Polyfunctional gene editing

Abstract

Polyfunctional editors comprising: (a) at least one epigenetic effector domain; operably linked to (b) an endonuclease, for use in gene editing and epigenetic modification.

Description

[0001] POLYFUNCTIONAL GENE EDITING

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 740732, filed December 31, 2024, entitled “GENE EDITING AND EPIGENETIC EDITING FOR HYPERCHOLESTEROLEMIA,” U.S. Provisional Application No. 63 / 857972, filed August 5, 2025, entitled “GENE EDITING AND EPIGENETIC EDITING FOR HYPERCHOLESTEROLEMIA,” U.S. Provisional Application No. 63 / 740762, filed December 31, 2024, entitled “GENE EDITING AND EPIGENETIC EDITING FOR METABOLIC DYSFUNCTION-ASSOCIATED STEATOHEPATITIS,” U.S. Provisional Application No. 63 / 740793, filed December 31, 2024, entitled “GENE EDITING AND EPIGENETIC EDITING FOR HYPERCHOLESTEROLEMIA,” U.S. Provisional Application No. 63 / 740825, filed December 31, 2024, entitled “GENE EDITING AND EPIGENETIC EDITING FOR METABOLIC DYSFUNCTION-ASSOCIATED STEATOHEPATITIS,” each of which is incorporated herein by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (C169870060WO00-SEQ-AXW.xml; Size: 1,197,116 bytes; and Date of Creation: December 18, 2025) are herein incorporated by reference in their entirety.

[0006] FIELD

[0007] The present disclosure provides polyfunctional editors for gene editing and epigenetic modification.

[0008] BACKGROUND

[0009] There is a need for new and improved approaches for modulating the expression of multiple genes simultaneously. The use of gene editing approaches that involve cutting the DNA of the target gene can be associated with risks of undesired mutations, including translocations, when two or more genes are targeted at once, and adapting such approaches to mitigate the risk of unwanted mutations can be cumbersome or unpractical. Accordingly, an unmet need remains for the development of multiplexed approaches that avoid such risks, and in particular the risk of translocations between multiple cut sites.

[0010] 1

[0011] #14686672vlFor example, hypercholesterolemia raises the risk of heart disease and other cardiovascular problems. One promising candidate for addressing hypercholesterolemia is the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene. PCSK9 is a key target in the treatment of heart disease, the leading cause of mortality worldwide (Berberich et al., Nature Rev Cardiol. (2019) 16(l):9-20). The human PCSK9 gene, located on chromosome 1, has approximately 94% and 80% homology with its cynomolgus and mouse counterparts, respectively. The gene has CpG islands in the promoter region and is distal from other genes and cis-regulatory features. The PCSK9 protein is produced predominantly by the liver.

[0012] In view of the role of PCSK9 in the pathogenesis of hypercholesterolemia and cardiovascular disease, there is a need for new and improved therapies that target the expression of PCSK9.

[0013] Other genes relevant to addressing hypercholesterolemia include LPA and ANGPTL3. In view of the role of ANGPTL3 and LPA in the pathogenesis of hypercholesterolemia and cardiovascular disease, there is a need for new and improved therapies that target the expression of ANGPTL3 and LPA.

[0014] Another promising indication for biological therapy is metabolic dysfunction-associated steatohepatitis, or MASH (previously known as non-alcoholic steatohepatitis, or NASH). MASH / NASH develops from metabolic dysfunction-associated steatotic liver disease (MASLD) or nonalcoholic fatty liver disease (NAFLD), with progression predominantly driven by obesity or type 2 diabetes. About a quarter of patients with NAFLD progress to MASH / NASH, and there are currently no approved therapies.

[0015] In view of the therapeutic gap in treating MASH / NASH, there is a need for new and improved therapeutic approaches to modifying expression of one or more relevant gene targets.

[0016] SUMMARY

[0017] The present disclosure provides polyfunctional editors for gene editing and epigenetic modification.

[0018] In some aspects, the present disclosure provides polyfunctional editors for use in multiplexing methods for modifying the expression of at least two target genes relevant for addressing hypercholesterolemia and / or MASH, wherein the expression of a first target gene is modified by gene editing, and the expression of second target gene is modified by epigenetic modification, including during gene therapy applications.

[0019] 2

[0020] #14686672vlIn humans, PCSK9 plays a key role in regulating the circulating level of low-density lipoprotein (LDL) particles as a result of its binding to the LDL receptor (LDLR). LDLR reduces the circulating concentration of LDL particles by mediating their endocytosis and degradation in the cell. In the absence of PCSK9, or if the interaction of PCSK9 with LDLR is blocked, the rate of recycling of LDLR to the cell surface is increased and recycled LDLR proteins continue to remove LDL particles from the extracellular fluid (Tombling et al., Atherosclerosis (2021) 330:52-60). By contrast, when the endocytosed LDLR is bound to PCSK9, LDLR is degraded along with its passenger LDL particle. Clinical and genetic studies have established 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 pharmacologic reduction of PCSK9 decreases 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). Lowering PCSK9 expression can help to increase the recycling of LDLR, which would lead to lower blood LDL particle concentrations.

[0021] Lipoprotein(a), or LP(a), expressed from the LPA gene, is another valuable target for gene editing or epigenetic editing to address hypercholesterolemia. A high level of LP(a), an apoBlOO-containing lipoprotein, is an independent and causal risk factor for atherosclerotic cardiovascular diseases through mechanisms associated with increased atherogenesis, inflammation, and thrombosis. Reyes-Soffer et al., Arteriosclerosis, Thrombosis, and Vascular Biology (2021) 42(1): e48-e60.

[0022] The angiopoietin-like 3 (ANGPTI.3) gene is also a promising candidate for gene editing and epigenetic silencing. ANGPTL3 is a key target in the treatment of heart disease, the leading cause of mortality worldwide (see, e.g., Dewey et al., TV Engl J Med. (2017) 377(3):211-21; and Berberich et al., Nature Rev Cardiol. (2019) 16(l):9-20). The human ANGPTL3 gene, located on chromosome 1, has CpG islands in the promoter region and has approximately 80% homology with its mouse counterpart. The ANGPTL3 protein is expressed predominantly in the liver.

[0023] In humans, ANGPTL3 plays a key role in lipid and lipoprotein metabolism as well as angiogenesis. ANGPTL3 regulates plasma levels of triglycerides (TG), triglyceride-rich lipoproteins, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) by inhibiting the activity of lipoprotein lipase (LPL) and / or endothelial lipase (EL). ANGPTL3 deficiency or pharmacological inhibition is associated with a marked reduction of plasma triglyceride 3

[0024] #14686672vland cholesterol levels, and an increased activity of LPL and EL. Loss-of-function (LOF) mutations in the ANGPTL3 gene have been found to lead to low plasma levels of LDL-C, VLDL-C, HDL-C, and TG, and genome-wide association studies suggest that these mutations are associated with lower risk of cardiovascular disease (CVD).

[0025] Multiplexed editing of all or a subset of the above targets, along with other targets for hypercholesterolemia known in the art, is an attractive clinical option and the subject of the present disclosure. While promising, these multiplexing gene editing approaches (i.e., disruption of multiple genes per cell) come with two related issues:

[0026] (i) Induction of multiple DNA breaks per cell may over-activate cellular DNA damage responses, ultimately leading to apoptosis or poor performance / fitness of the transplanted cells. In this regard, triple editing has been posed as the upper limit for multiplexing, above which significant cell toxicity can be observed.

[0027] (ii) Chromosomal translocations may occur between or among multiple DNA breaks (including on- and off-target sites of the nucleases and spontaneous breaks), further jeopardizing safety of the approach. Clinical and preclinical studies of multiplexing gene editing have reported alarming levels of genomic translocations (up to 5%), even when dualgene editing approaches were used (L. Poirot etal., Cancer Res. 2015 Sep 15;75(18):3853-64; W. Qasim et al., Sci Transl Med; 2017 Jan 25;9(374); E. Stadtaumer et al., Science 2020 Feb 28;367(6481)).

[0028] Targeted epigenetic modification (such as epigenetic silencing (“epi-silencing”)) may represent a safer alternative to gene editing approaches for multiplexing. Epi-silencing exploits epigenetics, rather than DNA breaks, to inactivate its intended target gene, for example through DNA methylation at CpG sites (A. Amabile etal., Cell. 2016 Sep 22;167(I):219-232).

[0029] In some aspects, epi-silencing is achieved by the transient delivery of fusion proteins comprising, for example, a catalytically disabled Cas9 (dCas9) or a transcription activatorlike effector (TALE) or a zinc-finger protein (ZFP) fused to epigenetic domains from naturally occurring epigenetic effector proteins (such as KRAB, DNMT3L and DNMT3 A). The application of such fusion proteins in silencing individual as well as multi-copy genes in cell lines and in primary T lymphocytes was reported by A. Amabile supra and T. Mlambo et al., Nucleic Acids Res. 2018 May 18;46(9):4456-4468. However, the activity of fusion proteins with these domains appears to preferably occur at genes that possess a CpG island (CGI), thus excluding several potentially relevant targets.

[0030] 4

[0031] #14686672vlAccordingly, there remains a need for the development of technologies capable of modifying multiple genes within the same cell. Technologies which reduce the number of multiple DNA breaks per cell, compared to multiplexing gene editing strategies, may be a safer approach and may avoid cellular DNA damage responses and undesired chromosomal translocations.

[0032] The present disclosure provides the development of a combined gene and epigenetic editing strategy to modify multiple genes within the same cell. In particular, it exploits fusions proteins which comprise an epigenetic effector domain operably linked to an endonuclease (such as a catalytically active Cas9) and guide ribonucleic acids (gRNAs) of different lengths to promote permanent epigenetic editing (e.g., silencing) of one or more genes and genetic or gene editing (e.g., inactivation) of another gene.

[0033] This orthogonal approach overcomes the genotoxic risks associated with the use of nuclease-mediated genome editing technologies to inactivate multiple genes per cell. In some aspects, the present disclosure enables targeting of genes that may be more challenging to achieve with targeted epigenetic modification, enabling targeting of both genes having a CpG island (CGI) and genes which do not have a CGI in one multiplexing strategy.

[0034] The present disclosure provides a combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising: (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins; (b) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and (c) one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs. In some embodiments, (1) the one or more gRNAs in (b) has a spacer sequence of 15 to 17 nucleotides; and / or (2) the one or more gRNAs in (c) has a spacer sequence of 18 to 24 nucleotides. In some embodiments, the combination comprises one to three fusion proteins. In some embodiments, the combination further comprises a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene. In some embodiments, the endonuclease domain is derived from a Cas9 protein. In some embodiments, the endonuclease domain is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein. In some embodiments, the first gene is PCSK9, and / or the second gene is selected from LPA and ANGPTL3. In some embodiments, the first gene is a 5

[0035] #14686672vlPCSK9 gene, optionally wherein the one or more gRNAs targeting the PCSK9 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 17-86 and 831-834. In some embodiments, the second gene is an LPA gene, optionally wherein the one or more gRNAs targeting the LPA gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 87-677 and 835. In some embodiments, the second gene is an ANGPTL3 gene, optionally wherein the one or more gRNAs targeting the ANGPTL3 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 678-794. In some embodiments, the one or more fusion proteins collectively further comprise a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3. In some embodiments, the DNMT3 A domain comprises an H3 tail. In some embodiments, the one or more fusion proteins comprises one or more NLS. In some embodiments, the one or more fusion proteins comprises one or more linkers. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS -XTEN16 - KRAB. In some embodiments, the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 - activeCas9 -NLS - XTEN16 - KRAB - BipartiteNLS. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 - activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 819-822 and 836. In some embodiments, the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0036] Aspects of the present disclosure also provide a combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising: (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins; (b) one or more guide RNAs (gRNAs) having a spacer sequence mismatch pattern that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and (c) one or more gRNAs having a spacer sequence mismatch pattern that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or 6

[0037] #14686672vlmore gRNAs. In some embodiments, (1) the one or more gRNAs in (b) has a spacer sequence mismatch patterns of 1 to 10 nucleotides; and / or (2) the one or more gRNAs in (c) has a spacer sequence with no mismatches. In some embodiments, the combination comprises one to three fusion proteins. In some embodiments, the combination further comprises a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene. In some embodiments, the endonuclease domain is derived from a Cas9 protein. In some embodiments, the endonuclease domain is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein. In some embodiments, the first gene is PCSK9; and / or the second gene is selected from LPA and ANGPTL3. In some embodiments, the first gene is a PCSK9 gene, optionally wherein the one or more gRNAs targeting the PCSK9 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 17-86. In some embodiments, the second gene is a LPA gene, optionally wherein the one or more gRNAs targeting the LPA gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 87- 677. In some embodiments, the second gene is an ANGPTL3 gene, optionally wherein the one or more gRNAs targeting the ANGPTL3 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 678-794. In some embodiments, the one or more fusion proteins collectively further comprise a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3. In some embodiments, the DNMT3A domain comprises an H3 tail. In some embodiments, the one or more fusion proteins comprises one or more NLS. In some embodiments, the one or more fusion proteins comprises one or more linkers. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail -Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS - XTEN16 - KRAB. In some embodiments, the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 - activeCas9 - NLS - XTEN16 - KRAB -BipartiteNLS. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 - activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 819-822. In some embodiments, the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0038] 7

[0039] #14686672vlThe present disclosure also provides polyfunctional editors for use in multiplexing methods for modifying the expression of at least two target genes relevant for addressing metabolic dysfunction-associated steatohepatitis (MASH), wherein the expression of a first target gene is modified by gene editing and the expression of second target gene is modified by epigenetic modification, including during gene therapy applications.

[0040] Several gene targets hold promise for the treatment of this indication through either silencing or activation, independently or in combination, including DGAT2, MARC 1, PNPLA3, CIDEB, FGF21, FASN, PSD3, and ENPP2. DGAT2, MARC 1, PNPLA3, and CIDEB have human genetic and / or pharmacological validation that they modulate key pathways contributing to fibrosis and steatohepatitis, such as triglyceride synthesis, mitochondrial functions, fatty acid oxidation, and enlargement of lipid droplets. FGF21 analogues are known to decrease steatosis, inflammation, and fibrosis.

[0041] Multiplexed editing of all or a subset of the above targets, along with other targets for addressing MASH known in the art, is an attractive clinical option and the subject of the present disclosure. While promising, these multiplexing gene editing approaches (i.e., disruption of multiple genes per cell) come with two related issues:

[0042] (i) Induction of multiple DNA breaks per cell may over-activate cellular DNA damage responses, ultimately leading to apoptosis or poor performance / fitness of the transplanted cells. In this regard, triple editing has been posed as the upper limit for multiplexing, above which significant cell toxicity can be observed.

[0043] (ii) Chromosomal translocations may occur between or among multiple DNA breaks (including on- and off-target sites of the nucleases and spontaneous breaks), further jeopardizing safety of the approach. Clinical and preclinical studies of multiplexing gene editing have reported alarming levels of genomic translocations (up to 5%), even when dualgene editing approaches were used (L. Poirot et al., Cancer Res. 2015 Sep 15;75(18):3853-64; W. Qasim et al., Sci Transl Med; 2017 Jan 25;9(374); E. Stadtaumer et al., Science 2020 Feb 28;367(6481)).

[0044] Targeted epigenetic modification (such as epigenetic silencing (“epi-silencing”)) may represent a safer alternative to gene editing approaches for multiplexing. Epi-silencing exploits epigenetics, rather than DNA breaks, to inactivate its intended target gene, for example through DNA methylation at CpG sites (A. Amabile et al., Cell. 2016 Sep 22;167(I):219-232).

[0045] In some aspects, epi-silencing is achieved by the transient delivery of fusion proteins comprising, for example, a catalytically disabled Cas9 (dCas9) or a transcription activator- 8

[0046] #14686672vllike effector (TALE) or a zinc-finger protein (ZFP) fused to epigenetic domains from naturally occurring epigenetic effector proteins (such as KRAB, DNMT3L and DNMT3 A). The application of such fusion proteins in silencing individual as well as multi-copy genes in cell lines and in primary T lymphocytes was reported by A. Amabile supra and T. Mlambo et al., Nucleic Acids Res. 2018 May 18;46(9):4456-4468. However, the activity of fusion proteins with these domains appears to preferably occur at genes that possess a CpG island (CGI), thus excluding several potentially relevant targets.

[0047] The present disclosure provides the development of a combined gene and epigenetic editing strategy to modify multiple genes within the same cell. In particular, it exploits fusions proteins which comprise an epigenetic effector domain operably linked to an endonuclease (such as a catalytically active Cas9) and guide ribonucleic acids (gRNAs) of different lengths to promote permanent epigenetic editing (e.g., silencing) of one or more genes and genetic or gene editing (e.g., inactivation) of another gene.

