-[ED1]-[DBD]-[ED2]-[C'

[0246] -N']-[ED1]-[DBD]-[ED2]-[ED3]-[C'

[0247] -N']-[ED1]-[ED2]-[DBD]-[ED3]-[C'

[0248] or

[0249] -N']-[ED1]-[ED2]-DBD]-[ED3]-[ED4]-[C'.

[0250] In some embodiments, the epigenetic editor comprises a DNA binding domain (DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repression ("repressor") domain that inhibits or silences target gene expression. The DBD, DNMT, and transcriptional repression domains can be any domain in any combination as described herein. The DBD, DNMT domain, and repression domain can be in any configuration, for example, having any of the domains at the N-terminus, C-terminus, or in the middle of the fusion protein. In some embodiments, the epigenetic editor comprises a fusion protein having the following configuration:

[0251] N']-[DNMT domain]-[DBD]-[repression domain]-[C'

[0252] N']-[repression domain]-[DBD]-[DNMT domain]-[C'

[0253] N']-[DNMT domain]-[repressor domain]-[DBD]-[C'

[0254] or

[0255] N']-[repression domain]-[DNMT domain]-[DBD]-[C'.

[0256] In some embodiments, the connecting structure "]-[" in any one of the epigenetic editor structures is a linker, such as a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and / or a promoter or regulatory sequence. In the epigenetic editor structure, multiple connecting structures "]-[" may be the same, or may each be a different linker, tag, NLS or peptide bond. In some embodiments, the DNMT domain may include any one of the domains in Table 6, or any combination or homolog thereof. In specific embodiments, the DNMT domain includes DNMT3A or a truncated version thereof, DNMT3L or a truncated version thereof, or both. In specific embodiments, the DBD is a catalytically inactive polynucleotide-guided DNA binding domain (e.g., dCas9) or a ZFP domain. In certain embodiments, the repression domain includes any one of the domains shown in Table 4 or 5, or any combination or homolog thereof. For example, the repression domain may be a KRAB domain. In certain embodiments, the repression 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 the domains (e.g., a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB). In specific embodiments, the repression domain is a KRAB domain from ZFP28, ZN627, ZIM3 or KOX1.

[0257] In some embodiments, the epigenetic editor comprises a configuration selected from:

[0258] N']-[DNMT3A-DNMT3L]-[DBD]-[repressor]-[C'

[0259] N']-[repressor]-[DBD]-[DNMT3A-DNMT3L]-[C'

[0260] N']-[repressor]-[DBD]-[DNMT3A]-[C'

[0261] N']-[DNMT3A]-[DBD]-[repressor]-[C'

[0262] N']-[repressor]-[DBD]-[DNMT3A]-[DNMT3L]-[C'

[0263] N']-[DNMT3A]-[DNMT3L]-[DBD]-[repressor]-[C'

[0264] N']-[DNMT3A]-[DBD]-[C'

[0265] N']-[DBD]-[DNMT3A]-[C'

[0266] N']-[DNMT3L]-[DBD]-[C'

[0267] N']-[DBD]-[DNMT3L]-[C'

[0268] Wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure]-[ is any one of a linker, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence as described herein. The DBD, repressor, DNMT3A, and DNMT3L domains can be any domain in any combination as described herein. For example, the DNMT3A and DNMT3L domains can be selected from those in Table 6. In a specific embodiment, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain; the repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.

[0269] In some embodiments, the epigenetic editor comprises a configuration selected from:

[0270] N']-[DNMT3A]-[DBD]-[SETDB1]-[C'

[0271] N']-[DNMT3A]-[DNMT3L]-[DBD]-[SETDB1]-[C'

[0272] N']-[DNMT3A-DNMT3L]-[DBD]-[SETDB1]-[C'

[0273] N']-[SETDB1]-[DBD]-[DNMT3A]-[DNMT3L]-[C'

[0274] N']-[SETDB1]-[DBD]-[DNMT3A]-[C'

[0275] Wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure]-[ is any of a linker, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence as described herein. The DBD, SETDB1, DNMT3A, and DNMT3L domains can be any domain described herein in any combination. In specific 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.

[0276] Specific constructs contemplated herein include:

[0277] DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1KRAB (configuration 1),

[0278] DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1KRAB (configuration 2),

[0279] NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1 KRAB-NLS (configuration 3),

[0280] NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1KRAB-NLS (configuration 4),

[0281] NLS-NLS-DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-KOX1KRAB-NLS-NLS (configuration 5), and

[0282] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1KRAB-NLS-NLS (Configuration 6).

[0283] 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 one of configurations 1-6 is also contemplated, in which the KOX1 KRAB domain is replaced by a ZFP28, ZN627, or ZIM3 KRAB domain. In some embodiments, ZFP28, ZN627, and ZIM3 are human ZFP28, human ZN627, and human ZIM3, respectively. In a specific embodiment, the fusion construct may have the following configurations:

[0284] NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-KOX1KRAB-NLS-NLS (Configuration 7),

[0285] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-KOX1KRAB-NLS-NLS (configuration 8),

[0286] NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZFP28KRAB-NLS-NLS (configuration 9),

[0287] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZFP28KRAB-NLS-NLS (configuration 10),

[0288] NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZN627KRAB-NLS-NLS (configuration 11),

[0289] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZN627KRAB-NLS-NLS (configuration 12),

[0290] NLS-NLS-hDNMT3A-hDNMT3L-XTEN80-dCas9-XTEN16-ZIM3KRAB-NLS-NLS (configuration 13), or

[0291] NLS-NLS-DNMT3A-DNMT3L-XTEN80-ZFP domain-XTEN16-ZIM3KRAB-NLS-NLS (Configuration 14).

[0292] In specific embodiments, the fusion constructs described herein can have Configuration 1 and comprise 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 the following SEQ ID NO: 658, the XTEN linker has Underline , W connector belt Bold, underlined, and italic body , NLS sequences are in bold, DNMT3A sequences are in italics, and DNMT3L sequences are in Underline and italics , dCas9 domain is in bold and italics, KOX1 KRAB domain is in Underline and bold :

[0293]

[0294] DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFD

[0295] SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK

[0296] DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP

[0297] EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA

[0298] ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS

[0299] QLGGDPKKKRKV SGSETPGTSESATPES TG RTLVTFKDVFVDFTREEWKLLDTAQQI

[0300] VYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP (SEQ ID NO:658)

[0301] In specific embodiments, the fusion constructs described herein can comprise the sequence provided below (SEQ ID NO: 1495), or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto. In the following SEQ ID NO: 1495, the XTEN linker is Underline , W connector belt Bold, Underline Lines and italics , NLS sequences are in bold, DNMT3A sequences are in italics, and DNMT3L sequences are in Underline and italics , dCas9 domain is in bold and italics, KOX1 KRAB domain is in Underline and bold :

[0302]

[0303]

[0304] In specific embodiments, the fusion constructs described herein can have Configuration 2 and comprise 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 the following SEQ ID NO: 659, the XTEN linker has Underline , W connector belt Bold, underlined, and italic body , NLS sequence band Bold and Underline , DNMT3A sequence is italicized, DNMT3L sequence is Underline and italics , the ZFP domain is in bold, and the KOX1 KRAB domain is in Underline and bold The variable amino acids represented by X are the amino acids of the DNA recognition helix of the zinc finger, and the italicized XX can be TR, LR, or LK.

[0305]

[0306] In certain embodiments, the six "XXXXXXX" regions in SEQ ID NO: 659 sequentially comprise the F1-F6 amino acid sequences shown 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.

[0307] In specific embodiments, the fusion constructs described herein can comprise the sequence provided below (SEQ ID NO: 1496), or a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In the following SEQ ID NO: 1496, the XTEN linker is Underline , W connector in bold ,Down Strikethrough and italics , NLS sequence band Bold and Underline , DNMT3A sequence is italicized, DNMT3L sequence is Underline and italics , ZFP domains are in bold, KOX1 KRAB domains are in Underline and bold The variable amino acids represented by X are the amino acids of the DNA recognition helix of the zinc finger, and the italicized XX can be TR, LR, or LK.

[0308]

[0309] In certain embodiments, the six "XXXXXXX" regions in SEQ ID NO: 1496 sequentially comprise the F1-F6 amino acid sequences shown 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.

[0310] In some embodiments, the fusion protein may further comprise a Dnmt3A ADD domain, such as downstream of the Dnmt3A domain sequence 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 located at the C-terminus of the Dnmt3A domain. In some embodiments, the Dnmt3A sequence and the ADD sequence are separated by a linker (e.g., a linker disclosed herein). In some embodiments, the ADD sequence and the Dnmt3L sequence are separated by a linker (e.g., a linker disclosed herein). In some embodiments, the ADD domain comprises the sequence:

[0311] MAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSL FEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL(SEQ ID NO:1497).