[0048] This orthogonal approach overcomes the genotoxic risks associated with the use of nuclease-mediated genome editing technologies to inactivate multiple genes per cell. In some aspects, the present disclosure enables targeting of genes that may be more challenging to achieve with targeted epigenetic modification, enabling targeting of both genes having a CpG island (CGI) and genes which do not have a CGI in one multiplexing strategy.

[0049] The present disclosure provides a combination for modifying transcription, expression, and / or activity of one or more gene in a cell, the combination comprising: (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins; (b) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and (c) one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs. In some embodiments, (1) the one or more gRNAs in (b) has a spacer sequence of 15 to 17 nucleotides; and / or (2) the one or more gRNAs in (c) has a spacer sequence of 18 to 24 nucleotides. In some embodiments, the combination comprises one to three fusion proteins. In some embodiments, the combination further comprises a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene. In some embodiments, the endonuclease domain is derived from a Cas9 protein. In some embodiments, the

[0050] 9

[0051] #14686672vlendonuclease domain is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein. In some embodiments, the first gene is selected from CIDEB, PNPLA3, MARC 1, and / or DGAT2; and / or the second gene is selected from CIDEB and HSD17B13. In some embodiments, the first gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 837-1011. In some embodiments, the first gene is a PNPLA3 gene, optionally wherein the one or more gRNAs targeting the PNPLA3 each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1012-1028. In some embodiments, the first gene is a MARC 1 gene, optionally wherein the one or more gRNAs targeting the MARC 1 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1029-1033. In some embodiments, the first gene is a DGAT2 gene, optionally wherein the one or more gRNAs targeting the DGAT2 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1034-1065. In some embodiments, the second gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1066-1189. In some embodiments, the second gene is a HSD17B13 gene, optionally wherein the one or more gRNAs targeting the HSD17B13 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1190-1281. In some embodiments, the one or more fusion proteins collectively further comprise a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3. In some embodiments, the DNMT3 A domain comprises an H3 tail. In some embodiments, the one or more fusion proteins comprises one or more NLS. In some embodiments, the one or more fusion proteins comprises one or more linkers. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS -XTEN16 - KRAB. In some embodiments, the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 - activeCas9 -NLS - XTEN16 - KRAB - BipartiteNLS. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 - activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 486-489. In some embodiments, the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0052] 10

[0053] #14686672vlAspects of the present disclosure also provide a combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising: (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins; (b) one or more guide RNAs (gRNAs) having a spacer sequence with a mismatch pattern that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and (c) one or more gRNAs having a spacer sequence with a mismatch pattern that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs. In some embodiments, (1) the one or more gRNAs in (b) has a spacer sequence mismatch pattern of 1 to 10 nucleotides; and / or (2) the one or more gRNAs in (c) has a spacer sequence with no mismatches. In some embodiments, the combination comprises one to three fusion proteins. In some embodiments, the combination further comprises a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene. In some embodiments, the endonuclease domain is derived from a Cas9 protein. In some embodiments, the endonuclease domain is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein. In some embodiments, the first gene is selected from CIDEB, PNPLA3, MARC 1, and / or DGAT2; and / or the second gene is selected from CIDEB and HSD17B13. In some embodiments, the first gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 837-1011. In some embodiments, the first gene is a PNPLA3 gene, optionally wherein the one or more gRNAs targeting the PNPLA3 each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1012-1028. In some embodiments, the first gene is a MARC 1 gene, optionally wherein the one or more gRNAs targeting the MARC 1 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1029-1033. In some embodiments, the first gene is a DGAT2 gene, optionally wherein the one or more gRNAs targeting the DGAT2 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1034-1065. In some embodiments, the second gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1066-1189. In some embodiments, the second gene is a HSD17B13 gene, optionally wherein the one or more gRNAs targeting the HSD17B13 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1190- 1281. In some embodiments, the one or more fusion proteins collectively further comprise a 11

[0054] #14686672vltranscriptional repressor domain. In some embodiments, the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3. In some embodiments, the DNMT3A domain comprises an H3 tail. In some embodiments, the one or more fusion proteins comprises one or more NLS. In some embodiments, the one or more fusion proteins comprises one or more linkers. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS - XTEN16 - KRAB. In some embodiments, the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker - horseDNMT3L -XTEN80 - activeCas9 - NLS - XTEN16 - KRAB - BipartiteNLS. In some embodiments, the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 -activeCas9 - NLS. In some embodiments, the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 486-489. In some embodiments, the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate intemucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0055] Aspects of the present disclosure provide a pharmaceutical composition comprising a combination of the present disclosure.

[0056] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human hepatocyte.

[0057] Aspects of the present disclosure provide a method of modifying transcription, expression and / or activity of one or more gene in a human hepatocyte, comprising introducing a combination of the present disclosure into the cell.

[0058] Aspects of the present disclosure provide a method of treating a human in need thereof, comprising administering to the human a pharmaceutical composition of the present disclosure. Other features, objects, and advantages of the disclosure are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the disclosure, is given by way of illustration only, not limitation. Various changes and modification within the scope of the disclosure will become apparent to those skilled in the art from the detailed description.

[0059] 12

[0060] #14686672vlDESCRIPTION OF THE DRAWINGS

[0061] FIGs. 1A-1B show Day 7 (FIG. 1A) and Day 14 (FIG. IB) results for different truncations of guide RNAs targeting different targets within PCSK9, modified with guide modification pattern 2 (MOD002). On the lefthand panel, for each position, from left to right, 20 nts, 18 nts, 17 nts, and 16 nts guides are shown. Fold change is indicated. The righthand panel shows the results for controls.

[0062] FIGs. 2A-2B show Day 7 (FIG. 2 A) and Day 14 (FIG. 2B) results for different truncations of guide RNAs targeting different targets within PCSK9, modified with guide modification pattern 86 (MOD086, 2 end modifications). On the lefthand panel, for each position, from left to right, 20 nts, 18 nts, 17 nts, and 16 nts guides are shown. Fold change is indicated. The righthand panel shows the results for controls.

[0063] FIG. 3 shows Day 5 results for guide RNAs truncated to 16 nucleotides and transfected with active Cas9 CHARM (aCHARM). Fold change relative to the untreated control is shown.

[0064] FIG. 4 shows Day 5 results for full-length (20 nt; left bar in each set of bars) and truncated (16 nt; right bar in each set of bars) guide RNAs transfected with aCHARM. The indel rates (% indel) of each construct are shown.

[0065] FIG. 5 shows the potencies (measured as % PCSK9+ cells) of guide RNAs truncated to 16 nucleotides at different dose amounts (log scale).

[0066] FIGs. 6A-6B show targeting human Lp(a) with aCHARM and full length Lp(a) guide RNAs. FIG. 6A shows a schematic illustration of a full length guide RNA interacting with its target and FIG. 6B shows human Lp(a) exon 2 editing as measured by the percentage of indels using aCHARM with Krab (Active Krab) and without Krab (Active krabless) and full-length Lp(a) guide RNAs. A CD151 targeted gRNA (gRNA 454) was used as a negative control. For each of the Active Krab and Active Krabless conditions, the gRNAs tested were, from left to right, gRNA454 (CD151 targeted), gRNA9591, gRNA9590 (CRISPR TX), gRNA9507, gRNA9586, gRNA9506, and gRNA9505.

[0067] FIG. 7 shows the relative PCSK9 expression with different doses of polyfunctional editors. An Lp(a) targeted gRNA was used as a negative control; Lp(a) and wild-type Cas9 was used as positive control for Lp(a) silencing; and the combination of guides RNA041 and RNA049 with CHARM was used as a positive control for PCSK9 silencing.

[0068] 13

[0069] #14686672vlFIGs. 8A-8B show the relative PCSK9 expression (left axis) and the percentage of indels (right axis) with dual guide epigenetic editing (FIG. 8A) and with the parent single guide epigenetic editing (FIG. 8B).

[0070] FIGs. 9A-9D show relative PCSK9 expression (left axis) and the percentage of indels (right axis) using a truncated single guide with dCHARM (FIG. 9A), a wild-type Cas9 and Lp(a) guide RNA (FIG. 9B), and a polyfunctional editor with two guides: a PCSK9 truncated guide and a long Lp(a) guide (FIG. 9C). The arrow in FIG. 9C indicates the dose where dual editing was observed at both targets. FIG. 9D shows the dilution effect of the polyfunctional editor on indel percentage.

[0071] FIG. 10 shows potency, measured as relative PCSK9 expression, when using active Cas9 CHARM (aCHARM) or dead Cas9 CHARM (dCHARM) with truncated (16 nt) and full-length (20 nt) guide RNAs. The results from lipid only (negative control) and CRISPRi (positive control) are also shown.

[0072] FIG. 11 shows potency, measured as the percentage of PCSK9+ cells, when using active Cas9 CHARM (aCHARM) with dual truncated (16 nt) guide RNAs, or dead Cas9 CHARM (dCHARM) with dual full-length (20 nt) guide RNAs.

[0073] FIG. 12 shows the percentage of PCSK9+ cells obtained using different single truncated (16 nt) guide RNAs or dual full-length (20 nt) guide RNAs. The results from effector only and CRISPRi controls are also shown.

[0074] FIGs. 13A-13B show the potency of epi-silencing using spacers of different lengths and a KRABless-CHARM editor at Day 6 (FIG. 13A) and Day 20 (FIG. 13B) in Hep3B cells. The percentage of PCSK9-tdTomato positive cells is shown. Four different gRNAs were tested.

[0075] FIG. 14 shows the potency of epi-silencing using spacers of different lengths at Day 20, and the results using controls: lipid only, effector only, CRISPRi with dual guides RNA041 and RNA049 (“gRNA1590” and “1591”, respectively, on the graph), and CHARM with dual guides RNA041 and RNA049 in Hep3B cells.

[0076] FIGs. 15A-15C show the percentage of PCSK9+ cells (left axis) and relative ANGPTL3 expression (right axis) at Day 7 under different treatment conditions: dCHARM with full-length RNA041 and RNA049 (FIG. 15A); wild-type Cas9 with ANGPTL3 guide (FIG. 15B); and CHARM (nuclease-active CHARM) with truncated guide and ANGPTL3 guide (FIG. 15C).

[0077] 14

[0078] #14686672vlFIG. 16 shows PCSK9 silencing, measured as PCSK9+ cell percentage, after treatment with wild-type Cas9 and an ANGPTL3guide (“ANG”); dCHARM with dual full-length guides; and aCHARM with PCSK9 (16 nt) and ANGPTL3 (20 nt) guides.

[0079] FIG. 17A-17B show ANGPTL3 silencing, measured as relative ANGPTL3 expression, at Day 7 (FIG. 17A) and Day 14 (FIG. 17B) after treatment with wild-type Cas9 and an ANGPTL3guide (“ANG”); dCHARM with dual full-length guides; and aCHARM with PCSK9 (16 nt) and ANGPTL3 (20 nt) guides.

[0080] FIGs. 18A-18B show PCSK9 silencing (FIG. 18A) and ANGPTL3 silencing (FIG.

[0081] 18B) at Day 14 after treatment with wild-type Cas9 and an ANGPTL3guide (“ANG”); dCHARM with 16 nt PCSK9 guide; and aCHARM with PCSK9 (16 nt) and ANGPTL3 (20 nt) guides.

[0082] FIG. 19 shows silencing of PCSK9, measured as PCSK9+ cell percentage, on Day 7 after a sub-saturating dose of the dual guide combinations (16 nt) and single guides in a Hep3B TdTomato PCSK9 cell line. The controls included untreated cells, CRISPRi (high dose), and RNA041+RNA049 (two guides known to direct epigenetic editing of PCSK9; the “gold standard”).

[0083] FIG. 20 shows ANGPTL3 expression on Days 7 and 14 with effector and guide RNA491, relative to effector only.

[0084] FIG. 21 shows relative ANGPTL3 expression on Days 7 and 14 after control treatment (lipid only) or treatment with dCHARM and ANGPTL3 guide (data from two replicate plates is shown).

[0085] FIGs. 22A-22B show a schematic of distal guide RNA mismatches and impact on cutting ability for a polyfunctional CHARM editor using a catalytically active Cas9 domain (aCHARM) (FIG. 22 A) versus perfect guide match and impact on cutting ability (FIG. 22B).

[0086] FIG. 23 shows the potency of epi-silencing using spacers of different lengths at Day 21, for a guide targeting RFXAP in T cells (gRNA2978) using KRABless CHARM.

[0087] FIG. 24 shows the dose response of guides using spacers between 4 and 20 nucleotides in length for epi-silencing of PCSK9 (in Hep3B cells) using guides with MOD002 modification.

[0088] FIGs. 25A-25C show the percentage of PCSK9+ cells (left axis) and relative ANGPTL3 expression (right axis) under different polyfunctional and orthogonal multiplexed editing conditions. FIG. 25A shows gene expression after treatment of cells with a polyfunctional (aCHARM) editing construct comprising a catalytically active Cas9 and an epigenetic repressor domain and targeting PCSK9 and ANGPTL3 (FIG. 25A); orthogonal 15

[0089] #14686672vlediting of PCSK9 using dSpCas9:CHARM for epigenetic editing (sp:EE) and of ANGPTL3 using catalytically active SaCas9 (Sa: Cut) for gene editing (FIG. 25B); and orthogonal editing of PCSK9 using wild-type SpCas9 (Sp:Cut) for gene editing and ANGPTL3 using dSaCas9:CHARM (Sa:EE) for epigenetic editing (FIG. 25C).

[0090] DETAILED DESCRIPTION

[0091] Polyfunctional editors

[0092] In some aspects, the present disclosure provides one or more polyfunctional editors, comprising one or more fusion proteins that comprise a) at least one epigenetic effector domain, operably linked to b) an endonuclease. In some aspects, polyfunctional editors also comprise one or more guide RNAs. As used herein, "polyfunctional editor" may refer to the fusion protein component of the disclosure, the gRNA component of the disclosure (if required), or both the fusion protein and gRNA components.

[0093] In some aspects, polyfunctional editors of the disclosure are agents that enable multiplexing of gene editing and epigenetic editing of different target genes. In some embodiments, polyfunctional editors according to the present disclosure enable repression (e.g., silencing) of transcription and / or expression of multiple different target genes, wherein one gene is repressed (e.g., silenced) by genetic or gene editing and at least one gene is repressed (e.g., silenced) by epigenetic repression (e.g., epi-silencing). In some embodiments, with use of the system described above, there is no reciprocal translocation between the simultaneously edited genes, thus greatly improving the safety of multiplex gene editing. In some embodiments, application of such a poly-functional editing approach allows performance of orthogonal edits in one step, without the need for sequential engineering procedures, thus greatly facilitating product manufacturing and reducing associated costs and cell toxicity. In some embodiments, the target gene selected for gene editing is also used as a target site for insertion of exogenous expression cassettes.

[0094] In some aspects, the polyfunctional editors described herein are considered programmable multi-editors. In some embodiments, the composition of the editor and selection of endonuclease(s), as well as the design of gRNAs, allows a polyfunctional editor to be programmed to modify the sequence or modify transcription, expression and / or activity of multiple targets in the same cell. In some embodiments, the polyfunctional editors are chimeric or fusion proteins that comprise at least one (such as one, two, or three) endonuclease operably linked to at least one effector domain (e.g., a KRAB domain, a DNMT3 A or a DNMT3L domain, or homologues thereof; wherein the domains may be full- 16

[0095] #14686672vllength proteins or functional fragments thereof and may be referred to herein as “KRAB,” “DNMT3 A,” or “DNMT3L,” respectively). In some embodiments, the one or more effector domains comprises a full-length protein or functional fragment thereof. In some embodiments, the effector domain comprises a domain that functions to recruit one or more endogenous proteins. In some embodiments, the one or more effectors comprises a full-length protein, of functional fragment thereof, and a domain that functions to recruit an endogenous protein. In some embodiments, an H3 tail is used to recruit DNMT3 A. In some embodiments, the epigenetic effector domain has a catalytic activity which enables modification (such as repression) of transcription of a target gene or target genes. In some embodiments, the effector domain recruits additional agents within a cell to a target gene or target genes, which results in the modification (such as repression) of transcription of the target gene. In some embodiments, the effector domain, or combination of effector domains, has both a catalytic activity and recruits additional agents within a cell to a target gene or target genes.

[0096] In some embodiments, the endonuclease enables cleavage of specific DNA sequence(s). In some embodiments, the endonuclease is chosen or engineered to bind to nucleic acid sequence(s) of choice. In some embodiments, the endonuclease comprises endonucleases from multiple species, e.g. S. pyogenes and S. aureus, and enables cleavage of multiple specific DNA sequence(s) in the same cell.

[0097] As used herein “operably linked”, means that the individual components are linked together in a manner which enables them to carry out their function (e.g., cleavage of DNA, binding to DNA, catalyzing a reaction or recruiting additional agents from within a cell) substantially unhindered. In some embodiments, an endonuclease is conjugated to an epigenetic effector domain, for example to form a fusion protein. Methods for conjugating polypeptides are known in the art, for example through the provision of a linker amino acid sequence connecting the polypeptides (e.g., a linker comprising glycine and / or serine residues). Alternative methods of conjugating polypeptides known in the art include chemical and light-induced conjugation methods (e.g., using chemical cross-linking agents). In some embodiments, the endonuclease and epigenetic effector domain (e.g., KRAB domain, DNMT3 A domain and / or DNMT3L domain, or homologue thereof) of the polyfunctional editor form a fusion protein.

[0098] In some aspects, the polyfunctional editor comprises an RNA binding domain. In some embodiments, the RNA binding domain binds to a gRNA which is complementary to a

[0099] 17

[0100] #14686672vlgenomic target site. Thus, in some embodiments, the RNA binding domain directs the polyfunctional editor to a target gene.

[0101] In some aspects, the polyfunctional editor is a fusion protein comprising a) at least one epigenetic effector domain; and b) an endonuclease.

[0102] In some aspects, the polyfunctional editor is a bi-partite fusion protein. In some embodiments, the polyfunctional editor comprises two effector domains fused to the same endonuclease.

[0103] In some aspects, the polyfunctional editor is a tri-partite fusion. In some embodiments, the polyfunctional editor comprises three effector domains fused to the same endonuclease.

[0104] In some aspects, the polyfunctional editor comprises four, five, six, or more effector domains fused to the same endonuclease.

[0105] In some embodiments, the polyfunctional editor comprises multiple effector domains, and the effector domains are different. In some embodiments, the polyfunctional editor comprises multiple effector domains, and the effector domains are the same.

[0106] In some aspects, a polyfunctional editor according to the present disclosure comprises or consists of a Cas9-KRAB, Cas9-DNMT3 A or Cas9-DNMT3L fusion protein.

[0107] In some embodiments, a polyfunctional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of endonuclease, KRAB and DNMT3 A domains. In some embodiments, a polyfunctional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of endonuclease, DNMT3L and DNMT3 A domains. In some embodiments, a polyfunctional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of endonuclease, DNMT3L and KRAB domains. In some embodiments, a poly functional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of endonuclease, DNMT3L, KRAB and DNMT3A domains.

[0108] In some embodiments, a polyfunctional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of Cas (e.g., Cas9), KRAB, and DNMT3 A domains. In some embodiments, a polyfunctional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of Cas (e.g., Cas9), DNMT3L and DNMT3 A domains. In some embodiments, a polyfunctional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of Cas (e.g., Cas9), DNMT3L and KRAB domains. In some

[0109] 18

[0110] #14686672vlembodiments, a poly functional editor according to the present disclosure comprises or consists of a fusion protein comprising or consisting of Cas (e.g., Cas9), DNMT3L, KRAB and DNMT3 A domains.

[0111] In some aspects, the polyfunctional editor comprises an endonuclease-KRAB fusion protein such as a Cas-KRAB, e.g., Cas9-KRAB domain fusion protein. In some aspects, the polyfunctional editor consists of an endonuclease-KRAB fusion protein such as a Cas-KRAB, e.g., Cas9-KRAB domain fusion protein.

[0112] In some embodiments, alternatives to the HA tag and glycine-serine linker shown in exemplary polyfunctional editors may are used according to the present disclosure.

[0113] In some aspects, the polyfunctional editor comprises an endonuclease-DNMT3A fusion protein such as a Cas-DNMT3A, e.g., a Cas9-DNMT3A domain fusion protein. In some aspects, the polyfunctional editor consists of an endonuclease-DNMT3 A fusion protein such as a Cas-DNMT3 A, e.g., a Cas9-DNMT3 A domain fusion protein.

[0114] In some aspects, the polyfunctional editor comprises an endonuclease-DNMT3L fusion protein such as a Cas-DNMT3L, e.g., a Cas9-DNMT3L domain fusion protein. In some aspects, the polyfunctional editor consists of an endonuclease-DNMT3L fusion protein such as a Cas-DNMT3L, e.g., a Cas9-DNMT3L domain fusion protein.

[0115] In some aspects, the polyfunctional editor comprises or consists of a CRISPROff fusion protein or a CRISPROff variant fusion protein. In some aspects, the polyfunctional editor comprises or consists of a CRISPROff fusion protein or a CRISPROff variant fusion protein that does not comprise a KRAB domain (CRISPROff KRABLESS).

[0116] In some aspects, the polyfunctional editor comprises or consists of a Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM) fusion protein or a CHARM variant fusion protein. In some aspects, the polyfunctional editor comprises or consists of a Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM) fusion protein or a CHARM variant fusion protein that does not comprise a KRAB domain (CHARM KRABLESS).

[0117] Epigenetic effector domains

[0118] As used herein, the term “epigenetic effector domain,” refers to the part of the polyfunctional editor which provides for the epigenetic effect on a target gene, for example by catalyzing a reaction on the DNA or chromatin (e.g., methylation of DNA), or by recruiting an additional agent from within a cell, e.g., resulting in the repression of the transcription of a gene.

[0119] 19

[0120] #14686672vlAs used herein, “domain” refers to a part of the polyfunctional editor that has a certain function. In some embodiments, the domain is an individual domain (e.g., a catalytic domain) isolated from a natural protein. In some embodiments, the domain is an entire, full-length natural protein. Put another way, either the full-length protein or a functional fragment thereof can be used as an effector domain. As used herein, for example, “Kriippel-associated box (KRAB) domain” or “KRAB domain” refers to the part of the polyfunctional editor that comprises an amino acid sequence with the function of a KRAB domain.

[0121] Chromatin remodeling enzymes that are known to be involved in the permanent epigenetic silencing of endogenous retroviruses (ERVs; Feschotte, C. etal. (2012) Nat. Rev. Genet. 13: 283-96; Leung, D.C. etal. (2012) Trends Biochem. Set. 37: 127-33) may provide suitable effector domains for exploitation in the present disclosure.

[0122] In some aspects, the epigenetic effector domain is capable of repressing transcription and / or expression of at least one target gene. A factor capable of repressing transcription of a gene is also called a transcriptional repressor. In some aspects, the epigenetic effector domain is a repressor domain, e.g., a transcriptional repressor domain.

[0123] In some aspects, the epigenetic effector domain initiates chemical modification of chromatin and / or chromatin remodeling.

[0124] In some aspects, the epigenetic effector domain initiates DNA modification, such as DNA methylation. In some aspects, the epigenetic effector domain is a DNA methyltransferase and / or is capable of recruiting a DNA methyltransferase.

[0125] In some aspects, the epigenetic effector domain initiates histone modification, such as histone methylation or histone acetylation. In some aspects, the epigenetic effector domain is a histone methyltransferase or histone acetyltransferase.

[0126] In some aspects, the at least one epigenetic effector domain comprises a Kriippel-associated box (KRAB) domain, a DNA methyltransferase (DNMT) domain, a DNMT-like domain, or a histone methyltransferase (HMT) domain.

[0127] In some aspects, the at least one epigenetic effector domain is an antibody or derivative thereof, such as a nanobody, which binds an epigenetic regulator, such as a chromatin regulator which may chemically modify chromatin and / or remodel chromatin.

[0128] See, for example, Van et al., Nat Commun. 2021 Jan 22;12(1)537, which describes nanobody-mediated control of gene expression and epigenetic memory.

[0129] 20

[0130] #14686672vlKRAB

[0131] In some aspects, the at least one epigenetic effector domain comprises a KRAB domain. The family of the Kriippel-associated box containing zinc finger proteins (KRAB-ZFP; Huntley, S. etal. (2006) Genome Res. 16: 669-77) plays an important role in the silencing of endogenous retroviruses. These transcription factors bind to specific ERV sequences through their ZFP DNA binding domain, while they recruit the KRAB Associated Protein 1 (KAP1) with their conserved KRAB domain. KAP1 in turn binds a large number of effectors that promote the local formation of repressive chromatin (Iyengar, S. et al. (2011) J. Biol. Chem. 286: 26267-76).

[0132] In some embodiments, a polyfunctional editor of the present disclosure comprises a KRAB domain. Various KRAB domains are known in the family of KRAB-ZFP proteins. In some embodiments, a polyfunctional editor of the present disclosure comprises the KRAB domain of human zinc finger protein 10 (ZNF 10; Szulc, J. et al. (2006) Nat. Methods 3 : 109- 16):

[0133] ALSPQHSAVTQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLD TAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETH PDSETAFEIKSSV (SEQ ID NO: 1).

[0134] Further examples of suitable KRAB domains for use in the present disclosure include:

[0135] MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQG ETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESL

[0136] (the KRAB domain of the human ZIM3 protein; SEQ ID NO: 2),

[0137] ITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLVAVGYQASKPDAL FKLEQGEQLWTIEDGIHSGACS

[0138] (the KRAB domain of the ZNF350 protein; SEQ ID NO: 3),

[0139] VMFEEVSVCFTSEEWACLGPIQRALYWDVMLENYGNVTSLEWETMTENEEV TSKPSSSQRADSHKGTSKRLQG

[0140] (the KRAB domain of the ZNF 197 protein; SEQ ID NO: 4),

[0141] VSFKDVAVDFTQEEWQQLDPDEKITYRDVMLENYSHLVSVGYDTTKPNVIIK LEQGEEPWIMGGEFPCQHSP

[0142] (the KRAB domain of the RBAK protein; SEQ ID NO: 5),

[0143] 21

[0144] #14686672vlVKIEDMAVSLILEEWGCQNLARRNLSRDNRQENYGSAFPQGGENRNENEEST SKAETSEDSASRGETTGRSQKE

[0145] (the KRAB domain of the ZKSCAN1 protein; SEQ ID NO: 6),

[0146] LTFKDVFVDFTLEEWQQLDSAQKNLYRDVMLENYSHLVSVGYLVAKPDVIF RLGPGEESWMADGGTPVRTCA

[0147] (the KRAB domain of the KRB0X4 protein; SEQ ID NO: 7), and

[0148] VTFEDVTLGFTPEEWGLLDLKQKSLYREVMLENYRNLVSVEHQLSKPDVVS QLEEAEDFWPVERGIPQDTIP

[0149] (the KRAB domain of the ZNF274 protein; SEQ ID NO: 8).

[0150] The above KRAB domains are illustrative only. Functional variants thereof are also contemplated herein. For example, the ZIM3 KRAB domain shown in SEQ ID NO: 4481 and 4482 (see Examples 3 and 4 below) may also be used. That ZIM3 KRAB domain has the following sequence:

[0151] MGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTK PDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESL (SEQ ID NO: 9).

[0152] DNMT

[0153] In some aspects, the epigenetic effector domain comprises a DNA methyltransferase (DNMT) domain. DNMTs catalyze the transfer of a methyl group to DNA. Examples of DNMTs are DNMT1, DNMT3A and DNMT3L.

[0154] In some embodiments, a polyfunctional editor of the present disclosure comprises a domain of human DNA methyltransferase 3 A (DNMT3A; Law, J. A. el al. (2010) Nat. Rev. Genet. 11: 204-20), e.g., the catalytic domain. In some embodiments, a polyfunctional editor of the present disclosure comprises the sequence:

[0155] TYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGI ATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHI QEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDD RPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLP GMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFM NEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAP LKEYFACV

[0156] (the catalytic domain of human DNMT3A; SEQ ID NO: 10), and / or

[0157] 22

[0158] #14686672vlMAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICIC CGSLQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLIC GNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRK WRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESG SDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQF HRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGG SLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNC FLPLREYFKYF STELTS SL

[0159] (human DNMT3L; SEQ ID NO: 11).

[0160] In some aspects, at least two epigenetic effector domains are utilized, one based on, for example, the KRAB domain (e.g., the initiator of the epigenetic cascade occurring at ERVs in embryonic stem cells), and the other based on, for example, DNMT3A (e.g., the final lock of this process). In some embodiments, this approach allows recapitulating on a pre-selected target gene those repressive chromatin states established at ERVs in the preimplantation embryo and then permanently inherited throughout mammalian development and adult life.

[0161] In some embodiments, the polyfunctional editor of the present disclosure comprises an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 respectively.

[0162] In some embodiments, the polyfunctional editor of the present disclosure is encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or a protein that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the polynucleotide encodes a protein that substantially retains the natural function of the protein represented by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, respectively. In some embodiments, the coding sequence is codon optimized. In some embodiments, the coding sequence is codon-optimized for optimal expression in human cells.

[0163] Endonuclease

[0164] In some aspects, the polyfunctional editor of the disclosure comprises an endonuclease.

[0165] 23

[0166] #14686672vlIn some embodiments, the endonuclease is site-specific. As used herein, “sitespecific endonuclease” refers to an enzyme which induces site-directed double-strand breaks in DNA. The site-specific endonuclease enables the activity of the polyfunctional editor to be targeted to specific sites in a polynucleotide, for example the genome of a cell. In some embodiments, the endonuclease is site-specific when used in combination with gRNAs. In some embodiments, the endonuclease is capable of inducing site-directed DNA breaks when used in combination with gRNAs.

[0167] In some aspects, the endonuclease has exonuclease activity in addition to endonuclease activity.

[0168] In some embodiments, the endonuclease binds to binding sites within a target gene or within regulatory sequences for the target gene. In some embodiments, a regulatory sequence comprises a promoter or enhancer sequence.

[0169] In some embodiments, the endonuclease binds to a binding site within splicing sites. In some embodiments, splicing variants of a given gene are regulated by DNA methylation / demethylation at splicing sites. In some embodiments, these modifications cause exon exclusion / inclusion in the mature transcript. In some embodiments, this exclusion / inclusion has therapeutic relevance, such as in the case of Duchenne Muscular Dystrophy, in which exclusion (by genetic ablation or exon skipping) from the mature mRNA of an exon bearing the most frequent disease-causing mutation has been proposed for therapy (Ousterout, D.G. et al. (2015) Mol. Ther. 23: 523-32; Ousterout, D.G. et al. (2015) Nat. Commun. 6: 6244; Kole, R. et al. (2015) Adv. Drug Deliv. Rev. 87: 104-7; Touznik, A. et al. (2014) Expert Opin. Biol. Ther. 14: 809-19).

[0170] A number of suitable endonucleases are known in the art. In some embodiments, CRISPR / Cas systems (Sander, J.D. et al. (2014) Nat. BiotechnoL 32: 347-55) are employed as suitable endonucleases in the polyfunctional editors of the present disclosure.

[0171] As used herein, “CRISPR / Cas system” refers to a clustered regularly interspaced short palindromic repeats / CRISPR associated nuclease system.

[0172] Clustered Regularly Interspaced Short Palindromic Repeats consist of short sequences that originate from viral genomes and have been incorporated into the bacterial genome. CRISPR associated proteins (Cas) process these sequences and cut matching viral DNA sequences. By introducing Cas and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired position.

[0173] In some aspects, the CRISPR / Cas system is an RNA-guided DNA binding system (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92), wherein the guide RNA (gRNA)

[0174] 24

[0175] #14686672vlis selected to enable a polyfunctional editor comprising a Cas domain to be targeted to a specific sequence. Thus, to employ the CRISPR / Cas system as an endonuclease in the present disclosure, it is to be understood that an epigenetic effector domain may be operably linked to a Cas endonuclease such as a Cas9 endonuclease. In some embodiments, the polyfunctional editor comprising the Cas endonuclease is delivered to a target cell in combination with one or more gRNAs. In some embodiments, the gRNAs are designed to target the polyfunctional editor to a target gene of interest or a regulatory element (e.g., a promoter, enhancer, or splicing site) of the target gene. Methods for the design of gRNAs are known in the art. Furthermore, fully orthogonal Cas9 proteins, as well as Cas9 / gRNA ribonucleoprotein complexes and modifications of the gRNA structure / composition to bind different proteins, have been developed to simultaneously and directionally target different effector domains to desired genomic sites of cells (Esvelt et al. (2013) Nat. Methods 10: 1116-21; Zetsche, B. et al. (2015) Cell 163(3) :759-71; Zalatan, J.G. et al. (2015) Cell 160: 339-50; Paix, A. et al. (2015) Genetics 201: 47-54), and are suitable for use in the present disclosure.