[0312] In specific embodiments, the fusion construct described herein can have configuration 7 and comprise 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.

[0313] In specific embodiments, the fusion construct described herein can have configuration 9 and comprise 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.

[0314] In specific embodiments, the fusion construct described herein can have configuration 11 and comprise 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.

[0315] In specific embodiments, the fusion construct described herein can have configuration 13 and comprise 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.

[0316] In some embodiments, the fusion construct described herein (e.g., a fusion construct of any one of Configurations 1-14) is within an expression construct comprising 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 of the polyadenylation site. In specific embodiments, the expression construct comprises a fusion construct (e.g., any one of Configurations 1-14) and a WPRE sequence in the 3' non-coding region upstream of the polyadenylation site.

[0317] In some embodiments, the fusion constructs described herein can have the sequence of any of the fusion proteins 1-12 as shown in Example 12.

[0318] A variety of fusion proteins can be used to achieve the activation or inhibition of a target gene or multiple target genes.For example, an epigenetic editor fusion protein comprising a DNA binding domain (e.g., dCas9 domain) and an effector domain can be delivered together with two or more guide polynucleotides (e.g., gRNA), each guiding polynucleotide targeting different target DNA sequences.The target sites of two DNA binding domains can be identical or near each other, or separated by, for example, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs or about 600 or more base pairs. In addition, when targeting double-stranded DNA such as endogenous loci, guiding polynucleotides can target the same or different chains (one or more for positive strands and / or one or more for negative strands).

[0319] V. Target sequence

[0320] The epigenetic editor herein can be directed to a target sequence in PCSK9 to achieve 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 the gene of interest but near the gene of interest, wherein methylation of the target sequence or binding of a repressor inhibits gene expression. In some embodiments, the target sequence may be a hypomethylated or hypermethylated nucleic acid sequence.

[0321] The target sequence can be in any part of the target gene. In some embodiments, the target sequence is a part of the non-coding sequence of the gene or near it. In some embodiments, the target sequence is a part of the exon of the gene. In some embodiments, the target sequence is a part of the transcriptional regulatory sequence (such as a promoter or enhancer) of the gene or near it. In some embodiments, the target sequence is adjacent to, overlaps or covers a CpG island. In certain embodiments, the target sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking the PCSK9 TSS. In certain embodiments, the target sequence is within 500 bp flanking the PCSK9 TSS. In certain embodiments, the target sequence is within 1000 bp flanking the PCSK9 TSS.

[0322] In some embodiments, the target sequence can be hybridized with a guide polynucleotide sequence (e.g., gRNA) compounded with a fusion protein, the fusion protein comprising a DNA binding domain (e.g., CRISPR protein, such as dCas9) and an effector domain guided by a polynucleotide. The guide polynucleotide sequence can be designed to have complementarity with the target sequence, or to have identity with 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 original spacer sequence in the target sequence. In a specific embodiment, the guide polynucleotide comprises a spacer sequence that is 100% identical to the original spacer sequence in the target sequence.

[0323] In some embodiments, wherein the DNA binding domain of the epigenetic editor described herein is a zinc finger array, the target sequence can be recognized by the zinc finger array.

[0324] In some embodiments, wherein the DNA binding domain of the epigenetic editor described herein is a TALE, the target sequence can be recognized by the TALE.

[0325] The target sequence described herein can be specific to a copy of the target gene, or can be specific to an allele of the target gene. Therefore, epigenetic modification and its expression regulation can be specific to a copy or an allele of the target gene. For example, an epigenetic editor can suppress the expression of a specific copy (e.g., a copy associated with a disease or condition, or a copy containing a mutation associated with a disease or condition) of a target sequence identified by a DNA binding domain.

[0326] In some embodiments, the target PCSK9 genomic region may fall within the sequence shown below (chr1: 55038548-55040548), with or without the terminal A:

[0327]

[0328] In some embodiments, the target sequence can be GRCh38 Chr1:55039228-55040296, as shown below:

[0329]

[0330] VI. Epigenetic modification

[0331] Epigenetic editors described herein can carry out sequence-specific epigenetic modification (e.g., chemically modified changes) to target genes containing target sequences. This epigenetic regulation can be safer and more easily reversible than the regulation (e.g., by producing DNA double-strand breaks) caused by gene editing. In some embodiments, epigenetic regulation can reduce or silence target genes. In some embodiments, modification is at a specific site of the target sequence. In some embodiments, modification is at a specific allele of the target gene. Therefore, epigenetic modification can cause a copy of a target gene containing a specific allele rather than another copy of the target gene to be regulated (e.g., reduced). In some embodiments, specific alleles are related to diseases, conditions, or illnesses.

[0332] In some embodiments, epigenetic modification reduces or eliminates the transcription of a target gene containing a target sequence. In some embodiments, epigenetic modification reduces or eliminates the transcription of a target gene copy containing a specific allele recognized by an epigenetic editor. In some embodiments, an epigenetic editor reduces the level of a protein encoded by a target gene or eliminates its expression. In some embodiments, an epigenetic editor reduces the level of a protein encoded by a target gene copy containing a specific allele recognized by an epigenetic editor or eliminates its expression. The target PCSK9 gene can be epigenetically modified in vitro, in vitro, or in vivo.

[0333] The effector domain of the epigenetic editor described herein can change (for example, place or remove) the chemical modification at the nucleotide of the target gene or the histone associated with the target gene. The chemical modification can be changed at a single nucleotide or a single histone, or can be changed 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.

[0334] In some embodiments, the effector domain of the epigenetic editor described herein can change the CpG dinucleotides in the target gene. In some embodiments, compared with the original state of the gene or the gene in comparable cells not contacted with the epigenetic editor, all CpG dinucleotides within 2000, 1500, 1000, 500 or 200bp of the target sequence flank (e.g., in the change site as described herein) are changed according to the modification type described herein. 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 CpG dinucleotides are altered compared to the original state of the gene or the gene in a comparable cell that has not been contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the CpG dinucleotides are altered compared to the original state of the gene or the gene in a comparable cell that has not been contacted with an epigenetic editor. In some embodiments, a single CpG dinucleotide is altered compared to the original state of the gene or the gene in a comparable cell that has not been contacted with an epigenetic editor.

[0335] The effector domain of the epigenetic editor described herein can change the histone modification state of the histone associated with or combined with the target gene.For example, the effector domain can be placed on one or more lysine residues of the histone tail of the histone associated with the target gene.In some embodiments, the effector domain can cause the deacetylation of one or more histone tails of the histone associated with the target gene, thereby reducing or silencing the 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 (for example, one or more of H3K9me2, H3K9me3, H3K27me2, H3K27me3 and H4K20me3 methylation) at one or more histone tails associated with the target gene, thereby reducing or silencing the expression of the target gene.

[0336] In some embodiments, compared to the original state of the chromosome or the chromosome in comparable cells not contacted with the epigenetic editor, all histone tails of the histone bound to the DNA nucleotides within 2000, 1500, 1000, 500 or 200bp of the target sequence flank are changed according to the modification type described herein. In some embodiments, compared to the original state of the chromosome or the chromosome in comparable cells not contacted with the epigenetic editor, 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 changed. In some embodiments, compared to the original state of the chromosome or the chromosome in a comparable cell that has not been contacted with an epigenetic editor, 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 changed. For example, compared to the original state of the chromosome or the chromosome in a comparable cell that has not been contacted with an epigenetic editor, a single histone tail of the bound histone can be changed. As another example, compared to the original state of the chromosome or the chromosome in a comparable cell that has not been contacted with an epigenetic editor, a single bound histone octamer can be changed.

[0337] The chemical modification placed at the target gene DNA nucleotide or histone residue can be located at or adjacent to the target sequence in the target gene. In some embodiments, the effector domain of the epigenetic editor described herein changes the chemical modification state of the nucleotides of 100-200, 200-300, 300-400, 400-55, 500-600, 600-700 or 700-800 nucleotides 5' or 3' of the target sequence in the target gene or the histone tail bound thereto. In some embodiments, the effector domain changes the chemical modification state of nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides flanking the target sequence, or the histone tails to which it is bound. As used herein, "flanking" refers to the nucleotide positions from the 5' to the 5' end and from the 3' to the 3' end of a particular sequence (e.g., a target sequence).

[0338] In some embodiments, the effector domain mediates or induces a chemical modification change in a nucleotide away from the target sequence or in a histone tail bound thereto. Such a modification may start near the target sequence and may subsequently spread to one or more nucleotides away from 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 within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nucleotides flanking the target sequence, or one or more histone residues bound thereto, and the change in the chemical modification state can spread to one or more nucleotides in the target gene that are 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 upstream or downstream of the target sequence. In certain embodiments, the chemical modification may be initiated at less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides in the target gene and diffuse to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides in the target gene. In some embodiments, the chemical modification extends to nucleotides throughout the target gene. Additional proteins or transcription factors, such as transcription repressors, methyltransferases, or transcriptional regulatory scaffold proteins, may participate in the diffusion of chemical modifications. Alternatively, an epigenetic editor may be involved alone.

[0339] In some embodiments, compared to a control cell, a control tissue, or a control subject (e.g., in the absence of an epigenetic editor), the epigenetic editor described herein reduces the expression of the target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or more, as measured by transcription of the target gene in a cell, tissue, or subject. In some embodiments, compared to a control cell, a control tissue, or a control subject, the epigenetic editor described herein reduces the expression of the 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, 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 recognized by the epigenetic editor. In a specific embodiment, the epigenetic modified copy encodes a functional protein, so the epigenetic editor disclosed herein can reduce or eliminate the expression and / or function of the protein. For example, compared to a control cell, a control tissue or a control subject, the epigenetic editor described herein can reduce the expression and / or function of a protein encoded by a target gene in a cell, tissue or subject by at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times or at least 100 times.

[0340] Modulation of target gene expression can be determined by determining any parameter that is indirectly or directly affected by target gene expression. Such parameters include, for example, changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in transcription or activity of reporter genes (such as, for example, luciferase, CAT, β-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 Ca); 2+) concentration changes; changes in cell growth; changes in neovascularization; and / or changes in any functional effect of gene expression. Measurements can be performed in vitro, in vivo, and / or ex vivo, and can be performed by conventional methods, such as measuring RNA or protein levels, measuring RNA stability, and / or identifying downstream or reporter gene expression. Readout can be by, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3), changes in intracellular calcium levels; cytokine release, etc.

[0341] Methods for determining the expression level of a gene (e.g., a target of an epigenetic editor) can include, for example, reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blotting, RNA sequencing, DNA sequencing (e.g., sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA); Next-generation (Next-Gen) sequencing, nanopore sequencing, pyrophosphate sequencing, or nanostring sequencing to determine the transcript level of a gene. Protein levels expressed from genes can be determined, for example, by Western blotting, enzyme-linked immunosorbent assay, mass spectrometry, immunohistochemistry, or flow cytometry analysis. Gene expression product levels can be standardized to internal standards, such as total messenger ribonucleic acid (mRNA) or the expression level of a specific gene (e.g., housekeeping gene).

[0342] In some embodiments, the effect of the epigenetic editor in regulating the expression of the target gene can be checked using a reporter system. For example, the epigenetic editor can be designed to target a reporter gene encoding a reporter protein (such as a fluorescent protein). The expression of the reporter gene in such a model system can be monitored by, for example, flow cytometry, fluorescence activated cell sorting (FACS) or fluorescence microscopy. In some embodiments, a cell group can be transfected with a vector containing a reporter gene. A vector can be constructed so that the reporter gene is expressed when the vector is transfected with cells. Suitable reporter genes include genes encoding fluorescent proteins, such as green, yellow, cherry, cyan or orange fluorescent proteins. The cell group carrying the reporter system can be transfected with DNA, mRNA or a vector encoding an epigenetic editor targeting a reporter gene.

[0343] VII. Pharmaceutical composition

[0344] In one aspect, the present disclosure provides a pharmaceutical composition comprising one or more epigenetic editors described herein or its components (e.g., fusion proteins and / or guidance polynucleotides) as active ingredients (or as the only active ingredient), or nucleic acid molecules encoding the epigenetic editors or their components. For example, the pharmaceutical composition may include a nucleic acid molecule encoding a fusion protein (and guidance polynucleotides, if applicable) of an epigenetic editor described herein. In some embodiments, a separate pharmaceutical composition comprises a fusion protein and a guidance polynucleotide. The pharmaceutical composition may also include cells that have undergone epigenetic modification mediated or induced by an epigenetic editor provided herein.

[0345] Typically, the epigenetic editors or components thereof described herein, or nucleic acid molecules encoding the epigenetic editors or components thereof, of the present disclosure are suitable for administration as a formulation in combination with one or more pharmaceutically acceptable excipients (e.g., as described below).

[0346] The term "excipient" is used herein to describe any ingredient other than the compounds of the present disclosure. The choice of excipient will depend to a large extent on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Another example of a pharmaceutically acceptable substance is a wetting agent or a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative, or a buffer, which enhances the shelf life or effectiveness of the antibody.

[0347] The preparation of the pharmaceutical composition suitable for parenteral administration generally comprises the active ingredient in combination with a pharmaceutically acceptable carrier (e.g., sterile water or sterile isotonic saline). Such preparations can be prepared, packaged or sold in a form suitable for bolus administration or continuous administration.

[0348] VIII. Delivery Method

[0349] In some embodiments, an epigenetic editor or its components are introduced into a target cell in the form of a nucleic acid molecule encoding an epigenetic editor or its components; therefore, the pharmaceutical composition herein comprises a nucleic acid molecule. Such nucleic acid molecules may be, for example, DNA, RNA or mRNA, and / or a modified nucleic acid sequence (e.g., with chemical modification, 5' cap or one or more 3' modifications). In some embodiments, nucleic acid molecules may be delivered as naked DNA or RNA, for example, by transfection or electroporation, or may be conjugated with a molecule (e.g., N-acetylgalactosamine) that promotes uptake by target cells. In some embodiments, nucleic acid molecules may be in a nucleic acid expression vector, which may include expression control sequences, such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art. The vector may also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), which is associated with a sequence encoding a protein (e.g., inserted or fused).

[0350] Examples of vectors include, but are not limited to, plasmid vectors; viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., murine leukemia virus or spleen necrosis virus, vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, 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 epigenetic editors or components thereof as 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 change target tissue specificity.

[0351] In certain embodiments, an epigenetic editor or a component thereof as described herein is encoded by a nucleic acid sequence present in one or more viral vectors or a suitable capsid protein of any viral vector. Examples of viral vectors include adeno-associated viral vectors (e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and / or variants thereof); retroviral vectors (e.g., Moloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., vectors based on HIV and FIV), and herpes virus vectors (e.g., HSV-2).

[0352] In some embodiments, delivery involves an adeno-associated virus (AAV) vector. When the DNA binding domain of the epigenetic editor fusion protein is a zinc finger array, AAV vector delivery may be particularly useful. Without wishing to be bound by any theory, compared to larger DNA binding domains such as Cas protein domains, smaller-sized zinc finger arrays may allow such fusion proteins to be conveniently packaged in viral vectors such as AAV vectors.

[0353] Any AAV serotype, such as a human AAV serotype, can be used for the AAV vectors as described herein, including but not limited to AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10) and AAV serotype 11 (AAV11) 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 with wild-type AAV. In certain embodiments, the AAV variant can be engineered so that its capsid protein has reduced immunogenicity or enhanced transduction ability in humans. In some cases, one or more regions of at least two different AAV serotype viruses are shuffled and recombined to produce chimeric variants. For example, a chimeric AAV may comprise an inverted terminal repeat (ITR) having a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV may have different antigenic reactivity or recognition compared to its parental serotype. In some embodiments, chimeric variants of AAV include amino acid sequences from 2, 3, 4, 5 or more different AAV serotypes.

[0354] 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 biotrajectory, lipid-mediated transfection, transfection by heat shock, transfection mediated by compressed DNA, lipofection, cationic agent-mediated transfection, and transfection with liposomes, immunoliposomes, exosomes, or cationic surface amphiphiles (CFA). In certain embodiments, one or more mRNAs encoding epigenetic editor fusion proteins as described herein can be co-electroporated with one or more guiding polynucleotides (e.g., gRNA) as described herein.An important category of non-viral nucleic acid vectors is nanoparticles, which can be organic (e.g., lipids) or inorganic (e.g., gold).For example, in certain embodiments of the present disclosure, organic (e.g., lipids and / or polymers) nanoparticles may be suitable for use as delivery vehicles.

[0355] In some embodiments, lipid nanoparticles (LNP) are used to deliver. The size of LNP compositions is usually in micrometer or smaller order of magnitude, and can include lipid bilayer. In some embodiments, LNP refers to any particle with a diameter less than 1000nm, 500nm, 250nm, 200nm, 150nm, 100nm, 75nm, 50nm or 25nm. In some embodiments, the size range of nanoparticles can be 1-1000nm, 1-500nm, 1-250nm, 25-200nm, 25-100nm, 35-75nm or 25-60nm. Nanoparticle compositions include lipid nanoparticles (LNP), liposomes (e.g., lipid vesicles) and lipid complexes.

[0356] LNP as described herein can be made of cations, anions or neutral lipids. In some embodiments, LNP can include neutral lipids, such as fusogenic phospholipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or membrane component cholesterol, as auxiliary lipids to enhance transfection activity and nanoparticle stability. In some embodiments, LNP 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 LNP. Lipid can be combined in any molar ratio to produce LNP. In some embodiments, LNP is a LNP targeting liver (e.g., preferentially or specifically targeting liver).