[0176] In some aspects, the polyfunctional editor comprises at least one endonuclease derived from type II CRISPR bacterial immune systems. In some embodiments, the polyfunctional editor comprises a Type II Cas. Examples of Cas Type II enzymes include Cas9, Csn2 and Cas4. In some embodiments, the polyfunctional editor comprises a Cas9. In some embodiments, the polyfunctional editor comprises a Csn2. In some embodiments, the polyfunctional editor comprises a Cas4.

[0177] Cas9 endonucleases typically comprise Reel, RecII, bridge helix, RuvC, HNH and PAM interacting domains. The HNH and RuvC domains are nuclease domains. The Reel domain binds gRNA. The bridge helix initiates cleavage upon binding of target DNA. The PAM-interacting domain confers PAM specificity and is responsible for initiating binding to target DNA.

[0178] In some aspects, the endonuclease comprises or consists of a Cas endonuclease. In some embodiments, the endonuclease has nuclease activity. In some embodiments, the endonuclease is a catalytically active nuclease, binds gRNA, and binds to target DNA.

[0179] In some aspects, the endonuclease comprised in a polyfunctional editor according to the disclosure is a catalytically active endonuclease. In some embodiments, the polyfunctional editor is capable of cleaving a target sequence, such as target DNA.

[0180] In some aspects, the endonuclease is catalytically active Cas nuclease.

[0181] 25

[0182] #14686672vlIn some aspects, the endonuclease is a modified or a variant endonuclease, such as a modified Cas or modified Cas9 enzyme. In some embodiments, the endonuclease is modified to recognize a specific PAM site suitable for a target gene. In some embodiments, the modified PAM is different from the PAM naturally recognized by the endonuclease.

[0183] In some aspects, the polyfunctional editor according to the present disclosure does not comprise only catalytically inactive, or catalytically dead (dCas) nuclease. In some aspects, the polyfunctional editor according to the present disclosure does not comprise a catalytically inactive, or catalytically dead (dCas) nuclease, such as dCas9.

[0184] In some aspects, the endonuclease comprises or consists of a catalytically active Cas9 nuclease.

[0185] In some aspects, the endonuclease comprises or consists of a catalytically active Cas9 nuclease from Streptococcus pyogenes (SpCas9). In other aspects, the endonuclease comprises or consists of a catalytically active Cas9 nuclease from Staphylococcus aureus (SaCas9). In some aspects, the endonuclease comprises or consists of both SpCas9 and SaCas9. In some embodiments, the endonuclease comprises endonucleases derived from more than one species.

[0186] Methods for determining whether a protein is a catalytically active nuclease include, but are not limited to, gel assays, Kunitz assays, radiolabel assays and fluorescence-based methods. Gel assays may be performed using purified recombinant target DNA as a substrate in an assay buffer. The protein to be tested may be incubated with the substrate, for example incubated at 37°C for 1 hour. The reaction products can be separated by electrophoresis, for example, on an agarose gel with ethidium bromide to visualize the products of the nuclease reaction. Other methods include, for example, fluorescence real-time quantification of DNA and RNA nuclease activity as reported in Sheppard, E.C., et al. Sci Rep 9, 8853 (2019) and cell free detection of Cas nucleases as reported in J. Cox et al., Chem Sci. 2019 Mar 7;

[0187] 10(9): 2653-2662.

[0188] In some embodiments, a polyfunctional editor of the present disclosure comprises the following catalytically active Cas9 sequence:

[0189] DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV EEDI<I<HERHPIFGNIVDEVAYHEI<YPTIYHLRI<I<LVDSTDI<ADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ

[0190] 26

[0191] #14686672vlEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFL KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPK LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHY LDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDS

[0192] (SEQ ID NO: 12).

[0193] In some embodiments, the polyfunctional editor of the present disclosure comprises an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 12. In some embodiments, the amino acid sequence substantially retains the natural function (e.g., endonuclease function) of the protein represented by SEQ ID NO: 12.

[0194] In some embodiments, the polyfunctional editor of the present disclosure is encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NO: 12, or a protein that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to SEQ ID NO: 12. In some embodiments, the polynucleotide encodes a protein that substantially retains the natural function of the protein represented by SEQ ID NO: 12. In some embodiments, the coding sequence is codon-optimized. In some embodiments, the coding sequence is codon optimized for optimal expression in human cells.

[0195] In some embodiments, the sequence of a catalytically dead Cas9 (dCas9) is:

[0196] DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV EEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ

[0197] 27

[0198] #14686672vlEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFL KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPK LESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHY LDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDS

[0199] (catalytically dead Cas9; dCas9; SEQ ID NO: 13).

[0200] The above sequence contains D9A and H839A substitutions relative to its catalytically active (i.e., live) counterpart (SEQ ID NO: 12). In some aspects, a catalytically dead Cas9 (e.g., the above dCas9) is used in the polyfunctional editor for epi-editing of one or more target genes, without simultaneous genetic editing of another gene in a cell. In some embodiments, the polyfunctional editor comprises an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 13. In some embodiments, the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 12, except for the endonuclease function. In some embodiments, the polyfunctional editor is encoded by a polynucleotide comprising a nucleic acid sequence which encodes the protein of SEQ ID NO: 13, or a protein that has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to SEQ ID NO: 13. In some embodiments, the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 12 but for the endonuclease function. In some embodiments, the coding sequence is codon-optimized. In some embodiments the coding sequence is codon-optimized for optimal expression in human cells.

[0201] gRNA

[0202] In some aspects, the present disclosure provides guide RNAs (gRNAs).

[0203] 28

[0204] #14686672vlIn some aspects, the gRNA targets the polyfunctional editor to a target gene. In some aspects, the gRNA is an RNA sequence which recognizes the target DNA region of interest and directs the endonuclease within the polyfunctional editor to that region.

[0205] A gRNA is typically made up of two parts:

[0206] a) a spacer sequence (which may also be referred to as a targeting domain, guide sequence, or complementarity region, and which may constitute a CRISPR RNA (crRNA)); and

[0207] b) a scaffold sequence (which may also be referred to as a tracrRNA in a CRISPR / Cas system).

[0208] In some embodiments, the spacer and the scaffold sequences are provided as separate molecules. In some embodiments, the spacer and the scaffold sequences are linked, such as via a linker loop or other sequence or may be fused together.

[0209] In some embodiments, the gRNA comprises two separate molecules, e.g., the spacer (crRNA) and the scaffold (tracrRNA). In some embodiments, the 3’ end of the spacer (crRNA) is complementary to the 5’ end of the scaffold (tracrRNA). In some embodiments, this complementarity leads to dimerization of the two molecules.

[0210] In some embodiments, the spacer (crRNA) and the scaffold (tracrRNA) are fused, for example via a linker loop. In some embodiments, the artificial configuration is also known as a single guide RNA (sgRNA).

[0211] In some aspects, variants of the scaffold (tracrRNA) are used. In some embodiments, the tetraloop and stem loop of the scaffold (tracrRNA) sequence are modified to include RNA aptamers, which can be bound by specific protein domains. In some aspects, such modified gRNAs are used to facilitate the recruitment of repressive or activating domains fused to the protein-interacting RNA aptamers.

[0212] Exemplary tracrRNA sequences include, without limitation:

[0213] 5’- GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3’ (SEQ ID NO: 14), and

[0214] 5 ’ -GUUUUAGAGCUAGAAAUAGC AAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3’(SEQ ID NO: 15).

[0215] 29

[0216] #14686672vlAs used herein, a “spacer” or “spacer sequence” refers to a sequence that may be fully complementary to a target domain (i.e., region) within a target sequence.

[0217] The 3’ end of the genomic target sequence generally comprises a proto-spacer adjacent motif (PAM) sequence. A “PAM” sequence is typically a 2 to 6 base pair DNA sequence immediately following the DNA sequence targeted by the nuclease. The PAM sequence is required for cleavage but is not part of the target of the gRNA sequence. The PAM sequence varies depending on the species of the nuclease. For example, the canonical PAM associated with the Cas9 nuclease of Streptococcus pyogenes is the sequence 5 ’-NOGS’ where “N” is any nucleobase. Nuclease enzymes derived from different organisms or which have been engineered may recognize different PAM sequences.

[0218] For example, the Cas9 of Francisella novicida recognizes the canonical PAM sequence 5'-NGG-3', but can be engineered to recognize 5'-YG-3' (where "Y" is

[0219] a pyrimidine), thus adding to the range of possible Cas9 targets. The Casl2a (or Cpfl) nuclease of Francisella novicida recognizes the PAM 5'-TTTN-3' or 5'-YTN-3'.

[0220] The nucleotides upstream (towards the 5’ end of the target sequence) of the PAM sequence is the protospacer sequence.

[0221] A Cas9 nuclease will typically cleave approximately three bases upstream of the PAM.

[0222] It will be appreciated that one may choose a suitable nuclease of a particular context based on PAM specificity and the genomic target.

[0223] A non-limiting example of a Cas9 target site, comprising a 22 nucleotide target domain, and an NGG PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target sequence (and thus base pairs with full complementarity with the DNA strand complementary to the strand comprising the target sequence and PAM) is provided below:

[0224] [ target domain (DNA) ] [ PAM ]

[0225] 5 ' -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-G-G-3 ' ( DNA)

[0226] 3 ' -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-C-C-5 ' (DNA)

[0227] I I I I I I I I I I I I I I I I I I I I I I

[0228] 5 ' -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N- [ gRNA scaffold] -3 ' (RNA) [ targeting domain ( RNA) ] [ binding domain ]

[0229] A non-limiting example of a Casl2a target site, comprising a 22 nucleotide target domain, and a TTN PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target sequence (and thus base pairs with full complementarity with 30

[0230] #14686672vlthe DNA strand complementary to the strand comprising the target sequence and PAM) is provided below:

[0231] [ PAM ] [ target domain ( DNA) ] 5 ' -T-T-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3 ' (DNA) 3 ' -A-A-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-5 ' ( DNA) I I I I I I I I I I I I I I I I I I I I I I

[0232] 5 ' - [gRNA scaffold] -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3 ' (RNA) [ binding domain ] [ targeting domain ( RNA) ]

[0233] A “scaffold” or “scaffold sequence” is a sequence necessary for endonuclease binding e.g., Cas binding.

[0234] In some aspects, the present disclosure provides single guide RNAs (sgRNAs). In some aspects, the gRNA according to the present disclosure is a sgRNA. sgRNAs are single RNA molecules which contain a crRNA sequence fused to the scaffold tracrRNA sequence. In nature, crRNAs and tracrRNAs exist as two separate RNA molecules, but sgRNAs have become a common format for CRISPR gRNAs in research.

[0235] In some aspects, the gRNA comprises a spacer sequence which is 11 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 12 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 13 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 14 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 15 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 16 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 17 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 18 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 19 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 20 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 21 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 22 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 23 nucleotides in length. In some aspects, the gRNA comprises a spacer sequence which is 24 nucleotides in length.

[0236] Without wishing to be bound by theory, certain gRNAs (e.g., gRNAs comprising a spacer sequence of around 20 nucleotides in length) may be used to induce gene editing by a polyfunctional editor while gRNAs comprising shorter spacer sequences (e.g., gRNAs comprising spacer sequences of around 16 nucleotides in length) may favor epigenetic editing

[0237] 31

[0238] #14686672vlsuch as epi-silencing by a polyfunctional editor. A non-limiting example of this concept is shown in FIG. 6A.

[0239] In some embodiments, the gRNA comprises a spacer sequence which is less than or equal to 15, 16, or 17 (e.g., less than or equal to 17 or 16) nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 11 to 16 nucleotides in length (11, 12, 13, 14, 15, or 16 nucleotides in length), such as 12 to 16, 13 to 16, 14 to 16, 15 to 16, 12 to 17, 13 to 17, 14 to 17, 15 to 17, 16, or 17 nucleotides in length.

[0240] In some embodiments, the gRNA comprises a spacer sequence which is greater than or equal to 16, 17, or 18 (e.g., greater than or equal to 17 or 18) nucleotides in length, such as 18 or more, 19 or more, or 20 or more nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 17 to 30 nucleotides in length (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length), such as 18 to 30, 19 to 30 or 20 to 30 nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 17 to 25 nucleotides in length, such as 18 to 25, 19 to 25 or 20 to 25 nucleotides in length. In some embodiments, the gRNA comprises a spacer sequence which is 17 to 20 nucleotides in length, such as 18 to 20 or 19 to 20 nucleotides in length.

[0241] Without wishing to be bound by theory, certain gRNAs (e.g., gRNAs comprising a spacer sequence of around 20 nucleotides in length) may be used to induce gene editing by a polyfunctional editor whilst gRNAs comprising mismatches may favor epigenetic editing such as epi-silencing by a polyfunctional editor. A non-limiting example of this concept is shown in FIGs. 22A-22B.

[0242] In some embodiments, the gRNA spacer sequence is mismatched with the target sequence. In some embodiments, the gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the mismatches are located at the distal end of the guide RNA relative to the target cutting site.

[0243] In some aspects, the polyfunctional editor according to the present disclosure is capable of modifying the transcription, expression and / or activity (e.g., repressing transcription and / or expression) of multiple target genes within the same cell by epigenetic editing and by gene editing.

[0244] The present disclosure enables the selection of gRNAs which promote either gene editing or epigenetic editing of a target. In this manner, it is possible to choose to perform gene editing on gene targets which are not susceptible to epigenetic editing while simultaneously epigenetically targeting genes which are susceptible to epigenetic editing in a multiplexing approach.

[0245] 32

[0246] #14686672vlIn some aspects, a gRNA is capable of promoting epigenetic editing of a target. In some embodiments, epigenetic editing is measured using a known method. For example, the level of expression of a reporter gene may be measured as a model of epigenetic editing.

[0247] In some aspects, a gRNA is capable of promoting gene editing of a target. In some embodiments, gene editing is measured using a known method.

[0248] The present disclosure also provides variations of exemplified gRNAs in which the spacer sequences are truncated at the 5’ end. In some embodiments, spacer sequences are truncated by 1 to 9 nucleotides at the 5’ end (1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides, e.g., 3 to 9 nucleotides). The present disclosure also provides gRNAs in which the spacers (full-length or truncated versions) described herein are linked to the above-exemplified tracr RNA.

[0249] The present disclosure also provides variations of exemplified gRNAs in which the spacer sequences are mismatched by, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the distal end. The present disclosure also provides gRNAs in which the spacers (fully matched or mismatched) described herein are linked to the above-exemplified tracr RNA.

[0250] In some aspects, the present disclosure provides a gRNA which comprises a spacer sequence which targets a sequence set forth in any one of SEQ ID NOs: 17-86, 87-677, 678-794, or 837-1281, or a homologue thereof.

[0251] In some aspects, a gRNA is chemically modified. In some embodiments, chemical modification increases the stability of the gRNA once administrated in a target cell as described for example in (Yin etal., Nat Biotechnol. 2017 Dec;35(12): 1179-1187). Such chemical modifications are known in the literature and can comprise but are not limited to locked nucleic acids (LNA), phosphorothioate modified oligonucleotides, 2'-O-methoxyethyl modified oligonucleotides, and 2' O-methyl modified oligonucleotides.

[0252] In some aspects, the first three nucleosides and the last three nucleosides of a gRNA, regardless of the gRNA’s length, are 2’ -O-methyl modified nucleosides. In some aspects, the first three internucleoside linkages and the last three internucleoside linkages of a gRNA, regardless of the gRNA’s length, are phosphorothioate linkages.

[0253] In some embodiments, the gRNA sequences have the tracr RNA of SEQ ID NO: 15 (which is 80 nucleotides in length), and the tracr sequence portion of the full-length gRNA is modified as follows (with nucleoside 1 being at the 5’ end of the tracr RNA sequence, and nucleoside 80 being at the 3’ end of the tracr RNA sequence):

[0254] nucleosides 1-8: unmodified RNA nucleosides,

[0255] nucleosides 9-20: 2’-0-Me modified nucleosides,

[0256] nucleosides 21-48: unmodified RNA nucleosides, and

[0257] 33

[0258] #14686672vlnucleosides 49-80: 2’-0-Me modified nucleosides.

[0259] In some embodiments, in such a modified tracr sequence, the internucleoside linkages between nucleosides 77 and 78, 78 and 79, and 79 and 80 (i.e., the last three intemucleoside linkages) are phosphorothioate linkages. In some embodiments, a spacer RNA is attached at the 5’ end of this modified tracr sequence to form a full-length gRNA. In this full-length gRNA, the tracr portion of the gRNA sequence is modified as described above, and the spacer portion of the gRNA sequence is modified as follows:

[0260] the first three nucleosides of the spacer sequence are 2’-0-Me nucleosides, and the first three internucleoside linkages are phosphorothioate linkages.

[0261] The general schematic for this full-length gRNA is shown below, wherein lowercase letters represent 2’-0-Me nucleosides, capital letters represent unmodified RNA nucleosides, s represents a phosphorothioate linkage, each X independently represents an A, C, G, or U nucleoside, and each x represents a 2’-0-Me A, C, G, or U nucleoside:

[0262] 5'-xsxsxs[X7-Xi7]GUUUUAGAgcuagaaauagcAAGUUAAAAUAAGGCUAGU CCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3' (SEQ ID NO: 16).

[0263] In some embodiments, for gRNA sequences having full-length spacer RNAs (i.e., 20 nucleotides) and the tracr RNA of SEQ ID NO: 15 (which is 80 nucleotides in length, for a gRNA of 100 nucleotides in length), the gRNA is modified as follows (with nucleoside 1 being at the 5’ end of the oligonucleotide, and nucleotide 100 being at the 3’ end of the oligonucleotide):

[0264] nucleosides 1-3: 2’-0-Me modified nucleosides,

[0265] nucleosides 4-28: Unmodified RNA nucleosides,

[0266] nucleosides 29-40: 2’-0-Me modified nucleosides,

[0267] nucleosides 41-68: Unmodified RNA nucleosides, and

[0268] nucleosides 79-100: 2’-0-Me modified nucleosides.

[0269] In some embodiments, in such a modified gRNA, the internucleoside linkages between nucleosides 1 and 2, 2 and 3, 3 and 4, 97 and 98, 98 and 99, and 99 and 100 (i.e., the first three internucleoside linkages and the last three internucleoside linkages) are phosphorothioate linkages. In some embodiments, the remainder of the internucleoside linkages are phosphate linkages.

[0270] In some embodiments, similar modifications are made to truncated gRNAs (e.g., a gRNA with a spacer that is 11 to 19 nucleotides). In some embodiments the first three and the last three internucleoside linkages of the gRNA are phosphorothioate linkages, and / or some or all of the nucleotides are chemically modified, e.g., 2’-O-methyl nucleotides.

[0271] 34

[0272] #14686672vlIn some aspects, the present disclosure utilizes two or more gRNAs.

[0273] In some embodiments, the two or more gRNAs target the polyfunctional editor to different target genes based on the length of the spacer sequences. In some embodiments, the two or more gRNAs comprise spacer sequences of different lengths. In some embodiments, the spacer sequences of different lengths target the endonuclease of the poly functional editor to different target genes.

[0274] In some aspects, at least one of the at least two gRNAs comprises a spacer sequence which is 18, 19 or 20 nucleotides in length.

[0275] In some aspects, at least one of the at least two gRNAs comprises a spacer sequence which is less than or equal to 17 nucleotides in length, such as 16 nucleotides in length, 15 nucleotides in length, such as 14 nucleotides in length, such as less than 13 nucleotides in length, such as 12 nucleotides in length, such as 11 nucleotides in length, or such as 10 nucleotides in length.

[0276] In some embodiments, the two or more gRNAs target the polyfunctional editor to different target genes based on mismatches in one or more of the gRNAs. In some aspects, at least one of the at least two gRNAs comprises a spacer sequence which has no mismatches with its target sequence. In some aspects, at least one of the at least two gRNAs comprises a spacer sequence which has one or more mismatches with its target sequence. In some embodiments, the mismatches in the spacer sequences target the endonuclease of the polyfunctional editor to different target genes.

[0277] In some aspects, two or more gRNAs target the same target gene. In some embodiments, it is beneficial to target the same gene with two gRNAs for optimal epigenetic modification e.g., epigenetic silencing.

[0278] Multiplexing - modifying multiple genes in the same cell

[0279] The present disclosure provides the development of a combined gene editing and epigenetic editing strategy to modify the expression and / or activity of multiple target genes within the same cell. In some aspects, the strategy uses a polyfunctional editor which comprises an epigenetic effector domain and an endonuclease and gRNAs comprising spacer sequences of different lengths to promote epigenetic editing of one or more genes and genetic editing of another gene.

[0280] As used herein “modify the expression and / or activity” refers to increasing or decreasing (e.g., decreasing) the expression and / or activity of a target gene.

[0281] In some aspects, transcription and / or expression of a target gene are repressed.

[0282] 35

[0283] #14686672vlIn some aspects, a target gene is silenced.

[0284] In some aspects, a target gene is enhanced. In some embodiments, enhanced means that the expression of the target gene is increased. In some embodiments, the expression of an endogenous target gene is increased.

[0285] In some embodiments, an endogenous target (e.g., gene) is modified (e.g., mutated) by gene editing and the expression of the modified target (e.g., gene) is increased.

[0286] The effect of a polyfunctional editor or combination of polyfunctional editors may be studied by comparing the transcription or expression of the target gene, for example a gene endogenous to a cell, in the presence and absence of the polyfunctional editor or combination of polyfunctional editors. Methods of analyzing transcription or expression of a gene are well known in the art.

[0287] The effect of a polyfunctional editor may also be studied using a model system wherein the expression of a reporter gene, for example a gene encoding a fluorescent protein, is monitored. Suitable methods for monitoring expression of such reporter genes include flow cytometry, fluorescence-activated cell sorting (FACS) and fluorescence microscopy.

[0288] In some embodiments, a population of cells is transfected with a vector comprising a reporter gene. In some embodiments, the vector is constructed such that the reporter gene is expressed when the vector transfects a cell. Suitable reporter genes include, but are not limited to, genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins. In some embodiments, the population of cells is transfected with vectors encoding the polyfunctional editor s of interest and / or gRNAs. Subsequently, the number of cells expressing and not expressing the reporter gene, as well as the level of expression of the reporter gene may be quantified using a suitable technique, such as FACS. In some embodiments, the level of reporter gene expression is then be compared in the presence and absence of the polyfunctional editor and / or gRNAs.

[0289] Methods for determining the transcription of a gene, for example the target of a polyfunctional editor, are known in the art. Suitable methods include, but are not limited to, reverse transcription PCR and Northern blot-based approaches. In addition to the methods for determining the transcription of a gene, methods for determining the expression of a gene are known in the art. Suitable additional methods include, but are not limited to, Western blotbased or flow cytometry approaches.

[0290] 36

[0291] #14686672vlTarget gene transcription and expression

[0292] In some aspects, the product (e.g., poly functional editor and / or gRNA) according to the present disclosure is used in a method which represses transcription and / or expression of at least one target gene. In some embodiments, the target gene is an endogenous gene.

[0293] In some aspects, the target gene transcription and / or expression is repressed by epigenetic editing. In some aspects, the target gene transcription and / or expression is repressed by gene editing.

[0294] In some aspects, the product (e.g., poly functional editor and / or gRNA) according to the present disclosure is used in a method which represses transcription and / or expression of at least two target genes. In some embodiments, at least one or both of the target genes is an endogenous gene.

[0295] In some aspects, transcription and / or expression of only one gene is repressed by gene editing.

[0296] In some embodiments, following administration of a polyfunctional editor of the disclosure (e.g., with suitable gRNA(s)), the level of transcription or expression of the target gene is reduced compared to the level of transcription or expression in the absence of the polyfunctional editor. In some embodiments, the level of transcription or expression of the target gene is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% compared to the level of transcription or expression in the absence of the polyfunctional editor. In some aspects, the product (e.g., polyfunctional editor and / or gRNA) according to the present disclosure is used in a method which silences at least one target gene. In some embodiments, the target gene is an endogenous gene. In some embodiments, the target gene is an exogenous gene, such as a viral gene.

[0297] In some aspects, the target gene is silenced by epigenetic editing. In some aspects, the target gene is silenced by gene editing.

[0298] In some aspects, the product (e.g., polyfunctional editor fusion protein and / or gRNA) according to the present disclosure is used in a method which silences at least two target genes. In some embodiments, at least one or both of the target genes is an endogenous gene.

[0299] In some aspects, only one gene is silenced by gene editing.

[0300] Without wishing to be bound by theory, restricting gene editing activity to one gene may reduce the potential for undesirable genomic translocations.

[0301] 37

[0302] #14686672vlAs used herein, “silencing a target gene”, means that the expression of the target gene is reduced to an extent sufficient to achieve a desired effect. In some embodiments, the reduced expression is sufficient to achieve a therapeutically relevant effect, such as the prevention or treatment of a disease. In some embodiments, a dysfunctional target gene which gives rise to a disease is repressed such that there is either no expression of the target gene, or the residual level of expression of the target gene is sufficiently low to ameliorate or prevent the disease state. In some embodiments, the reduced expression allows for purification of the cells having gene silencing.

[0303] In some embodiments, the reduced expression is sufficient to enable investigations to be performed into the gene’s function by studying cells reduced in or lacking that function.

[0304] In some embodiments, the repression of the target gene occurs following transient delivery or expression of the polyfunctional editors of the present disclosure to or in a cell (e.g., along with suitable gRNAs).

[0305] Enhancing a target gene

[0306] As used herein “enhancing a target gene”, means that the expression of the target gene is increased to an extent sufficient to achieve a desired effect. In some embodiments, the increased expression is sufficient to achieve a therapeutically relevant effect, such as the prevention or treatment of a disease. In some embodiments, a dysfunctional target gene which gives rise to a disease is enhanced to an extent that there is sufficient expression of the target gene to ameliorate or prevent the disease state. In some embodiments, increased expression of the target gene compensates for the dysfunctional activity of a disease-related gene. In some embodiments, increased expression of the target gene allows for selection of the cells expressing de novo that specific target gene.

[0307] In some embodiments, following administration of a polyfunctional editor of the disclosure (e.g., with suitable gRNA(s)), the level of transcription or expression of the target gene is increased compared to the level of transcription or expression in the absence of the polyfunctional editor. In some embodiments, the level of transcription or expression of the target gene is increased by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500% compared to the level of transcription or expression in the absence of the polyfunctional editor.

[0308] 38

[0309] #14686672vlIn some embodiments, the enhancement of the target gene occurs following transient delivery or expression of the polyfunctional editors of the present disclosure to or in a cell (along with suitable gRNAs).

[0310] Transient expression

[0311] As used herein, “transient expression”, means that the expression of the polyfunctional editor is not stable over a prolonged period of time. In some embodiments, the polynucleotide encoding the polyfunctional editor does not integrate into the host genome. In some embodiments, transient expression is expression which is substantially lost within 20 weeks following introduction of the polynucleotide encoding the polyfunctional editor into the cell. In some embodiments, expression is substantially lost within 12, 6, 4, or 2 weeks following introduction of the polynucleotide encoding the polyfunctional editor into the cell.

[0312] Similarly, “transient delivery”, means that the polyfunctional editor substantially does not remain in the cell (i.e., is substantially lost by the cell) over a prolonged period of time. In some embodiments, transient delivery results in the polyfunctional editor being substantially lost by the cell within 20 weeks following introduction of the polyfunctional editor into the cell. In some embodiments, the polyfunctional editor is substantially lost within 12, 6, 4, or 2 weeks following introduction of the polyfunctional editor into the cell.

[0313] In some aspects, the polyfunctional editor and / or gRNA is delivered transiently. In some embodiments, transient delivery results in permanent changes. In some embodiments, transient delivery of the polyfunctional editor and / or gRNA leads to DNA methylation of a repressive regulatory element which in turn leads to gene activation (e.g., given the stability of this epigenetic modification, permanent gene activation).

[0314] In some embodiments, the target gene is repressed, silenced, or enhanced permanently. As used herein, “permanent repression”, “permanent silencing” or “permanent enhancement” of a target gene, means that transcription or expression of the target gene is reduced or increased (e.g., reduced or increased by at least 60%, at least 70%, at least 80%, at least 90% or 100%) compared to the level of transcription or expression in the absence of the polyfunctional editor for at least 2 months, at least 6 months, at least 1 year, at least 2 years or the entire lifetime of the cell / organism. In some embodiments, a permanently repressed, silenced, or enhanced target gene remains repressed, silenced, or enhanced for the remainder of the cell’s life.

[0315] 39

[0316] #14686672vlIn some aspects, the polyfunctional editor and / or gRNA is stably expressed. In some embodiments, stable expression is required to achieve permanent gene activation of some targets. In some embodiments, the target gene remains repressed, silenced, or enhanced in the progeny of the cell to which the product of the disclosure has been administered (i.e., the repression, silencing or enhancement of the target gene is inherited by the cell’s progeny). In some embodiments, the polyfunctional editor and gRNAs of the disclosure are administered to a stem cell (e.g., a hematopoietic stem cell) to repress or silence a target gene in a stem cell and also in the stem cell’s progeny. In some embodiments, a stem cell’s progeny includes cells that have differentiated from the stem cell.

[0317] Target gene

[0318] In some embodiments, the target gene gives rise to a therapeutic effect when modified, e.g., repressed or silenced. In some embodiments, modifying the target gene produces a therapeutic effect in a subject in need thereof.

[0319] In some embodiments, the products of the present disclosure are used to modify, e.g., repress or silence, genes with and without CpG islands (CGI).

[0320] Table 1. Non-limiting examples of gRNA target sequences for epigenetic editing oiPCSK9.

[0321]

[0322] 40

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[0325] 41

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[0327]

[0328] Table 2. Non-limiting examples of gRNA target sequences for gene editing of LPA.

[0329]

[0330] 42

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[0338]

[0339] 45

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[0341]

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[0348] 48

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[0351] 49

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[0359]

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[0363] 53

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[0366] 54

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[0369] Table 3. Non-limiting examples of gRNA target sequences for gene editing of ANGPTL3.

[0370]

[0371] 55

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[0373]

[0374] 56

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[0377] 57

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[0379]

[0380] Table 4. Non-limiting examples of gRNA target sequences for epigenetic editing of CIDEB.

[0381]

[0382] 58

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[0384]

[0385] 59

[0386] #14686672vl

[0387]

[0388] 60

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[0390]

[0391] 61

[0392] #14686672vl

[0393]

[0394] #14686672vlTable 5. Non-limiting examples of gRNA target sequences for epigenetic editing of PNPLA3.

[0395]

[0396] Table 6. Non-limiting examples of gRNA target sequences for epigenetic editing of MARC 1 (al so known as MT ARC 1 ) .

[0397]

[0398] Table 7. Non-limiting examples of gRNA target sequences for epigenetic editing c DGAT2.

[0399]

[0400] 63

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[0402]

[0403] Table 8. Non-limiting examples of gRNA target sequences for gene editing of CIDEB.

[0404]

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[0407]

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[0413]

[0414] Table 9. Non-limiting examples of gRNA target sequences for gene editing of HSD17B13.

[0415]

[0416] 67

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[0418]

[0419] 68

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[0421]

[0422] Table 10. Additional components of polyfunctional editor fusion proteins

[0423]

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[0426] <<<<

[0427]

[0428] 70

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[0440]

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[0443]

[0444] Table 11. Non-limiting examples of polyfunctional editor fusion proteins and encoding sequences.

[0445]

[0446] #14686672vl <<<<<

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[0449] #14686672vl <<<<< <<<<

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[0540]

[0541] Cell

[0542] In some aspects, the present disclosure provides a cell comprising a polyfunctional editor according to the present disclosure, at least one gRNA according to the present disclosure, a combination according to the present disclosure, a polynucleotide according to the present disclosure, a nucleic acid construct according to the present disclosure, a vector according to the present disclosure or a kit of polynucleotides according to the present disclosure.

[0543] In some embodiments, the cell is any cell which can be used to express the product of the disclosure.

[0544] In some embodiments, cells are generated by introducing DNA or RNA coding for the polyfunctional editor of the present disclosure by one of any means including transduction with a viral vector or transfection with DNA or RNA.

[0545] In some aspects, the cell further comprises a polynucleotide, such as an integrating vector, which encodes an agent:

[0546] i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the polynucleotide or a cell which does not comprise the polynucleotide; and / or

[0547] ii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises the polynucleotide or a cell which does not comprise the polynucleotide and / or

[0548] iii) which enables selection of a cell, such as a cell which comprises the polynucleotide or a cell which does not comprise the polynucleotide.

[0549] Combinations

[0550] In some aspects, the present disclosure provides a combination (e.g., a system) comprising a polyfunctional editor according to the present disclosure, and at least one gRNA which targets the endonuclease of the polyfunctional editor to a target gene.

[0551] 107

[0552] #14686672vlIn some embodiments, the combination comprises at least two gRNAs (e.g., at least three, at least four, at least five, at least six, at least seven, or at least eight gRNAs). In some embodiments, the combination comprises two gRNAs, three gRNAs, four gRNAs, five gRNAs, six gRNAs, seven gRNAs, eight gRNAs, nine gRNAs, ten gRNAs, eleven gRNAs, twelve gRNAs, thirteen gRNAs, fourteen gRNAs, or fifteen gRNAs.