[0357] Any type of cell can be targeted to deliver an epigenetic editor or its components as described herein. For example, the cell can be eukaryotic or prokaryotic. In some embodiments, the cell is a mammalian (eg, human) cell. Human cells can include, for example, hepatocytes, bile duct epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.

[0358] In some embodiments, the epigenetic editor described herein or its components are delivered to the host cell for transient expression, for example, via a transient expression vector. The transient expression of the epigenetic editor or its components can result in extended or permanent epigenetic modification of the target gene. For example, after the epigenetic editor is introduced into the host cell, the epigenetic modification can be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks or longer; or 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer. The epigenetic modification can be maintained after one or more mitotic and / or meiotic events of the host cell. In a specific embodiment, the epigenetic modification is maintained across generations in offspring produced or derived from the host cell.

[0359] IX. Therapeutic uses of epigenetic editors

[0360] The present disclosure also provides a method for treating or preventing a condition in a subject, comprising administering an epigenetic editor or pharmaceutical composition as described herein to the subject. The epigenetic editor can achieve epigenetic modification of a target polynucleotide sequence in a target gene associated with a disease, condition or disorder in a subject, thereby regulating the expression of the target gene to treat or prevent the disease, condition or disorder. In some embodiments, the epigenetic editor reduces the expression of the target gene to a degree sufficient to achieve the desired effect, such as a treatment-related effect, such as preventing or treating a disease, condition or disorder.

[0361] In some embodiments, a system for modulating (e.g., inhibiting) PCSK9 expression is administered to a subject, wherein the system comprises (1) a fusion protein as described herein and (where relevant) a guide polynucleotide, or (2) a nucleic acid molecule encoding the fusion protein and (where relevant) a guide polynucleotide.

[0362] "Treat", "treating" and "treatment" refer to a method of alleviating or eliminating at least one of a biological disorder and / or its associated symptoms. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder or condition. In addition, references to "treatment" herein include references to curative, palliative and preventive treatments. In some embodiments, alleviating symptoms may involve a reduction in symptoms measured by any standard technique of at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9% or 100% compared to an equivalent untreated control.

[0363] In some embodiments, the subject can be a mammal, such as a human. In some embodiments, the subject is selected from a non-human primate, such as a chimpanzee, a cynomolgus monkey, or a macaque, and other apes and monkey species.

[0364] In some embodiments, the human patient has a condition selected from hypercholesterolemia (e.g., familial hypercholesterolemia, such as heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), or established atherosclerotic cardiovascular disease (ASCVD)) or renal insufficiency (RI).

[0365] In some embodiments, the patient to be treated with an epigenetic editor of the present disclosure has received prior treatment for the condition to be treated (e.g., hypercholesterolemia (such as HeFH, HoFH, HF, or established ASCVD) or RI). In other embodiments, the patient has not received such prior treatment. In some embodiments, the patient has failed prior treatment for the condition (e.g., prior hypercholesterolemia treatment).

[0366] The epigenetic editor disclosed herein can be applied to patients with conditions described herein in a therapeutically effective amount. As used herein, "therapeutically effective amount" refers to the amount of the therapeutic agent administered, which will alleviate one or more symptoms of the treated condition to a certain extent, and / or result in the clinical endpoint desired by a healthcare professional. The effective amount for treatment can be measured by its ability to stabilize the patient's disease progression and / or improve symptoms and preferably reverse the ability to progress the disease. The ability of the epigenetic editor disclosed herein to reduce or silence PCSK9 expression can be assessed by in vitro assays (e.g., as described herein) and in suitable animal models predicted to be effective for humans. A suitable dosage regimen will be selected so as to provide the best therapeutic response in each specific case, for example, as a single bolus or as a continuous infusion, and the dosage may be adjusted according to the emergency situation of each case.

[0367] The epigenetic editor of the present disclosure can be administered without additional therapeutic treatment, i.e. as an independent therapy (monotherapy). Alternatively, treatment with the epigenetic editor of the present disclosure may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the additional therapeutic agent is any therapeutic agent known in the art for treating hypercholesterolemia or RI. Therapeutic agents include but are not limited to statins, fibrates, HMG-CoA reductase inhibitors, nicotinic acid, bile acid regulators or chelators, cholesterol absorption inhibitors or regulators, CETP inhibitors, MTTP inhibitors and PPAR agonists.

[0368] The epigenetic editor or its components (or nucleic acid molecules encoding epigenetic editors or their components) disclosed herein can be administered by any method accepted in the art, such as subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, intralymphatically or intraperitoneally. In a specific embodiment, the pharmaceutical composition of the present disclosure is administered intravenously to a subject.

[0369] X. definition

[0370] As used herein, the term "nucleic acid" refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form, and includes DNA and RNA. "Nucleotide" contains sugar deoxyribose (DNA) or ribose (RNA), bases and phosphate groups, and is linked together by the phosphate groups. "Bases" include purines and pyrimidines, which include natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs; and synthetic derivatives of purines and pyrimidines, including but not limited to modified forms of placing new reactive groups such as amines, alcohols, thiols, carboxylates, halogenated alkyls, etc. Nucleic acids may contain known nucleotide analogs and / or modified main chain residues or bonds, which may be synthetic, naturally occurring and non-naturally occurring. Such nucleotide analogs, modified residues and modified bonds are well known in the art, and can provide nucleic acid molecules with enhanced cellular uptake, reduced immunogenicity and / or increased stability in the presence of nucleases.

[0371] As used herein, an "isolated" or "purified" nucleic acid molecule is a nucleic acid molecule that is free from its natural environment. For example, an "isolated" or "purified" nucleic acid molecule (1) has been separated from the nucleic acid of the genomic DNA or cellular RNA from which it was derived; and / or (2) does not exist in nature. In some embodiments, an "isolated" or "purified" nucleic acid molecule is a recombinant nucleic acid molecule.

[0372] It should be understood that in addition to the specific proteins and nucleic acid molecules mentioned herein, the present disclosure also contemplates the use of variants, derivatives, homologs and fragments thereof. Variants of any given sequence may have a specific sequence of residues (whether amino acid residues or nucleic acid residues) that are modified in such a way that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. Variant sequences may be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue (in some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues) present in a naturally occurring sequence. For specific proteins described herein (e.g., KRAB, dCas9, DNMT3A and DNMT3L proteins described herein), the present disclosure also contemplates retaining at least one of its endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98% or 99% of its function) of any protein naturally occurring form or variant or homolog.

[0373] Some exemplary fusion proteins included in the present disclosure are provided herein. It will be understood by those skilled in the art that these exemplary proteins are non-limiting examples, and additional proteins are within the scope of the present disclosure. For example, in the case of providing an exemplary protein comprising a specific domain (e.g., mammalian DNMT3A, DNMT3L and / or KRAB domain (e.g., human or mouse DNMT3A, DNMT3L and / or KRAB domain), a person skilled in the art can determine that, in some embodiments, the present disclosure also includes a fusion protein having the same configuration but wherein one or more mammalian domains replace a homologous domain from another mammal (e.g., one or more mouse domains replace one or more human domains). For example, in the case of providing an exemplary fusion protein comprising a mouse DNMT3L domain, a fusion protein having the same structure but mouse DNMT3L replaces a human DNMT3L domain is also included.

[0374] As used herein, any polypeptide or homologue of the nucleic acid sequence considered herein includes a sequence with certain homology to wild-type amino acids and nucleic acid sequences.Homologous sequences can include sequences identical to subject sequences at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92% < 93%, 94%, 95%, 96%, 97%, 98% or 99%, such as amino acid sequences.In the context of amino acids or nucleotide sequences, the term "identity percentage" refers to the percentage of identical residues in two sequences when the maximum correspondence is compared.In some embodiments, the length of the reference sequence compared 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., sequence analysis software package of the University of Wisconsin Biotechnology Center Genetics Computer Group, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning homology to various substitutions, deletions and / or other modifications. In an exemplary method for determining the degree of identity, the BLAST program can be used, wherein the probability score between e-3 and e-100 indicates closely related sequences.

[0375] The percent identity of two nucleotide or polypeptide sequences is determined by e.g. Determined using default parameters (available at the National Center for Biotechnology Information website at the U.S. National Library of Medicine). 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%) of the reference sequence.

[0376] It will be appreciated that the numbering of specific positions or residues in a polypeptide sequence depends on the specific protein and numbering scheme used. The numbering may differ, for example, in the precursor of a mature protein and the mature protein itself, and differences in the sequence from species to species may affect the numbering. Those skilled in the art will be able to identify the corresponding residues in any homologous protein and the corresponding encoding nucleic acid by methods well known in the art, such as by sequence alignment and determination of homologous residues.