[0553] In some embodiments, the combination comprises gRNAs which target the endonuclease to at least two different target genes.

[0554] In some embodiments, one target gene is targeted with two or more gRNAs. In some embodiments, it is beneficial to target the same gene with several gRNAs for optimal epigenetic modification, e.g., epigenetic silencing.

[0555] In some embodiments, the combination comprises at least two gRNAs which comprise spacer sequences of different lengths. In some embodiments, at least one gRNA comprises a spacer sequence which is 15, 16, 17, 18, 19 or 20 nucleotides in length. In some embodiments, at least one of the at least two gRNAs comprises a spacer sequence which is less than or equal to 17 (e.g., less than or equal to 16) nucleotides in length. In some embodiments, at least one of the at least two gRNAs comprises a spacer sequence which is less than or equal to 17 (e.g., less than or equal to 16) nucleotides in length and at least one of the at least two gRNAs comprises a spacer sequence which is more than 17 nucleotides in length.

[0556] Without wishing to be bound by theory, the gRNAs comprising spacer sequences of different lengths may target the polyfunctional editor to different target genes, wherein a first target gene is modified by gene editing and at least a second target gene is modified by epigenetic editing.

[0557] In some aspects, the combination comprises at least one gRNA according to the present disclosure. In some embodiments, the combination comprises at least two gRNAs according to the present disclosure.

[0558] In some aspects, the combination comprises two or more gRNAs designed to effect epigenetic editing.

[0559] Polynucleotides

[0560] In some aspects, the present disclosure provides a polynucleotide encoding at least one polyfunctional editor according to the present disclosure.

[0561] Polynucleotides of the disclosure may comprise DNA or RNA. They may be singlestranded or double-stranded. It will be understood by a skilled person that numerous 108

[0562] #14686672vldifferent polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that the skilled person may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the disclosure to reflect the codon usage of any particular host organism in which the polypeptides of the disclosure are to be expressed.

[0563] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides of the disclosure.

[0564] Polynucleotides such as DNA polynucleotides may be produced recombinantly, synthetically or by any means available to those of skill in the art. They may also be cloned by standard techniques.

[0565] Longer polynucleotides will generally be produced using recombinant means, for example using PCR cloning techniques. This will involve making a pair of primers (e.g., of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0566] Constructs

[0567] In some aspects, the present disclosure provides a nucleic acid construct comprising a nucleic acid sequence encoding at least one polyfunctional editor according to the present disclosure.

[0568] In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence which encodes an agent:

[0569] i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / or

[0570] ii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which expresses said nucleic acid construct or a cell which does not express said nucleic acid construct; and / or

[0571] 109

[0572] #14686672vliii) which enables selection of a cell, such as a cell which comprises the nucleic acid construct or a cell which does not comprise the construct.

[0573] Proteins

[0574] As used herein, the term “protein” encompasses single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms “polypeptide” and “peptide” refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.

[0575] Variants, derivatives, analogues, homologues and fragments

[0576] In addition to the specific proteins and nucleotides mentioned herein, the present disclosure encompasses the use of variants, derivatives, analogues, homologues, and fragments thereof.

[0577] In the context of the present disclosure, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring protein.

[0578] The term “derivative” as used herein, in relation to proteins or polypeptides of the present disclosure, encompasses any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its endogenous functions.

[0579] The term “analogue” as used herein, in relation to polypeptides or polynucleotides, encompasses any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.

[0580] In some embodiments, amino acid substitutions are made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence substantially retains the required activity or ability. In some embodiments, amino acid substitutions include the use of non-naturally occurring analogues.

[0581] In some embodiments, proteins used in the present disclosure further comprise deletions, insertions or substitutions of amino acid residues which produce a silent change 110

[0582] #14686672vland result in a functionally equivalent protein. In some embodiments, deliberate amino acid substitutions are made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.

[0583] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and, in particular examples, in the same line in the third column may be substituted for each other:

[0584]

[0585] The term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term “homology” can be equated with “identity”.

[0586] In some aspects, a homologous sequence encompasses an amino acid sequence which is at least 50%, at least 55%, at least 65%, at least 75%, at least 85% or at least 90% identical, for example at least 95% or at least 97% or at least 99% identical, to the subject sequence. In some embodiments, a homologue comprises the same active sites, etc., as the subject amino acid sequence. In some embodiments homology is considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions).

[0587] In some embodiments, a homologous sequence encompasses a nucleotide sequence which is at least 50%, at least 55%, at least 65%, at least 75%, at least 85% or at least 90% identical, for example at least 95% or at least 97% or at least 99% identical, to the subject sequence. In some embodiments, homology is considered in terms of similarity.

[0588] In some embodiments, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to. In some embodiments, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to 111

[0589] #14686672vla sequence which has the stated percent identity over a portion of the length of the SEQ ID NO referred to.

[0590] Homology comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences.

[0591] Percentage homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time.

[0592] As used herein, “fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0593] Such variants may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the disclosure to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0594] Codon optimization

[0595] In some embodiments, the polynucleotides used in the present disclosure are codon-optimized. Codon optimization has previously been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. Conversely, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.

[0596] 112

[0597] #14686672vlVectors

[0598] In some aspects, the present disclosure provides a vector comprising a polynucleotide according to the present disclosure, or a nucleic acid construct according to the present disclosure.

[0599] As used herein, a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the present disclosure, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g., a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid, or facilitating the expression of the protein encoded by a segment of nucleic acid. Vectors may be non-viral or viral.

[0600] Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNAs), chromosomes, artificial chromosomes, and viruses. The vector may also be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, the vector may itself be a nucleotide of interest.

[0601] The vectors used in the disclosure may be, for example, plasmid, mRNA, or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.

[0602] Vectors comprising polynucleotides used in the disclosure may be introduced into cells using a variety of techniques known in the art, such as transfection, transformation, and transduction. Several such techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral (e.g., integration-defective lentiviral), adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation.

[0603] Non-viral delivery systems include but are not limited to DNA or RNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofection, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.

[0604] As used herein, the term “transfection” encompasses the delivery of polynucleotides to cells by both viral and non-viral delivery.

[0605] 113

[0606] #14686672vlProtein transduction

[0607] In some embodiments, as an alternative to the delivery of polynucleotides to cells, the products and polyfunctional editors of the present disclosure are delivered to cells by protein transduction.

[0608] In some embodiments, protein transduction is performed via vector delivery (Cai, Y. et al. (2014) Elife 3: e01911; Maetzig, T. et al. (2012) Curr. Gene Ther. 12: 389-409).

[0609] Vector delivery involves the engineering of viral particles (e.g., lentiviral particles) to comprise the proteins to be delivered to a cell. Accordingly, when the engineered viral particles enter a cell as part of their natural life cycle, the proteins comprised in the particles are carried into the cell.

[0610] In some embodiments, protein transduction is performed via protein delivery (Gaj, T. et al. (2012) Nat. Methods 9: 805-7). In some embodiments, protein delivery is achieved, for example, by utilizing a vehicle (e.g., liposomes) or even by administering the protein itself directly to a cell.

[0611] Composition

[0612] The present disclosure provides compositions comprising the products of the disclosure such as polyfunctional editors, gRNAs, combinations, polynucleotides, nucleic acid constructs, vectors, cells, and kits of polynucleotides. In some embodiments, the products of the disclosure are provided to a subject in need thereof in a composition.

[0613] In some embodiments, the products of the disclosure such as combinations, polyfunctional editors, gRNAs, polynucleotides, nucleic acid constructs, vectors, compositions, and cells of the present disclosure are formulated for administration to subjects with a pharmaceutically acceptable carrier, diluent, or excipient. Suitable carriers and diluents include, but are not limited to, isotonic saline solutions, for example, phosphate-buffered saline, and potentially contain human serum albumin.

[0614] In some aspects, there is provided a combination of chemically modified mRNA encoding for the fusion protein of a polyfunctional editor plus a chemically modified gRNA.

[0615] In other aspects, there is provided a ribonucleic complex of protein-RNA that includes the fusion protein of the polyfunctional editor attached to a chemically modified gRNA.

[0616] Kit

[0617] In some aspects, the present disclosure provides a kit of polynucleotides comprising:

[0618] 114

[0619] #14686672vla) at least one polynucleotide encoding at least one polyfunctional editor according to the present disclosure; and

[0620] b) a polynucleotide providing at least one gRNA as described herein; and optionally,

[0621] c) further comprising a nucleic acid sequence which encodes an agent:

[0622] i) which promotes the survival, proliferation and / or activity of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides; and / or

[0623] ii) which is detrimental to the survival, proliferation, activity, chemoresistance and / or chemotaxis of a cell, such as a cell which comprises said polynucleotides or a cell which does not comprise said polynucleotides; and / or

[0624] iii) which enables selection of a cell, such as a cell which comprises the polynucleotides or a cell which does not comprise the polynucleotides.

[0625] In some embodiments, the kit further includes instructions for use, for example instructions for the simultaneous, sequential, or separate administration of at least one polyfunctional editor and at least two gRNAs, to a subject in need thereof.

[0626] Use

[0627] In some aspects, the present disclosure provides the use of a poly functional editor according to the present disclosure, at least one gRNA according to the present disclosure, a combination according to the present disclosure, a polynucleotide according to the present disclosure, a nucleic acid construct according to the present disclosure, a vector according to the present disclosure or a kit of polynucleotides according to the present disclosure for modifying the activity and / or expression of at least one target gene. In some embodiments, the use is in vitro use. In some embodiments, the use is ex vivo use.

[0628] In some embodiments, the use represses (e.g., silences) transcription and / or expression of at least one target gene. In some embodiments, the use represses (e.g., silences) transcription and / or expression of at least two target genes. In some embodiments, transcription and / or expression of a first gene is repressed (e.g., silenced) by gene editing and transcription and / or expression of a second target gene is repressed (e.g., silenced) by epigenetic editing.

[0629] 115

[0630] #14686672vlIn some embodiments, the use enhances at least one target gene.

[0631] In other aspects, the present disclosure provides a method of repressing transcription and / or expression of (e.g., silencing) at least one target gene in a cell comprising the step of administering a polyfunctional editor according to the present disclosure, at least one gRNA according to the present disclosure, a combination according to the present disclosure, a polynucleotide according to the present disclosure, a nucleic acid construct according to the present disclosure, a vector according to the present disclosure or a kit of polynucleotides according to the present disclosure to a cell.

[0632] In some embodiments, transcription and / or expression of at least two target genes is repressed (e.g., silenced), wherein at least one of the at least two target genes is epigenetically repressed (e.g., silenced) and at least one of the at least two target genes is repressed (e.g., silenced) by gene editing, wherein at least one polyfunctional editor and at least two gRNAs are administered to said cell simultaneously, sequentially, or separately.

[0633] In other aspects, the present disclosure provides the products, polyfunctional editors, gRNAs, combinations, polynucleotides, nucleic acid constructs, vectors, kits of polynucleotides, cells, and pharmaceutical compositions of the present disclosure for use in therapy.

[0634] In some embodiments, the polyfunctional editor (or polynucleotide, nucleic acid construct, or vector encoding therefor) and gRNAs are administered simultaneously, in combination, sequentially or separately (as part of a dosing regimen).

[0635] As used herein, “simultaneously”, means that the two or more agents are administered concurrently, whereas the term “in combination” is used to mean that the agents are administered, if not simultaneously, then “sequentially” within a time frame such that they are both available to act therapeutically within the same time frame. In some embodiments, administration “sequentially” permits one agent to be administered within 5 minutes, 10 minutes, or a matter of hours after the other provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts. The time delay between administration of the components will vary depending on the exact nature of the components, the interaction there-b etween, and their respective halflives.

[0636] In contrast to “in combination” or “sequentially”, “separately” as used herein means that the gap between administering one agent and the other agent is significant, i.e., the first administered agent may no longer be present in the bloodstream in a therapeutically effective amount when the second agent is administered.

[0637] 116

[0638] #14686672vlIn other aspects, the present disclosure provides a method for treating and / or preventing a disease or condition, which comprises the step of administering any of the products of the disclosure (e.g., polyfunctional editors, gRNAs, combinations, polynucleotides, nucleic acid constructs, vectors, kits of polynucleotides, cells, or pharmaceutical compositions according to the present disclosure) to a subject in need thereof.

[0639] In some embodiments, the polyfunctional editor and gRNAs are administered to a subject simultaneously, sequentially, or separately.

[0640] In some embodiments, the nucleic acid construct or vector is introduced by transduction or transfection.

[0641] The skilled person will understand that they can combine any or all features of the disclosure disclosed herein without departing from the scope of the disclosure as disclosed.

[0642] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Non-limiting examples of methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms includes pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. As used herein the term “about” refers to a numerical range that is 10%, 5%, or 1% plus or minus from a stated numerical value within the context of the particular usage. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0643] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of a person of ordinary skill in the art. Such 117

[0644] #14686672vltechniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press.

[0645] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0646] EMBODIMENTS

[0647] Below is a list of non-limiting embodiments.

[0648] Embodiment 1. A combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising:

[0649] (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins;

[0650] (b) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and

[0651] (c) one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

[0652] Embodiment 2, The combination of embodiment 1, wherein:

[0653] 1) the one or more gRNAs in (b) has a spacer sequence of 15 to 17 nucleotides; and / or

[0654] 2) the one or more gRNAs in (c) has a spacer sequence of 18 to 24 nucleotides.

[0655] 118

[0656] #14686672vlEmbodiment 3, The combination of embodiment 1 or 2, wherein the combination comprises one to three fusion proteins.

[0657] Embodiment 4, The combination of any one of embodiments 1-3, further comprising a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene.

[0658] Embodiment 5, The combination of any one of embodiments 1-4, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.

[0659] Embodiment 6, The combination of any one of embodiments 1-5, wherein

[0660] the first gene is PCSK9; and / or

[0661] the second gene is selected from LPA and ANGPTL3.

[0662] Embodiment 7, The combination of any one of embodiments 1-6, wherein the first gene is a PCSK9 gene, optionally wherein the one or more gRNAs targeting the PCSK9 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 17-86 and 831-834.

[0663] Embodiment 8, The combination of embodiment 7 wherein the second gene is a LPA gene, optionally wherein the one or more gRNAs targeting the LPA gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 87-677 and 835.

[0664] Embodiment 9, The combination of embodiment 7, wherein the second gene is an ANGPTL3 gene, optionally wherein the one or more gRNAs targeting the ANGPTL3 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 678-794.

[0665] Embodiment 10. The combination of any one of embodiments 1-9, wherein the one or more fusion proteins collectively further comprise a transcriptional repressor domain.

[0666] Embodiment 11. The combination of embodiment 10, wherein the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3.

[0667] Embodiment 12, The combination of any one of embodiments 1-11, wherein the DNMT3A domain comprises an H3 tail.

[0668] 119

[0669] #14686672vlEmbodiment 13, The combination of any one of embodiments 1-12, wherein the one or more fusion proteins comprises one or more NLS.

[0670] Embodiment 14, The combination of any one of embodiments 1-13, wherein the one or more fusion proteins comprises one or more linkers.

[0671] Embodiment 15, The combination of any one of embodiments 1-14, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS - XTEN16 - KRAB.

[0672] Embodiment 16, The combination of any one of embodiments 1-14, wherein the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS - XTEN16 - KRAB - BipartiteNLS.

[0673] Embodiment 17, The combination of any one of embodiments 1-14, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS.

[0674] Embodiment 18, The combination of any one of embodiments 1-14, wherein the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS.

[0675] Embodiment 19, The combination of any one of embodiments 1-14, wherein the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 819-830 and 836.

[0676] Embodiment 20, The combination of any one of embodiments 1-19, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0677] Embodiment 21, A pharmaceutical composition comprising the combination of any one of embodiments 1-20.

[0678] 120

[0679] #14686672vlEmbodiment 22, The combination of any one of embodiments 1-20, wherein the cell is a mammalian cell, optionally a human cell, further optionally wherein the cell is a human hepatocyte.

[0680] Embodiment 23, A method of modifying transcription, expression and / or activity of one or more gene in a human hepatocyte, comprising introducing the combination of any one of embodiments 1-20 into the cell.

[0681] Embodiment 24, A method of treating a human in need thereof, comprising administering to the human the combination of any one of embodiments 1-20 or the pharmaceutical composition of embodiment 21.

[0682] Embodiment 25, A combination for modifying transcription, expression, and / or activity of one or more gene in a cell, the combination comprising:

[0683] (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins;

[0684] (b) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and

[0685] (c) one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

[0686] Embodiment 26, The combination of embodiment 25, wherein:

[0687] 1) the one or more gRNAs in (b) has a spacer sequence of 15 to 17 nucleotides; and / or 2) the one or more gRNAs in (c) has a spacer sequence of 18 to 24 nucleotides.