[0377] The term "regulate" or "change" refers to the change in the amount, degree or scope of a function. For example, an epigenetic editor as described herein can regulate the activity of a promoter sequence by binding to a motif in a promoter, thereby inducing, enhancing or inhibiting the transcription of a gene operably connected to a promoter sequence. As other examples, an epigenetic editor as described herein can block RNA polymerase transcription of a gene, or can inhibit the translation of an mRNA transcript. When used to refer to an epigenetic editor as described herein or its components, the term "inhibit (inhibit)", "repress (repress)", "suppress (suppress)", "silence" etc. refers to the activity of a nucleic acid sequence or protein relative to the absence of an epigenetic editor or its components, reducing or preventing a nucleic acid sequence (e.g., target gene) or protein activity (e.g., transcription). The term may include partially or completely blocking activity, or preventing or delaying activity. The inhibited activity can be, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the activity of the control, or can be, for example, at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold lower than the activity of the control.

[0378] The term "about" or "approximately" means within an acceptable error range 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, such as limitations of the measurement system. For example, by convention, "about" can mean within one or more than one standard deviation of a given value. Where specific values ​​are described in the application and claims, the term "about" should be assumed to mean an acceptable error range for the specific value unless otherwise indicated.

[0379] The scope provided herein should be understood as the abbreviation of all values ​​in the scope. For example, the scope of 1 to 50 should be understood as including any number, combination of numbers or subranges selected from the group consisting of the following: 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, and all intermediate decimal values ​​between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. About subrange, " nested subrange " extending from any end point of scope is particularly considered. For example, nested sub-ranges of the exemplary range 1 to 50 may 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.

[0380] Unless otherwise defined herein, scientific and technical terms used in conjunction 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, but methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure. In the event of a conflict, the present specification including the definition shall prevail. In addition, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout this specification and the examples, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" will be understood to imply the inclusion of the integer or integer group, but do not exclude any other integer or integer group. Unless otherwise stated, the description of the element list herein includes any element alone or in any combination. The description of the embodiment herein includes the 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 as a whole. To the extent that a reference incorporated by reference contradicts the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents becomes part of the common general knowledge in the art.

[0381] According to the present disclosure, the back-references in the dependent claims are intended to serve as a shorthand for directly and unambiguously disclosing each claim combination indicated by the back-reference. In addition, the headings herein are created for organizational convenience and are not intended to limit the scope of the claimed compositions and methods in any way.

[0382] Some protein sequences provided herein, such as some fusion protein sequences, include peptide tags, such as His6 tags or DYKDDDDK (SEQ ID NO: 1528) tags, which can be used to detect and / or purify tagged proteins without affecting protein function. These peptide tags and other suitable peptide tags are well known to those skilled in the art. It will be apparent to those skilled in the art that the disclosed tags can replace other suitable peptide tags, and fusion proteins with identical or highly similar sequences but not including such peptide tags (e.g., peptide tags have been cut therefrom or produced without peptide tags) are also suitable for practicing embodiments of the present disclosure.

[0383] In order to better understand the present disclosure, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present disclosure in any way. Example

[0384] Example 1: Design and synthesis of fusion protein

[0385] A fusion protein containing dCas9, DNMT3A, DNMT3L and KOX1KRAB ("CRISPR-off") was designed and constructed. From N-terminus to C-terminus, the protein has the domains DNMT3A-Linker-DNMT3L-XTEN80-NLS-dspCas9-NLS-XTEN16-KOX1KRAB (SEQ ID NOs: 658 and 1495). The CRISPR-off plasmid construct has been ( et al., Cell (2021) 184(9):2503-19) and ordered from Twist Biosciences.

[0386] A ZF fusion protein ("ZF-off") comprising DNMT3A, 3L and KOX1 KRAB was also constructed. The construct had the general structure DNMT3A-Linker-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1Krab (SEQ ID NOs: 659 and 1496).

[0387] Example 2: Selection of target PCSK9 sequences for gRNA epigenetic silencing

[0388] The Benchling gRNA platform (Benchling (2021), retrieved from benchling.com) was used to computationally design gRNAs targeting the PCSK9 TSS of + / -1 kb for human (GRCh38), mouse (mm10), and macaque (5.0) PCSK9. gRNAs containing polyTTTT sequences were first discarded. We used CasOFFinder for gRNA off-target analysis (Bae et al., Bioinformatics (2014) 30(10): 1473-5). If the gRNA matched multiple positions in the corresponding genome build of each independent species, the gRNA was discarded.

[0389] Human PCSK9 gRNAs were subjected to cross-reactivity sequence analysis to annotate sequence mismatches with macaque or mouse gRNA sequences. Specifically, gRNA sequence alignments were performed to identify the degree of DNA similarity at each nucleotide, including annotation of guides containing up to zero, one, or two nucleotide mismatches. A final set of 226 gRNA sequences were selected for PCSK9 primary screening in HeLa cells.

[0390] Example 3: Selection of ZF target sites and design of ZF proteins for epigenetic silencing

[0391] A library of two-finger ZFPs (2F units) that each recognize a 6bp DNA site is used to design a larger six-finger ZFP array targeting an 18bp DNA binding site. The source of the 2F unit is a set of three-finger zinc finger proteins that have been selected using the bacteria-2-hybrid (B2H) selection system to bind to specific target sites (Hurt et al., PNAS (2003) 100: 12271-6; Maeder et al., Mol Cell (2008) 31 (2): 294-301). A list of targetable DNA sites is created by generating all possible triplet combinations of the 6bp binding sites represented in the library and allowing 0 or 1bp between the 6bp target sites. In order to identify zinc finger target sites within PCSK9, sequences within + / - 1kb of TSS (human (GRCh38)) are queried for this list. For each identified ZF target site, a variety of ZF proteins can be designed. The design of the six recognition helices for producing the complete protein is carried out by selecting the two-finger unit and considering many factors, such as the known binding preference of zinc finger proteins, the frequency of selecting -1, 2, 3 and 6 amino acids to bind to the desired target base in the B2H selection system, avoiding the selection of -1, 2, 3 and 6 amino acids to bind to multiple different bases in B2H, and maintaining background dependence by matching flanking bases where possible. The complete ZF sequence is derived from the naturally occurring Zif268 protein, and the selected recognition helices are maintained in the sequence background in which they are selected in B2H (finger 1-2 or finger 2-3 from Zif268). The two-finger unit is connected by a linker TGSQKP (SEQ ID NO: 651) (wherein the 6bp binding site is continuous) and by a linker TGGGGSQKP (SEQ ID NO: 652) (wherein 1bp separates the 6bp binding site). The final collection of 209 ZFPs targeting 49 different binding sites was selected for the primary screening of PCSK9 in HeLa cells.

[0392] Figure 1 The overlap of gRNAs and zinc finger proteins mapped to the PCSK9 target region is shown.

[0393] Example 4: Guide RNA screening in HeLa cells

[0394] Primary screening of gRNAs targeting PCSK9 was performed in HeLa cells. gRNA sequences were ordered from TwistBiosciences as DNA fragments with the u6 promoter sequence preceding the gRNA coding sequence.

[0395] HeLa cells were transfected with gRNA and CRISPR-off in DNA format. TM Six 96-well plates (Sigma-Aldrich catalog number M2936) were seeded with 12,000 HeLa cells per well (ATCC catalog number CCL-2) in standard culture medium containing DMEM (Thermo Fisher catalog number 11-965-092) (Thermo Fisher catalog number 35050061) and 1x penicillin-streptomycin (Thermo Fisher catalog number 15140122). After plating, the cells were grown in an incubator at 37°C, 5% CO2 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 number 14190144) to a concentration of 7.5 ng / μL. In addition, 10 ng of EF1a:Puromycin resistance plasmid was added to the transfection mixture to achieve a total payload of 85 ng DNA. Following the manufacturer's instructions, the resuspended DNA components were added to the Transfection reagent (Mirus catalog number MIR2300) is used to produce transfection mixture. 10 μL of each transfection mixture is added in duplicate to a total of six screening plates. The positive control used is CRISPRi (dCas9-KRAB), which has two gRNAs targeting sites near TSS. The two CRISPRi positive control gRNAs used are gRNA004 and gRNA005, as annotated in the gRNA sequence table. These control conditions are respectively referred to as "CRISPRi-1" and "CRISPRi-2" in the primary screening data table. Negative controls are CRISPR-off without gRNA, CRISPR-off with non-PCSK9 locus (CD151) targeting gRNA and empty vector (pUC19; NEB catalog number N3041S).

[0396] 24 hours after transfection, puromycin resistance selection was performed. The cell culture medium was completely aspirated, and all wells were washed 3 times with DPBS buffer (Thermo Fisher catalog number 14190144), and 200 μL of 1 μg / μL puromycin was added to all screening plate wells.

[0397] 48 hours after transfection, cells were passaged. The cell culture medium was completely aspirated and all wells were washed 3 times with DPBS buffer (Thermo Fisher catalog number 14190144). Cells were enzymatically extracted by adding 25 μL trypsin-EDTA (0.25%) (ThermoFisher catalog number 25200056) and incubated in a 37°C incubator for 5 minutes. Trypsinized cells were resuspended in fresh standard culture medium at a ratio of 1:8 and re-plated at a ratio of 1:4 after changing the culture medium for 72 hours.

[0398] To measure the levels of secreted PCSK9 protein, culture medium was harvested 24 h after medium change and titrated using the Promega Cell Titer Glo according to the manufacturer's recommendations. TM Relative cell counts were performed using BioLegend's LEGEND MAX TM PCSK9 protein levels were assessed using the Human PCSK9 ELISA Kit (Cat. No. 443107). Harvested media was plated and all subsequent steps were performed exactly as recommended by the manufacturer. Nivo TM The final plate reading was performed at 450 nm on the F instrument. Functions were fitted to the standard curve and unknowns were interpolated using GraphPad Prism software. PCSK9 ELISA results were analyzed by cell titer Assay (Promega Cat. No. G7571) results were normalized to correct for any well-to-well variability in cell number.

[0399] More than 200 gRNAs were tested, of which 40 were identified as top sequences ( Figure 2 ; the leading sequences are designated as darker circles). The sequences and efficacy of the gRNAs tested are shown in Table 7 (SEQ: SEQ ID NO). Relative PCSK9 secretion ("Control PCSK9%") represents the average PCSK9 protein level of the treated samples, expressed as a percentage relative to the average of all non-targeting gRNA (CD151) negative control conditions. Robust silencing of PCSK9 (30-40% of negative control levels) was observed in cells treated with a variety of gRNA candidate treatments. The top 40 gRNAs with the best PCSK9 protein knockdown were selected as sgRNAs for further follow-up studies.

[0400] Table 7. Targeting sequences of tested gRNAs

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408] The best performing gRNA was found to be closely aligned with the transcription start site of the PCSK9 gene ( Figure 3 ).

[0409] After this primary screen, the top 40 gRNAs were subjected to secondary screening in RNA form. The top 40 guides were chemically synthesized and co-transfected with in vitro transcribed mRNA encoding CRISPR-off, CRISPRi or WT Cas9 constructs. Secreted PCSK9 levels were measured 7 and 28 days after transfection.

[0410] To generate in vitro transcribed CRISPR-off, CRISPRi, and WT Cas9 effector mRNA, use Plasmid constructs encoding these proteins were linearized with the MfeI restriction enzyme (Catalog No. R3589S). 1 μg of linearized template was used to generate the plasmid constructs using the CellScript T7 mScript TM The in vitro transcription reaction was set up using the standard mRNA production system (Cat. No. C-MSC100625). The resulting RNA had a cap 1 structure at the 5' end and was 3' polyadenylated. Mini Kit (Cat. No. 74104) was used to purify the transcribed RNA.

[0411] End-modified sgRNAs purified using standard desalting were obtained from Integrated DNA Technologies. The three nucleotides at the 5' end and the three nucleotides at the 3' end of each guide were 2'-O-methyl modified. The three internucleoside bonds at the 3' end and the three internucleoside bonds at the 5' end were phosphorothioate internucleoside bonds (Table 8; SEQ ID NO). In the table, mX (i.e., mA, mC, mG, or mU) represents a 2'-O-methyl modified ribonucleoside, rX (i.e., rA, rC, rG, or rU) represents a natural ribonucleoside, and * represents a phosphorothioate bond. All non-phosphorothioate internucleoside bonds are phosphate bonds.

[0412] Table 8. Targeting sequences of the top 40 gRNAs in the secondary HeLa cell screen

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421] Using Mirus HeLa cells were reverse transfected with 25 ng effector and 12.5 ng sgRNA using transfection reagent (Cat. No. MIR6003) in a 96-well plate format. Conditioned media was harvested weekly for up to four weeks and used for transfection using BioLegend’s LEGEND MAX TM The secreted PCSK9 level was measured using the Human PCSK9 ELISA Kit (Cat. No. 443107). ELISA data were normalized to cell number using the CRISPRi(dCas9-KRAB) kit (Cat. No. G7571). While PCSK9 silencing was transient in CRISPRi(dCas9-KRAB) and returned to baseline by day 28, several sgRNAs co-transfected with the CRISPR-off(DNMT3A-3L-dCas9-KRAB) construct showed robust and durable silencing ( Figure 4A ). 16 of the 40 guides tested with CRISPR-off showed silencing efficiency greater than WT Cas9 (Table 9).

[0422] Table 9. Relative PCSK9 expression in HeLa cells treated with modified gRNA and CRISPR-off

[0423]

[0424]

[0425] At the two-week time point, RNA was extracted using the Quick-RNA 96 kit (Cat. No. R1053) from Zymo Research. qPCR (PCSK9: Hs00545399_m1, PPIA: Hs99999904_m1) was performed using Quantabio's qScript XLT One-Step RT-qPCR ToughMix (Cat. No. 95134-500) and TaqMan assays. PCSK9 levels were standardized with PPIA. Relative quantification was performed using the delta-delta Ct method.

[0426] Inhibition of PCSK9 secretion was found to be associated with mRNA silencing at day 14 ( Figure 4B ).

[0427] ModRNA004 and modRNA111 were tested in HeLa cells to inhibit PCSK9 secretion over 60 days ( Figure 5 ). WTCas9 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 durable silencing of PCSK9, comparable to that achieved by gene editing with WT Cas9 in HeLa cells.

[0428] Furthermore, the results showed that epigenetic silencing was 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 was shown to not increase PCSK9 secretion ( Figure 6 ).

[0429] Example 5: Guide RNA assay in Huh7 liver cancer cell line

[0430] The Huh7 hepatoma cell line is suitable for high-throughput screening and transfection. The first 13 guides from the HeLa screen with complete homology or a single mismatch to the cynomolgus PCSK9 gene were tested in the Huh7 hepatoma cell line. Epigenetic silencing with CRISPR-off constructs was shown to be stable within 7 days ( Figure 7 ).

[0431] Example 6: Guide RNA assays in primary human and cynomolgus monkey hepatocytes

[0432] BioIVT's primary human and cynomolgus monkey Cultures were used to test the efficacy of gRNA in primary hepatocytes. They were maintained according to the manufacturer's recommendations. In short, Maintenance medium was thawed and replenished within 30 minutes of cell arrival. After medium change, cells were acclimated for two days in a 37°C, 10% CO2 incubator. The day after receipt, cells were cultured using SPARK TM (Precision Nanosystems) and hepato9mRNA LNP preparation kit (Catalog No. NWS0016) to prepare LNPs of CRISPR-off+sgRNA, GFP-mRNA, and WT CRISPR Cas9 at different concentrations. TM The RiboGreen RNA Assay Kit characterizes the encapsulation efficiency and total mRNA payload delivery of the LNPs. The LNPs are then added to the culture medium at a specified amount of total mRNA to achieve clinically relevant levels of PCSK9 silencing. The culture medium is replaced every other day for up to four weeks to assess the persistence and / or heritability of the silencing. PCSK9 silencing is assessed every seven days by ELISA to measure secreted PCSK9 levels in the culture medium. The human albumin ELISA (Thermo Fisher Scientific) was used to measure the PCSK9 levels in the culture medium. ) PCSK9 concentration was controlled to total hepatocytes. Data were then expressed as a percentage of total PCSK9 secretion to 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 isolating primary hepatocytes from the feeder layers, cells were processed for RNAseq evaluation and whole genome bisulfite sequencing.

[0433] The top 13 gRNAs in RNA form 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, (ii) whether they were 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 only, gRNA fragments only (modRNA003), and CRISPRi only. The positive control was CRISPRi co-transfected with modRNA004 (Table 10; SEQ: SEQ ID NO; NHP: non-human primate). All tested gRNAs were predicted to bind both human and non-human primate PCSK9.

[0434] Table 10. Screening of gRNAs in primary human hepatocytes

[0435]

[0436] Robust PCSK9 silencing was observed. For some gRNAs, a >70% reduction in secreted PCSK9 was observed at day 7 (depending on transfection efficiency).

[0437] Example 7: ZF assay in HeLa cells

[0438] A total of 209 zinc finger proteins (structure shown in SEQ ID NO: 659) were designed from the ZF library against 49 PCSK9 target sites (selected from GRCh38 chromosome 1, between 55038548 and 55040548). The target sites have no other exact matches in the human genome (GRCh38).