[0688] Embodiment 27, The combination of embodiment 25 or 26, wherein the combination comprises one to three fusion proteins.

[0689] Embodiment 28, The combination of any one of embodiments 25-27, further comprising a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene.

[0690] 121

[0691] #14686672vlEmbodiment 29, The combination of any one of embodiments 25-28, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.

[0692] Embodiment 30, The combination of any one of embodiments 25-29, wherein

[0693] the first gene is selected from CIDEB, PNPLA3, MARC 1, and / or DGAT2; and / or the second gene is selected from CIDEB and HSD17B13.

[0694] Embodiment 31 , The combination of any one of embodiments 25-30, wherein the first gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 837-1011.

[0695] Embodiment 32, The combination of any one of embodiments 25-30, wherein the first gene is a PNPLA3 gene, optionally wherein the one or more gRNAs targeting the PNPLA3 each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1012-1028.

[0696] Embodiment 33, The combination of any one of embodiments 25-30, wherein the first gene is a MARC_1 gene, optionally wherein the one or more gRNAs targeting the MARC_1 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1029-1033.

[0697] Embodiment 34, The combination of any one of embodiments 25-30, wherein the first gene is a DGAT2 gene, optionally wherein the one or more gRNAs targeting the DGAT2 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1034-1065.

[0698] Embodiment 35, The combination of any one of embodiments 25-34, wherein the second gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1066-1189.

[0699] Embodiment 36, The combination of any one of embodiments 25-34, wherein the second gene is a HSD17B13 gene, optionally wherein the one or more gRNAs targeting the HSD17B13 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1190-1281.

[0700] 122

[0701] #14686672vlEmbodiment 37, The combination of any one of embodiments 25-36, wherein the one or more fusion proteins collectively further comprise a transcriptional repressor domain.

[0702] Embodiment 38, The combination of embodiment 37, wherein the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3.

[0703] Embodiment 39, The combination of any one of embodiments 25-38, wherein the DNMT3A domain comprises an H3 tail.

[0704] Embodiment 40, The combination of any one of embodiments 25-39, wherein the one or more fusion proteins comprises one or more NLS.

[0705] Embodiment 41, The combination of any one of embodiments 25-40, wherein the one or more fusion proteins comprises one or more linkers.

[0706] Embodiment 42, The combination of any one of embodiments 25-41, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS - XTEN16 - KRAB.

[0707] Embodiment 43, The combination of any one of embodiments 25-41, wherein the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS - XTEN16 - KRAB - BipartiteNLS.

[0708] Embodiment 44, The combination of any one of embodiments 25-41, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS.

[0709] Embodiment 45, The combination of any one of embodiments 25-41, wherein the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS.

[0710] Embodiment 46, The combination of any one of embodiments 25-41, wherein the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 819-830 and 836.

[0711] 123

[0712] #14686672vlEmbodiment 47, The combination of any one of the embodiments 25-46, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0713] Embodiment 48, A pharmaceutical composition comprising the combination of any one of embodiments 25-47.

[0714] Embodiment 49, The combination of any one of embodiments 25-47, wherein the cell is a mammalian cell, optionally a human cell, further optionally wherein the cell is a human hepatocyte.

[0715] Embodiment 50, A method of modifying transcription, expression and / or activity of one or more gene in a human hepatocyte, comprising introducing the combination of any one of embodiments 25-47 into the cell.

[0716] Embodiment 51 , A method of treating a human in need thereof, comprising administering to the human the combination of any one of embodiments 25-47 or the pharmaceutical composition of embodiment 48.

[0717] Embodiment 52, A combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising:

[0718] (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins;

[0719] (b) one or more guide RNAs (gRNAs) having a spacer sequence mismatch pattern that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and

[0720] (c) one or more gRNAs having a spacer sequence mismatch pattern that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

[0721] 124

[0722] #14686672vlEmbodiment 53, The combination of embodiment 52, wherein:

[0723] 1) the one or more gRNAs in (b) has a spacer sequence mismatch patterns of 1 to 10 nucleotides; and / or

[0724] 2) the one or more gRNAs in (c) has a spacer sequence with no mismatches.

[0725] Embodiment 54, The combination of embodiment 52 or 53, wherein the combination comprises one to three fusion proteins.

[0726] Embodiment 55, The combination of any one of embodiments 52-54, further comprising a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene.

[0727] Embodiment 56, The combination of any one of embodiments 52-55, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.

[0728] Embodiment 57, The combination of any one of embodiments 52-56, wherein

[0729] the first gene is PCSK9; and / or

[0730] the second gene is selected from LPA and ANGPTL3.

[0731] Embodiment 58, The combination of any one of embodiments 52-57, wherein the first gene is a PCSK9 gene, optionally wherein the one or more gRNAs targeting the PCSK9 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 17-86 and 831-834.

[0732] Embodiment 59, The combination of any one of embodiments 52-58, wherein the second gene is a LPA gene, optionally wherein the one or more gRNAs targeting the LPA gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 87-677 and 835.

[0733] Embodiment 60, The combination of any one of embodiments 52-58, wherein the second gene is an ANGPTL3 gene, optionally wherein the one or more gRNAs targeting the ANGPTL3 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 678-794.

[0734] Embodiment 61, The combination of any one of embodiments 52-60, wherein the one or more fusion proteins collectively further comprise a transcriptional repressor domain.

[0735] 125

[0736] #14686672vlEmbodiment 62, The combination of embodiment 61, wherein the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3.

[0737] Embodiment 63, The combination of any one of embodiments 52-62, wherein the DNMT3A domain comprises an H3 tail.

[0738] Embodiment 64, The combination of any one of embodiments 52-63, wherein the one or more fusion proteins comprises one or more NLS.

[0739] Embodiment 65, The combination of any one of embodiments 52-64, wherein the one or more fusion proteins comprises one or more linkers.

[0740] Embodiment 66, The combination of any one of embodiments 52-65, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS - XTEN16 - KRAB.

[0741] Embodiment 67, The combination of any one of embodiments 52-65, wherein the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS - XTEN16 - KRAB - BipartiteNLS.

[0742] Embodiment 68, The combination of any one of embodiments 52-65, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS.

[0743] Embodiment 69, The combination of any one of embodiments 52-65, wherein the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS.

[0744] Embodiment 70, The combination of any one of embodiments 52-65, wherein the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 819-830 and 836.

[0745] Embodiment 71, The combination of any one of embodiments 52-70, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s)

[0746] 126

[0747] #14686672vlcomprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0748] Embodiment 72, A pharmaceutical composition comprising the combination of any one of embodiments 52-71.

[0749] Embodiment 73, The combination of any one of embodiments 52-71, wherein the cell is a mammalian cell, optionally a human cell, further optionally wherein the cell is a human hepatocyte.

[0750] Embodiment 74, A method of modifying transcription, expression and / or activity of one or more gene in a human hepatocyte, comprising introducing the combination of any one of embodiments 52-71 into the cell.

[0751] Embodiment 75, A method of treating a human in need thereof, comprising administering to the human the combination of any one of embodiments 52-71 or the pharmaceutical composition of embodiment 72.

[0752] Embodiment 76, A combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising:

[0753] (a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins;

[0754] (b) one or more guide RNAs (gRNAs) having a spacer sequence with a mismatch pattern that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and

[0755] (c) one or more gRNAs having a spacer sequence with a mismatch pattern that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

[0756] Embodiment 77, The combination of embodiment 76, wherein:

[0757] 1) the one or more gRNAs in (b) has a spacer sequence mismatch pattern of 1 to 10 nucleotides; and / or

[0758] 127

[0759] #14686672vl2) the one or more gRNAs in (c) has a spacer sequence with no mismatches.

[0760] Embodiment 78, The combination of embodiment 76 or 77, wherein the combination comprises one to three fusion proteins.

[0761] Embodiment 79, The combination of any one of embodiments 76-78, further comprising a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene.

[0762] Embodiment 80, The combination of any one of embodiments 76-79, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.

[0763] Embodiment 81, The combination of any one of embodiments 76-80, wherein

[0764] the first gene is selected from CIDEB, PNPLA3, MARC 1, and / or DGAT2; and / or the second gene is selected from CIDEB and HSD17B13.

[0765] Embodiment 82, The combination of any one of embodiments 76-81, wherein the first gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 837-1011.

[0766] Embodiment 83, The combination of any one of embodiments 76-81, wherein the first gene is a PNPLA3 gene, optionally wherein the one or more gRNAs targeting the PNPLA3 each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1012-1028.

[0767] Embodiment 84, The combination of any one of embodiments 76-81, wherein the first gene is a MARC_1 gene, optionally wherein the one or more gRNAs targeting the MARC_1 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1029-1033.

[0768] Embodiment 85, The combination of any one of embodiments 76-81, wherein the first gene is a DGAT2 gene, optionally wherein the one or more gRNAs targeting the DGAT2 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1034-1065.

[0769] 128

[0770] #14686672vlEmbodiment 86, The combination of any one of embodiments 76-85, wherein the second gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1066-1189.

[0771] Embodiment 87, The combination of any one of embodiments 76-85, wherein the second gene is a HSD17B13 gene, optionally wherein the one or more gRNAs targeting the HSD17B13 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1190-1281.

[0772] Embodiment 88, The combination of any one of embodiments 76-87, wherein the one or more fusion proteins collectively further comprise a transcriptional repressor domain.

[0773] Embodiment 89, The combination of any one of embodiments 88, wherein the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3.

[0774] Embodiment 90, The combination of any one of embodiments 76-89, wherein the DNMT3A domain comprises an H3 tail.

[0775] Embodiment 91, The combination of any one of embodiments 76-90, wherein the one or more fusion proteins comprises one or more NLS.

[0776] Embodiment 92, The combination of any one of embodiments 76-91, wherein the one or more fusion proteins comprises one or more linkers.

[0777] Embodiment 93, The combination of any one of embodiments 76-92, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS - XTEN16 - KRAB.

[0778] Embodiment 94, The combination of any one of embodiments 76-92, wherein the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS - XTEN16 - KRAB - BipartiteNLS.

[0779] 129

[0780] #14686672vlEmbodiment 95, The combination of any one of embodiments 76-92, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS.

[0781] Embodiment 96, The combination of any one of embodiments 76-92, wherein the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker -horseDNMT3L - XTEN80 - activeCas9 - NLS.

[0782] Embodiment 97, The combination of any one of embodiments 76-92, wherein the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 819-830 and 836.

[0783] Embodiment 98, The combination of any one of embodiments 76-97, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

[0784] Embodiment 99, A pharmaceutical composition comprising the combination of any one of embodiments 76-98.

[0785] Embodiment 100. The combination of any one of embodiments 76-98, wherein the cell is a mammalian cell, optionally a human cell, further optionally wherein the cell is a human hepatocyte.

[0786] Embodiment 101. A method of modifying transcription, expression and / or activity of one or more gene in a human hepatocyte, comprising introducing the combination of any one of embodiments 76-98 into the cell.

[0787] Embodiment 102, A method of treating a human in need thereof, comprising administering to the human the combination of any one of embodiments 76-98 or the pharmaceutical composition of embodiment 99.

[0788] Embodiment 103, The combination of any one of embodiments 1-20 or the pharmaceutical composition of embodiment 21 for use in therapy.

[0789] 130

[0790] #14686672vlEmbodiment 104, The combination of any one of embodiments 25-47 or the pharmaceutical composition of embodiment 48 for use in therapy.

[0791] Embodiment 105, The combination of any one of embodiments 52-47 or the pharmaceutical composition of embodiment 72 for use in therapy.

[0792] Embodiment 106, The combination of any one of embodiments 76-98 or the pharmaceutical composition of embodiment 99 for use in therapy.

[0793] Embodiment 107, The combination of any one of embodiments 1-20 or the pharmaceutical composition of embodiment 21 for use in treating hypercholesterolemia.

[0794] Embodiment 108, The combination of any one of embodiments 25-47 or the pharmaceutical composition of embodiment 48 for use in treating metabolic dysfunction-associated steatohepatitis (MASH).

[0795] Embodiment 109, The combination of any one of embodiments 52-47 or the pharmaceutical composition of embodiment 72 for use in treating hypercholesterolemia.

[0796] Embodiment 110. The combination of any one of embodiments 76-98 or the pharmaceutical composition of embodiment 99 for use in treating metabolic dysfunction-associated steatohepatitis (MASH).

[0797] Embodiment 111. Use of the combination of any one of embodiments 1-20 or the pharmaceutical composition of embodiment 21 in the manufacture of a medicament for use in therapy.

[0798] Embodiment 112, Use of the combination of any one of embodiments 25-47 or the pharmaceutical composition of embodiment 48 in the manufacture of a medicament for use in therapy.

[0799] Embodiment 113, Use of the combination of any one of embodiments 52-71 or the pharmaceutical composition of embodiment 72 in the manufacture of a medicament for use in therapy.

[0800] 131

[0801] #14686672vlEmbodiment 114, Use of the combination of any one of embodiments 76-98 or the pharmaceutical composition of embodiment 99 in the manufacture of a medicament for use in therapy.

[0802] Embodiment 115, Use of the combination of any one of embodiments 1-20 or the pharmaceutical composition of embodiment 21 in the manufacture of a medicament for the treatment of hypercholesterolemia.

[0803] Embodiment 116, Use of the combination of any one of embodiments 25-47 or the pharmaceutical composition of embodiment 48 in the manufacture of a medicament for the treatment of metabolic dysfunction-associated steatohepatitis (MASH).

[0804] Embodiment 117, Use of the combination of any one of embodiments 52-47 or the pharmaceutical composition of embodiment 72 in the manufacture of a medicament for the treatment of hypercholesterolemia.

[0805] Embodiment 118. Use of the combination of any one of embodiments 76-98 or the pharmaceutical composition of embodiment 99 in the manufacture of a medicament for the treatment of metabolic dysfunction-associated steatohepatitis (MASH).

[0806] EXAMPLES

[0807] Example 1. Testing truncated guides targeting PCSK9 in Hep3B cells

[0808] Guides targeting PCSK9 from Table 1 were tested using a CHARM KRABLESS fusion protein. Cells were transfected with 12.5 ng of fusion protein and 25 ng of guide. Controls included lipid only, CRISPRi+ RNA041 (a guide known to direct epigenetic editing of PCSK), KRABLESS CHARM + RNA041+RNA049 (two guides known to direct epigenetic editing of PCSK9), mouse(m)PCSK9, WT Cas9 + RNA041, effector only, and CRISPROff + RNA041 (50 ng total, 1:1 ratio), and GFP. Guides were designed to be truncated from the N terminus and were 20, 18, 17, and 16 nts long. Four different RNA modification patterns were used, including 2 end modifications, 1 end modifications, no modifications, and heavy modifications. Results of silencing were observed at Day 7 and

[0809] 132

[0810] #14686672vlDay 14. Results for two modification patterns are shown in FIGs. 1A-1B (Day 7) and FIGs.

[0811] 2A-2B (Day 14) The no modification and 1 end modification pattern lost durability and / or exhibited toxicity. Many guide truncations with MOD002 (heavy modifications, described in International PCT application No. PCT / US2024 / 035873) and MOD086 (2 end modifications) retained durable silencing up to Day 14.

[0812] Example 2. Further testing of truncated guides targeting PCSK9

[0813] Truncated guides targeting PCSK9 from Table 1 were tested in this Example.

[0814] Briefly, guide spacers were truncated down to 16 nucleotides in length and transfected with an active Cas9 containing CHARM (aCHARM). Five days later, PCSK9 expression was measured. The results, shown in FIG. 3, demonstrate that robust PCSK9 silencing is observed. In a similar experiment, the effect of truncation on indel rates was examined. Full-length and truncated guides were transfected with aCHARM and taken down at Day 5 and assayed for indel rate and silencing. The results, presented in FIG. 4, show that the indel rates for truncated guides are nearly ablated as compared to their full-length counterparts, and the majority showing 0 indels across all amounts used. Therefore, aCHARM can achieve high indel rates with full-length guides.

[0815] All 12 guides were run in a dose-response experiment to identify the most potent candidate in a first group of guides. As is shown in FIG. 5 and below, each of the guides tested showed reduced potency, which appears to be guide-dependent.

[0816]

[0817] In another experiment, amplicons were built to assess the indel rate at exon 2 of Lp(a). Active Cas9 CHARM (aCHARM) constructs that contain or do not contain KRAB (KRABless) were transfected into Hep3B cells. Indel rates of greater than 50% were observed for the KRABless aCHARM group (FIG. 6B).