[0439] HeLa cells were transfected with the ZF-off construct in DNA form. The cells were cultured in a 1x GlutaMAX medium supplemented with 10% fetal bovine serum (ThermoFisher catalog number A4766) v / v. TM Six 96-well plates (Sigma-Aldrich catalog number M2936) were seeded with 12,000 HeLa cells per well (ATCC catalog number CCL-2) in standard culture medium of DMEM (Thermo Fisher catalog number 11-965-092) supplemented with 1x penicillin-streptomycin (Thermo Fisher catalog number 15140122). After plating, the cells were grown in a 37°C incubator under 5% CO2 for 24 hours. 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 added to the transfection mixture to achieve a total payload of 85 ng DNA. The cells were cultured in serum-free OPTI-MEM medium (Thermo Fisher catalog number 14190144). Catalog number 31985062) and follow the manufacturer's instructions to add Transfection reagent (MIR2300) was used to construct the transfection mixture. 10 μL of transfection mixture was added to a total of six screening plates in duplicate. The positive control was CRISPR-off (SEQ ID NO: 658) with high-performance gRNA (gRNA009). The negative control was ZF-off with non-PCSK9 locus target (CLTA) and empty vector (pUC19; NEB catalog number N3041S).

[0440] ZF screening yielded hits with activity comparable to CRISPR ( Figure 8 ). Candidates with high silencing efficiency were taken to subsequent experiments. Fig. 9 The results of ZF screening divided by distance to TSS are shown. A total of 209 ZFs were screened, and their PCSK9 knockdown activities relative to negative controls are shown in Table 11 below.

[0441] Table 11. ZF-off construct activity

[0442]

[0443]

[0444] Compared with WTCas9 and CRISPR-off and gRNA003 combination, several ZF-off constructs showed more effective in silencing PCSK9. The target sites and ZF sequences (F1 to F6) of the ZFP domains in these ZF-off constructs are shown in Table 1.

[0445] Example 8: Full specificity screening of constructs in primary human hepatocytes

[0446] The specificity of CRISPR-off and ZF-off constructs for silencing PCSK9 will be tested in primary human hepatocytes. The readouts to assess specificity are RNAseq, methylation arrays, and whole-genome bisulfite sequencing assays. Whole-genome expression and methylation changes after epigenetic editing compared to negative controls will be analyzed.

[0447] Example 9: CpG methylation patterns

[0448] The CpG methylation pattern in human hepatocytes (e.g., primary cells or cell lines) treated with CRISPR-off or ZF-off is studied. A hybridization capture assay is performed on bisulfite-treated DNA to study the methylation pattern of CpG sites induced by CRISPR-off or ZF-off in a 1 kb region around the PCSK9 TSS.

[0449] Example 10: Stable PCSK9 silencing in mice with wild-type PCSK9 by epigenetic editing

[0450] The ability of CRISPR-off and ZF-off constructs to mediate epigenetic silencing of endogenous PCSK9 in vivo was tested. The constructs were delivered using a single IV administration of mRNA (and gRNA for CRISPR-off silencing) formulated into LNP. Silencing was tested for a period of two to six months in wild-type mice. The readout was serum PCSK9 levels and serum cholesterol levels. A subset of each cohort was selected for liver hematoxylin and eosin (H&E) staining RNAseq and analysis. For several constructs, robust, stable and heritable PCSK9 silencing was observed.

[0451] Example 11: Stable PCSK9 Silencing in Mice Expressing Transgenic Human PCSK9 by Epigenetic Editing

[0452] Three different mouse strains expressing transgenic human PCSK9 were used: hPCSK9-Tg (mPCSK9+ / -) heterozygous mice, hPCSK9-Tg (mPCSK9+ / +) homozygous mice, and hPCSK9-Tg (mPCSK9- / -) mice. The hPCSK9-Tg (mPCSK9- / -) mouse strain used was C57BL / 6J-Pcsk9- / -Tg (RP11-55M23-AbsI), which expresses human PCSK9 under the control of its own promoter ( Fig.10 ). See, for example, Weider et al., J Biol Chem (2016) 291(32): 16659-71.

[0453] CRISPR-off and ZF-off constructs were tested. Constructs were delivered via a single IV administration of mRNA / gRNA formulated into LNPs. Readouts were liver H&E staining, RNAseq and AST / ASL measurements measuring PCSK9 mRNA levels. Efficacy was also tested, including the persistence of PCSK9 silencing over three to four months as measured by serum PCSK9 protein levels. For some constructs, persistent and significant reductions in serum PCSK9 levels were observed.

[0454] Persistence was tested over six to twelve months. Readouts were serum PCSK9 levels and serum cholesterol levels. A subset of the cohort was selected for liver H&E and RNAseq analysis.

[0455] Example 12: Fusion proteins with variant NLS configurations

[0456] Several improved fusion protein constructs were developed using variant nuclear localization sequence (NLS) configurations to have significantly higher apparent silencing activity.

[0457] Several constructs with NLS domain variant configurations were constructed ( Fig.11A and 11B ) and tested in the PCSK9 locus of HeLa cells ( Figures 12A-12B ). In addition, in Hepa1-6 ( Fig.13 ) and HuH7( Figures 14A-14C and Fig.15 ) in the PCSK9 locus. Exemplary fusion protein construct amino acid and DNA sequences are shown below:

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[0494] Fig.14A The sequence in can be found below:

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[0496] Fig.15 The sequence in can be found below:

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[0501] Cell culture and transfection

[0502] HeLa (ATCC-CRM-CCL-2), Hepa1-6 (PCSK9-IRES-TdTomato), Huh7 (Sekisui XenoTech, LLC), and HEK293TGriptite (CLTA-GFP) cells were cultured in DMEM containing 10% FBS. All experiments were performed in HeLa and Huh7 cells using chemically synthesized guide RNAs and in vitro transcribed effector constructs. HeLa cells were reverse transfected using TransIT-X2 transfection reagent from Mirus (Cat. No. MIR6003). Huh7 cells were reverse transfected using MessengerMAX reagent from Invitrogen (Cat. No. LMRNA003). LEGEND MAX from Biolegend was used to reverse transfect Huh7 cells. TMThe secreted PCSK9 levels were measured at the indicated time points using the Human PCSK9 ELISA Kit (Cat. No. 443107). All ELISA data were normalized to cell number using Promega's CellTiter-Glo Kit (Cat. No. G7571).

[0503] HEK293T Griptite cells with GFP knocked into the CLTA locus as an in-frame CLTA fusion were co-transfected with plasmids encoding effector constructs and human CLTA guide RNA using Mirus' TransIT-X2 transfection reagent (Cat#MIR6003). GFP expression was measured by FACS as a surrogate for CLTA expression.

[0504] Plasmids encoding effector constructs and mouse PCSK9 guide RNA were co-transfected into Hepa1-6 cells using the SF cell line 96-well nuclear transfection kit (Catalog No. V4SC-2096, Program Code: CM-138) in Lonza's Amaxa 4D nuclear transfection device. At the indicated time points, cells were subjected to FACS analysis of TdTomato expression as a surrogate for PCSK9 levels.

[0505] In vitro transcription of effector constructs and synthetic gRNA

[0506] CellScript T7 mScript was used according to the manufacturer's instructions. TM The standard mRNA production system (Cat. No. C-MSC100625) was used to set up an in vitro transcription reaction with 1 μg of the linearized effector template to obtain RNA with a Cap 1 structure at the 5' end and polyadenylated at 3'. Terminally modified sgRNAs with three 2'O-methyl modified nucleotides with phosphorothioate bonds at both the 5' and 3' ends were obtained from Integrated DNA Technologies.

[0507] Methylation profile

[0508] 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 by the 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, xGen TMDNA libraries were prepared using the Methyl-Seq DNA Library Preparation Kit (IDT) and analyzed using xGen TM DNA library hybridization capture kit (IDT) was used for hybridization capture. For amplicon sequencing experiments, xGen TM DNA libraries were prepared using the Methyl-Seq DNA Library Preparation Kit (IDT) and analyzed using xGen TM DNA library hybrid capture (IDT) was used for hybrid capture. Using a platinum Taq kit (Invitrogen), bisulfite-converted DNA from each sample was used to inoculate PCR corresponding to each of the two VIM amplicons. Before sequencing by a commercial service (Azenta), the merged products were cleaned using an AMPure XP kit (Beckman Coulter) and the fragment size was assessed by a D1000 screening tape on a Tapestation 4200 (Agilent).

[0509] Example 13: Bacterial DNA methyltransferase

[0510] In this experiment, a panel of bacterial proteins were screened for DNA methyltransferase activity in mammalian cells. These bacterial DNA methyltransferases (Table 12) were N-terminally fused to the dCas9 domain using the experimental procedure of Example 1 to test epigenetic silencing activity. These constructs were then transfected into a reporter cell line expressing GFP under the control of the mammalian promoter of CTLA4.

[0511] Table 12: Bacterial DNA methyltransferases

[0512]

[0513] M.SssI DNA methyltransferase can effectively methylate DNA in mammalian cells ( Fig.16 ), stable silence for up to 30 days. Fig.16 The sequence in can be found below:

[0514] describe sequence SEQ ID NO: TAR087 target DNA sequence GGCGGAAGTGCTGCCTGATA 667 TAR089 target DNA sequence GCAACACCGCCTAGACCGAC 668 TAR090 target DNA sequence GGAGGTTCAGAAAGCCTAAG 669 TAR091 target DNA sequence GCAGCACTAGAGTCCCCTCA 670 TAR092 target DNA sequence GAACAACTCGTGACTGGGGT 671 TAR093 target DNA sequence GGGAGAGGAGGACAAGCGCC 672 TAR094 target DNA sequence GGACCCACCGACACCACGCC 673 TAR114 target DNA sequences CGCGGGAAAATGCAAGACGA 674

[0515] The methylation profiles of these cells were also analyzed on day 29, confirming 20% ​​methylation of the target genes ( Figure 17-18 ).

[0516] Three additional orthologous DNA methyltransferases predicted to be closely related to M.SssI were identified using the experimental procedure of Example 1 and tested for epigenetic silencing activity (Table 13).

[0517] Table 13: Bacterial methyltransferases

[0518] describe SEQ ID NO DNA cytosine methyltransferase Mycoplasmaales bacteria 601 DNA cytosine methyltransferase Mycoplasma marineum 602 DNA (cytosine-5-)-methyltransferase Spiroplasma sinensis 603

[0519] The DNA methyltransferases of Table 13 are predicted to have similar or improved functions to M.Sss I. The sequences are tested in the context of CRISPR-off, replacing murine DNMT3A / DNMT3L, and their functions are compared to those of the M.Sss I DNA methyltransferase in silencing the PCSK9 locus in the HeLa TdTomato system to identify new features and improved functions.

[0520] Example 14: Alternative KRAB domains

[0521] In this example, fusion proteins were constructed with alternative KRAB domains (Table 14) and showed improved activity compared to CRISPR-off when tested using the experimental procedure of Example 1 ( Figures 19A-19D ).

[0522] Table 14: Alternative KRAB domains

[0523]

[0524]

[0525] Example 15: ZIM 3 fusion construct

[0526] New fusions of ZIM3 and KOX1KRAB were generated. Both ZIM 3 and KOX1KRAB are KRAB family proteins with extensive homology. Therefore, sequences were designed that represent the midpoint between ZIM3 and KOX1KRAB. These KOX1KRAB and ZIM3 constructs encode small regions of KOX1KRAB and ZIM3 that are concentrated around the zinc finger domains of the proteins. While the regions used by KOX1KRAB and ZIM3 are very similar within the first 75 bp of their sequences, ZIM3 also has a small alpha-helical region at the C-terminus that is not present in KOX1KRAB. The KOX1KRAB-FL sequence includes a KOX1 KRAB sequence equivalent to this additional segment, while the ZIM3 truncation removes this additional segment from the ZIM3 sequence. The ZIM3 / KOX1KRAB chimera is a fusion of the N- and C-terminal segments of both proteins. ZIM3-like KOX1KRAB variants were all assembled by: first, BLAST of ZIM3 or KOX1KRAB proteins from non-human species to assemble the 100 closest homologs ("families") for each gene; second, identification of the three members of the KOX1KRAB family that were most similar to ZIM3 and the three members of the ZIM3 family that were most similar to KOX1KRAB; third, rationally modifying the KOX1KRAB-FL sequence to resemble three of each group (Table 15).

[0527] Table 15: ZIM-KOX1KRAB chimeric proteins

[0528]

[0529] sequence

[0530] Listed below are the SEQ ID NOs (SEQ) of the nucleotide (nt) and amino acid (aa) sequences described in the present disclosure.

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Claims

1. A system for inhibiting transcription of the human PCSK9 gene in human cells, optionally in human hepatocytes, the system comprising a) one or more fusion proteins, which together comprise a DNA methyltransferase (DNMT) domain and / or a domain that recruits DNMTs, optionally wherein the DNMT domain and / or the recruitment domain comprises a DNMT3A domain and / or a DNMT3L domain, and optionally wherein the recruited DNMT is DNMT3A, and Transcription repressor domain, Each domain is connected to a DNA binding domain, and the DNA binding domain binds to a target region in the human PCSK9 gene; or b) one or more nucleic acid molecules encoding said one or more fusion proteins.

2. The system of claim 1, wherein the DNA binding domain binds to a target sequence in SEQ ID NO: 1488 or 1489.

3. The system of claim 1 or 2, wherein 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.

4. The system of any one of claims 1-3, wherein the DNA binding domain comprises a death CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain.

5. The system of claim 4, wherein the DNA binding domain comprises a dCas9 domain, and the system further comprises (i) one or more guide RNAs comprising any one of SEQ ID NOs: 1262-1487, or (ii) a nucleic acid molecule encoding the one or more guide RNAs.

6. The system of claim 4 or 5, wherein the dCas domain comprises a dCas9 sequence, optionally a sequence having at least 90% identity to SEQ ID NO: 12 or 13.

7. The system of claim 4, wherein the ZFP domain targets a nucleotide sequence selected from the group consisting of SEQ ID NOs: 700-747.

8. The system of claim 7, wherein the ZFP domain comprises the F1-F6 amino acid sequence of any one of ZF001 to ZF048 as shown in Table 1.

9. The system of any one of claims 1-8, wherein the DNMT3A domain comprises a sequence that is at least 90% identical to SEQ ID NO: 574 or 575.

10. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 578-581.

11. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 582-603.

12. The system of any one of claims 1-8, wherein the DNMT domain comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 601-603.

13. The system of any one of claims 1-12, wherein the transcriptional repression domain comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 33-570.

14. The system of any one of claims 1-12, wherein the transcriptional repression domain comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627.

15. The system of claim 14, wherein the KRAB domain comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 89, 116, 245, and 255.

16. The system of any one of claims 1-12, wherein the transcriptional repression domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 571 or 572.

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

18. The system according to any one of claims 1 to 17, wherein the system comprises a) a fusion protein comprising the DNMT3A domain, the DNMT3L domain, the transcription repression domain and the DNA binding domain, Optionally, wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and Optionally, wherein the DNA binding domain is a death CRISPR Cas domain or a ZFP domain; or b) a nucleic acid molecule encoding the fusion protein.

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

20. The system of claim 19, wherein the fusion protein comprises, from N-terminus to C-terminus, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, a first nuclear localization signal (NLS), the DNA binding domain, a second NLS, the third peptide linker, and the transcription repression domain.

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

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

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

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

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

26. The system of claim 25, wherein the fusion protein comprises SEQ ID NO: 658 or a sequence at least 90% identical thereto, or SEQ ID NO: 1495 or a sequence at least 90% identical thereto.

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

28. The system of claim 27, wherein the fusion protein comprises SEQ ID NO: 659 or a sequence at least 90% identical thereto, or SEQ ID NO: 1496 or a sequence at least 90% identical thereto, optionally wherein the ZFP comprises the F1-F6 amino acid sequence of any one of ZF001 to ZF048 as shown in Table 1.

29. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

30. The system of claim 29, wherein the fusion protein comprises SEQ ID NO: 660 or a sequence at least 90% identical thereto.

31. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS, optionally wherein the fusion protein comprises SEQ ID NO: 1514, or a sequence at least 90% identical thereto, or SEQ ID NO: 1523, or a sequence at least 90% identical thereto.

32. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

33. The system of claim 32, wherein the fusion protein comprises SEQ ID NO: 661 or a sequence at least 90% identical thereto, or SEQ ID NO: 1516 or a sequence at least 90% identical thereto.

34. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

35. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

36. The system of claim 35, wherein the fusion protein comprises SEQ ID NO: 662 or a sequence at least 90% identical thereto, or SEQ ID NO: 1520 or a sequence at least 90% identical thereto.

37. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

38. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

39. The system of claim 38, wherein the fusion protein comprises SEQ ID NO: 663 or a sequence at least 90% identical thereto, or SEQ ID NO: 1518 or a sequence at least 90% identical thereto.

40. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first and a 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 a fourth NLS.

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

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

43. A human cell comprising the system of any one of claims 1-42, or a descendant of said cell, optionally wherein said cell is a hepatocyte.

44. A pharmaceutical composition comprising the system according to any one of claims 1 to 42 and a pharmaceutically acceptable excipient, optionally wherein 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.

45. A method of treating a patient in need thereof, said method comprising administering to said patient the system according to any one of claims 1-42 or the pharmaceutical composition according to claim 44, optionally intravenously.

46. ​​The method of claim 45, wherein the patient Suffering from heart disease, Have elevated low-density lipoprotein cholesterol (LDL-C) or hypercholesterolemia, At risk of developing myocardial infarction, stroke, or unstable angina, and / or Having primary hyperlipidemia, optionally heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH).

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

48. Use of a system according to any one of claims 1-42 for the preparation of a medicament for treating a patient in need thereof, optionally in a method according to claim 45 or 46.

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