[0818] Polyfunctional editing next was examined. Truncated gRNA RNA056 was used for PCSK9 epigenetic editing and a guide for Lp(a) editing (SEQ ID NO: 835). PCSK9 silencing with polyfunctional editors was measured (FIG. 7). Control samples demonstrated no genetic editing as shown by no indels at either Lp(a) or PCSK9 genome sequences (FIGs. 8A-8B) Combinations of a truncated single guide (truncated) with dead Cas9 CHARM 133

[0819] #14686672vl(dCHARM; FIG. 9A) and wild-type Cas9 with an Lp(a) guide (FIG. 9B) demonstrated epigenetic editing and no gene editing or Lp(a) gene editing with no epigenetic editing, respectively. The polyfunctional editor produced both epigenetic editing and Lp(a) gene editing (FIG. 9C) in the presence of both guides. Moreover, the dilution effect of the polyfunctional editor was examined (FIG. 9D) and demonstrated that the polyfunctional editor had a lower percentage of indels compared to wild-type Cas9 and Lp(a) guide at both dosage levels tested.

[0820] Truncated guides were further examined and it was determined that the majority of potency loss was due to truncation of the guide, rather than the activation of Cas 9 (FIG. 10 and FIG. 11).

[0821] Next, further guides were selected based on their respective off-target predictions. Previous guides tested were from a combination of original CRISPR-Off leads and hits from a CHARM screen. Five additional single guides were identified through a guide screening process. They were observed to have potency levels similar to those in guides RNA041 / 049 in Hep3B cells. Each was tested in full-length and in a truncated (16 nt) format.

[0822] CHARM

[0823]

[0824]

[0825] OFF

[0826]

[0827]

[0828] 134

[0829] #14686672vlAll five guides were run in dose response (truncation compared to full-length) and, of the five truncated guides, three were selected for follow up: RNA6798, RNA6789, and RNA6801. The truncated forms (16 nt) of the selected guides were then screened against the gold-standard 20 nt guide and RNA6801 was found to closely match the 20 nt guide (FIG. 12).

[0830] A dual guide screen for 16-nucleotide guides was performed in Hep3B TdTomato PCSK9 cell line at a sub-saturating dose (1.5 ng mRNA and 1.5 ng gRNA). The results demonstrated that dual guide combinations at sub-saturating doses silence comparably to the gRNA041 / 049 (“gold standard”) combination (FIG. 19).

[0831] Therefore, as described above, polyfunctional editors were demonstrated to work as evidenced by simultaneous epi-silencing at PCSK9 (without indels), and indels at Lp(a). Potency loss from spacer truncations was found to be guide-dependent, while the data suggests that activating Cas9 in CHARM does not affect potency.

[0832] Further experiments test new target(s) for gene editing with functional readouts and test dual guide and mRNA:guide ratios to further increase potency.

[0833] Example 3. Spacer length and epi-silencing with CHARM

[0834] This Example provides determining how potency and specificity of epi-silencing is impacted by guide spacer length. Spacers between four and 20 nucleotides in length were tested for silencing of PCSK9 (in Hep3B cells) using KRABless CHARM (SEQ ID NO:836). With a single dose of 12.5 ng mRNA and 6.25 gRNA, the potency of epi-silencing was maintained down to 11 nt spacers for PCSK9 at Day 6 (FIG. 13A). Note that none of the gRNA molecules used were modified. This effect was found to be durable, lasting through at least Day 13 (FIG. 13B) and Day 20 (FIG. 14).

[0835] Further experiments tested the potency and durability of guides using spacers between 4 and 20 nucleotides in length for epi-silencing of another gene target, RFXAP (in T cells) using a single dose of 1500 ng of guides with MOD002 modification with 1500 ng of mRNA encoding KRABless CHARM. Note that the gRNA molecules used were modified with MOD002. Similar to the former experiments with PCSK9, the potency of epi-silencing was maintained down to 11 nt spacers and was found to be durable, lasting through at least Day 21 (FIG. 23). Similar effects were shown for other gRNAs targeting the RFXAP gene.

[0836] Further experiments tested the dose response of guides using spacers between 4 and 20 nucleotides in length for epi-silencing of PCSK9 (in Hep3B cells) using guides with MOD002 modification with KRABless CHARM. Results of silencing were observed at Day 135

[0837] #14686672vl6. The shorter 12 and 14 nt truncated spacers for PCSK9 generally showed greater potency compared to 16 nt spacers.

[0838] Example 4. Second locus polyfunctional editing

[0839] This Example demonstrates polyfunctional editing at a second locus with a functional reduction in both biomarkers. The two targets were ANGPTL3 and PCSK9. The “gold standard” epigenetic editing combination (dCHARM with truncated RNA041 / 049 at a 2: 1 : 1 ratio) showed epigenetic editing and no gene editing (FIG. 15A). The “gold standard” gene editing combination (wild-type Cas9 with a full-length ANGPTL3 guide) showed ANG gene editing and no epigenetic editing (FIG. 15B). The polyfunctional editor (aCHARM), truncated guide (16nt, RNA6801), and the ANGPTL3 guide (full-length) at a 2:1:1 ratio) demonstrated both epigenetic editing of PCSK9 and gene editing of ANGPTL3 (FIG. 15C). A reduction in potency was observed in PCSK9 silencing when two guides were used in the polyfunctional editor at Day 7 (FIG. 16). Unexpectedly, the same result was not observed with respect to ANGPTL3 expression. Instead of an approximately 2-fold reduction in ANGPTL3 silencing due to the dilution effect from the presence of both guides, no difference was observed between the polyfunctional editor and wild-type Cas9 at Day 7 (FIG. 17A) or Day 14 (FIG. 17B). This data was recapitulated in a second experiment (FIGs. 18A and 18B) ANGPTL3 gene editing guides have been shown to silence ANGPTL3 at Day 7, but not at Day 14 (FIGs. 20, 21A, and 21B).

[0840] Example 5. Testing mismatched guides targeting PCSK9 in Hep3B cells

[0841] Guides targeting PCSK9 from Table 1 are tested using a CHARM KRABLESS fusion protein. Cells are transfected with 12.5 ng of fusion protein and 25 ng of guide.

[0842] Controls include lipid only, CRISPRi+ RNA041 (a guide known to direct epigenetic editing of PCSK9), KRABLESS CHARM + RNA041 + RNA049 (two guides known to direct epigenetic editing of PCSK9), mouse(m)PCSK9, WT Cas9 + RNA041, effector only, and CRISPROff + RNA041 (50 ng total, 1:1 ratio), and GFP. Guides are designed to be mismatched at the distal end and comprise 1-10 mismatched nucleotides per guide tested. Four different RNA modification patterns are used, including 2 end modifications, 1 end modification, no modifications, and heavy modifications. Results of silencing are observed at Day 7 and Day 14. Many guides with mismatches retain durable silencing up to Day 14.

[0843] 136

[0844] #14686672vlExample 6. Testing polyfunctional versus orthogonal editing

[0845] This Example demonstrates that polyfunctional editing showed better dual silencing of two gene targets compared with orthogonal editing. The two gene targets were PCSK9 and ANGPTL3. Poly functional editing and orthogonal editing were compared in doseresponse experiments using two orthogonal conditions to epigenetically silence both PCSK9 and ANGPTL3 expression using aCHARM (polyfunctional editing), epigenetically silence PCSK9 using dSpCas9:CHARM and edit ANGPTL3 using SaCas9 (orthogonal editing condition 1), or epigenetically silence PCSK9 using dSaCas9: CHARM and edit ANGPTL3 using SpCas9 (orthogonal editing condition 2).

[0846] Cells were transfected with the following reagents: For polyfunctional editing 1) The polyfunctional editor active sp:CHARM (aCHARM), gRNA1937 (14 nt), SpANGPTL3 at a mRNA:Guide ratio of= 2:1:1 (FIG. 25A); 2) for orthogonal editing (Sp:EE and Sa:Cut) dSpCas9: CHARM, wild-type SaCas9, RNA041 / 049, and SaANGPTL3 at a mRNA:Guide ratio 1 : 1 : 1 : 1 (FIG. 25B); or 3) orthogonal editing (Sp:Cut and Sa:EE) WT SpCas9 + dSaCas9: CHARM, SaPCCSK9, and SpANGPTL3 at a mRNA: Guide ratio of 1 : 1 : 1 : 1 (FIG.

[0847] 25C). The results, show that at high doses, all conditions robustly silenced both ANGPTL3 and PCSK9 targets. SpCas9 was shown to silence both targets better than SaCas9 regardless of platform. However, polyfunctional editing allowed for universal SpCas9 use and showed superior dual silencing.

[0848] Example 7. Testing truncated guides targeting DGAT2 in HepG23B cells

[0849] Guides targeting DGAT2 are tested using a CHARM KRABLESS fusion protein. HepG2 cells are transfected with 12.5 ng of fusion protein and 25 ng of guide. Controls included lipid only, CRISPRi+ RNA001, KRABLESS CHARM + RNA001+RNA002 (two guides to direct epigenetic editing of DGAT2), mouse(m) DGAT2, WT Cas9 + RNA001, effector only, and CRISPROff + RNA041 (50 ng total, 1 : 1 ratio), and GFP. Guides are designed to be truncated from the N terminus and are 20, 18, 17, and 16 nts long. Four different RNA modification patterns are used, including 2 end modifications, 1 end modifications, no modifications, and heavy modifications. Results of silencing are observed at Day 7 and Day 14. Many guides with truncations retain durable silencing up to Day 14.

[0850] 137

[0851] #14686672vlExample 8. Testing mismatched guides targeting DGAT2 in HepG23B cells Testing polyfunctional versus orthogonal editing

[0852] Guides targeting DGAT2 are tested using a CHARM KRABLESS fusion protein. HepG2 cells are transfected with 12.5 ng of fusion protein and 25 ng of guide. Controls include lipid only, CRISPRi+ RNA001 (a guide known to direct epigenetic editing of DGAT2), KRABLESS CHARM + RNA001 + RNA002 (two guides known to direct epigenetic editing of DGAT2), mouse(m)DGAT2, WT Cas9 + RNA001, effector only, and CRISPROff + RNA001 (50 ng total, 1:1 ratio), and GFP. Guides are designed to be mismatched at the distal end and comprise 1-10 mismatched nucleotides per guide tested. Four different RNA modification patterns are used, including 2 end modifications, 1 end modification, no modifications, and heavy modifications. Results of silencing are observed at Day 7 and Day 14. Many guides with mismatches retain durable silencing up to Day 14.

[0853] 138

[0854] #14686672vlLIST OF SEQUENCES

[0855] Sequences disclosed in the present disclosure are listed below.

[0856] Table 6. Sequence Description

[0857]

[0858] 139

[0859] #14686672vl

Claims

CLAIMS1. A combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising:(a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins;(b) one or more guide RNAs (gRNAs) having a spacer sequence with a length that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and(c) one or more gRNAs having a spacer sequence with a length that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

2. A combination for modifying transcription, expression and / or activity of one or more gene in a cell, the combination comprising:(a) one or more fusion proteins each comprising a catalytically active CRISPR / Cas endonuclease domain, wherein the one or more fusion proteins collectively comprise a DNMT3 A domain or a domain serving to recruit DNMT3 A and a DNMT3L domain, or polynucleotide(s) encoding the one or more fusion proteins;(b) one or more guide RNAs (gRNAs) having a spacer sequence mismatch pattern that allows epigenetic editing and not gene editing of a first gene in the cell, or polynucleotide(s) coding for the one or more gRNAs; and(c) one or more gRNAs having a spacer sequence mismatch pattern that allows gene editing of a second gene in the cell, or polynucleotide(s) coding for the one or more gRNAs.

3. The combination of claim 1, wherein:1) the one or more gRNAs in (b) has a spacer sequence of 15 to 17 nucleotides; and / or2) the one or more gRNAs in (c) has a spacer sequence of 18 to 24 nucleotides.

4. The combination of claim 2, wherein:140#14686672vl1) the one or more gRNAs in (b) has a spacer sequence mismatch patterns of 1 to 10 nucleotides; and / or2) the one or more gRNAs in (c) has a spacer sequence with no mismatches.

5. The combination of any one of claims 1-4, wherein the combination comprises one to three fusion proteins.

6. The combination of any one of claims 1-5, further comprising a donor DNA comprising 5’ and 3’ arms that are homologous to sequences in the second gene.

7. The combination of any one of claims 1-6, wherein the endonuclease domain is derived from a Cas9 protein, optionally SpCas9.

8. The combination of any one of claims 1-7, whereinthe first gene is PCSK9; and / orthe second gene is selected from LPA and ANGPTL3.

9. The combination of any one of claims 1-8, wherein the first gene is a PCSK9 gene, optionally wherein the one or more gRNAs targeting the PCSK9 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 17-86 and 831-834.

10. The combination of claim 9 wherein the second gene is a LPA gene, optionally wherein the one or more gRNAs targeting the LPA gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 87-677 and 835.

11. The combination of claim 9, wherein the second gene is an ANGPTL3 gene, optionally wherein the one or more gRNAs targeting the ANGPTL3 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 678-794.

12. The combination of any one of claims 1-6, whereinthe first gene is selected from CIDEB, PNPLA3, MARC 1, and / or DGAT2; and / or the second gene is selected from CIDEB and HSD17B13.141#14686672vl13. The combination of any one of claims 1-6 or claim 12, wherein the first gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 837-1011.

14. The combination of any one of claims 1-6 or claim 12, wherein the first gene is a PNPLA3 gene, optionally wherein the one or more gRNAs targeting the PNPLA3 each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1012-1028.

15. The combination of any one of claims 1-6 or claim 12, wherein the first gene is a MARC_1 gene, optionally wherein the one or more gRNAs targeting the MARC_1 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1029-1033.

16. The combination of any one of claims 1-6 or claim 12, wherein the first gene is a DGAT2 gene, optionally wherein the one or more gRNAs targeting the DGAT2 gene each comprise a spacer targeting the sequence of one of SEQ ID NOs: 1034-1065.

17. The combination of any one of claims 1-6 or claims 12-16, wherein the second gene is a CIDEB gene, optionally wherein the one or more gRNAs targeting the CIDEB gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1066-1189.

18. The combination of any one of claims 1-6 or claims 12-17, wherein the second gene is a HSD17B13 gene, optionally wherein the one or more gRNAs targeting the HSD17B13 gene comprise a spacer targeting the sequence of one of SEQ ID NOs: 1190-1281.

19. The combination of any one of claims 1-18, wherein the one or more fusion proteins collectively further comprise a transcriptional repressor domain.

20. The combination of claim 19, wherein the transcriptional repressor domain is a Kriippel-associated box (KRAB) domain, optionally derived from human Koxl or ZIM3.

21. The combination of any one of claims 1-20, wherein the DNMT3A domain comprises an H3 tail.142#14686672vl22. The combination of any one of claims 1-21, wherein the one or more fusion proteins comprises one or more NLS.

23. The combination of any one of claims 1-22, wherein the one or more fusion proteins comprises one or more linkers.

24. The combination of any one of claims 1-23, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS -XTEN16 - KRAB.

25. The combination of any one of claims 1-23, wherein the one or more fusion proteins comprises the configuration bipartiteNLS - cdDNMT3a - Linker - horseDNMT3L - XTEN80 - activeCas9 - NLS - XTEN16 - KRAB - BipartiteNLS.

26. The combination of any one of claims 1-23, wherein the one or more fusion proteins comprises the configuration H3 Tail - Maxiflex40 - AsDNMT3L - NLS - activeCas9 - NLS.

27. The combination of any one of claims 1-23, wherein the one or more fusion proteins comprises the configuration BipartiteNLS - cdDNMT3a - Linker - horseDNMT3L -XTEN80 - activeCas9 - NLS.

28. The combination of any one of claims 1-23, wherein the one or more fusion proteins comprises the sequence of one of SEQ ID NOs: 819-822 and 836.

29. The combination of any one of claims 1-28, wherein the gRNA(s) are chemically modified, optionally wherein the chemically modified gRNA(s) comprise phosphorothioate internucleoside linkages at the 5’ and / or 3’ ends, and / or 2’-O-methyl nucleotides.

30. A pharmaceutical composition comprising the combination of any one of claims 1-29.

31. The combination of any one of claims 1-29, wherein the cell is a mammalian cell, optionally a human cell, further optionally wherein the cell is a human hepatocyte.143#14686672vl32. A method of modifying transcription, expression and / or activity of one or more gene in a human hepatocyte, comprising introducing the combination of any one of claim 1-29 into the cell.

33. A method of treating a human in need thereof, comprising administering to the human the pharmaceutical composition of claim 30.144#14686672vl

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